Effects of Siberian fir terpenes extract Abisil on antioxidant activity, autophagy, transcriptome and proteome of human fibroblasts

Reagents and cell treatment

Abisil in the form of resin was kindly provided by Initium-Pharm LLC (Moscow, Russia; https://initium-pharm.com/). Abisil was dissolved in DMSO to obtain a stock for treatment of cell cultures.

All cells were maintained in Dulbecco's modified Eagle's medium (DMEM)(PanEco, Russia) supplemented with 10% fetal bovine serum (FBS, Biosera) and 50 U/ml penicillin and 50 μg/ml streptomycin (PanEco) in a humidified atmosphere containing 5% CO₂. Immediately prior to treatment, Abisil stock was diluted in serum-free DMEM; treatment was performed in DMEM containing 5% FBS when cells were 50-70% confluent. Negative controls were treated with an equal amount of DMSO diluted in DMEM supplemented with 5% FBS.

Oxidative stress assays

MRC5-SV40 cells were seeded at a density of 4×10⁴ cells/well in 12-well plates. Next day, Abisil treatment was performed in a wide range of concentrations (6-200 μg/ml). After 16 hours, cell oxidative stress was induced by treatment with 1 μM of rotenone (Sigma-Aldrich, USA) for 1.5 hours. After that, the cells were stained with 10 μM DHE (dihydroethidium, Sigma-Aldrich) for 35 min at 37° C. The cells were trypsinized and resuspended in PBS (PanEco) supplemented with 2% FBS. Before measurement cells were stained with 1 μg/ml of DAPI (Sigma-Aldrich) as a viability assay. Fluorescence was detected using a BD LSR Fortessa cytofluorimeter (Beckman Dickinson). The analysis was performed using Flowing Software 2.0 (Perttu Terho, Turku Center for Biotechnology). The results were based on three independent experiments, each with three biological replicates.

Mitochondrial potential assessment

MRC5-SV40 and LECH-4 cells were seeded at a density of 4×10⁴ cells/well in 12-well plates. Next day, Abisil treatment was performed with a wide range of concentrations (10-80 μg/ml). After 16 hours, the cells were stained with 100 nM and 200 nM of TMRM (Thermo Fisher Scientific, USA), respectively, for 30 min at 37° C. 10 μM of CCCP (Sigma-Aldrich) was used as positive control. Before measurement cells were stained for viability with 1 μg/ml of DAPI (Sigma-Aldrich). Fluorescence was detected using a BD LSR Fortessa cytofluorimeter (Beckman Dickinson). The analysis was performed using Flowing Software 2.0 (Perttu Terho, Turku Center for Biotechnology). The results were based on three independent experiments with three biological replicates.

Autophagy and mitophagy assays

Autophagy was assessed on a panel of cell lines stably expressing mCherry-GFP-LC3 reporter frame. Treatment with Abisil was carried out in concentration 50 μg/ml for 16 hours. Rapamycin (R0395, Sigma-Aldrich) was used as a positive control.

To detect mitophagy in MRC5-SV40 and LECH-4 cells, concentrations of 50 μg/ml of Abisil were used for treatment for 24 hours. After incubation, the cells were stained with 200 nM of MitoTracker Red CMXRos (Thermo Fisher Scientific) and 100 nM of LysoTracker Green (Thermo Fisher Scientific) for 20 minutes at 37° C.

Confocal microscopy was performed on the Nikon A1+MP confocal imaging system using an Apo TIRF 60x/1.49 oil DIC objective (numerical aperture=1.49; Nikon Japan), Apo LWD 40x/1.15 S water immersion objective (numerical aperture=1.15; Nikon Japan). Images were scanned sequentially using 488- and 561-nm diode lasers in combination with a DM405/488/561/633-nm dichroic beam splitter. Differential Interference Contrast Imaging (DIC) microscopy was used to visualize cell contours. The images were analyzed with NIS-elements AR (Nikon Japan).

Quantitative real-time PCR

To analyze the level of mitochondrial DNA, total cellular DNA was extracted from the cells using GeneJET Genomic DNA Purification Kit (Thermo Fisher Scientific) and subsequently quantified by qPCR. qPCRmix-HS SYBR kit (Evrogen) was used according to the manufacturer’s instructions, and the real-time PCR was performed on the Rotor-Gene Q Real-Time PCR system (Quigen). The real-time qPCR results were analyzed with 2^−ΔΔCt method traditionally used for gene expression studies. We used genomic sequence of B2M gene as an endogenous control. All primer pairs used for qPCR are presented in the Table 1.

Measurement of mitochondrial function using the Seahorse XF-24 extracellular flux analyzer

MRC5-SV40 cells were cultured in DMEM medium (Gibco, Thermo Fisher Scientific) containing 4.5 g/L glucose, supplemented with 10% bovine serum (BioSera) and 2 mM glutamine (PanEco). 40 hours before the experiment, the cells were seeded on 24-well Seahorse XFe24 Cell Culture Microplate (Agilent Technologies,) at a density of 12 000 cells per well in 500 μl of growth medium without affecting comparison wells A1, B3, C4 and D6 and incubated at +37° C in a CO₂ incubator with a relative humidity of 95% and a CO₂ content of 5%. When the cells reached 45% of the monolayer (24 hours after seeding) they were treated with Abisil at a concentration of 50 μg/ml and incubated for 16 hours at +37° C in a CO₂ incubator with a relative humidity of 95% and a CO₂ content of 5%. On the day of the MitoStress Test experiment, the growth medium was removed, the cells were washed with warm DMEM medium without sodium bicarbonate and supplemented with 25 mM glucose, 2 mM sodium pyruvate (PanEko, Moscow, Russia) and 2 mM glutamine (PanEko, Moscow, Russia) (2 times 300 μl), after which the experiment medium (described above, 500 μl per well) was added and incubated for 45 minutes at +37° C in a non-CO₂ incubator. The experiment medium was also added to the comparison wells. The following preparations were added to the ports of the Agilent Seahorse XF24 Sensor Cartridge prepared in accordance with the manufacturer's recommendations: Port A - 11 μM oligomycin (1 μM final concentration in well), Port B - 24 μM FCCP (2 μM final concentration in well) and Port C - 13 μM Rot/AA (1 μM final concentration in well). Then, the Oxygen Consumption Rates (OCR) were measured (3 measurements per injection) at +37° C on the Seahorse Bioanalyzer instrument. After the measurement, the experiment medium was removed, 25 μl of RIPA buffer was added and the protein concentration was measured using a Bradford Reagent (Sigma B6916). OCR measurements were normalized to the total protein level.

On the day of the GlycoTest experiment, the growth medium was removed, the cells were washed with warm DMEM w/o sodium bicarbonate and supplemented with 2 mM sodium pyruvate (PanEco) and 2 mM glutamine (PanEco; 2 times 300 μL), added experiment medium (500 μl per well) and incubated for 30 minutes at +37° C in a non-CO₂ incubator. The experiment medium was also added to the comparison wells. The following preparations were added to the ports of the Agilent Seahorse XF24 Sensor Cartridge prepared in accordance with the manufacturer's recommendations: Port A - 275 mM Glucose (25 mM final concentration in well), Port B - 12 μM oligomycin (1 μM final concentration in well) and Port C - 650 mM 2-deoxy-D-glucose (50 mM final concentration). Then, the Extracellular Acidification Rate (ECAR) was measured (3 measurements per injection) at +37° C on the Seahorse Bioanalyzer instrument. After the measurement, the experiment medium was removed, 25 μl of RIPA buffer was added, and the protein concentration was measured with a Bradford Reagent (Sigma B6916). ECAR measurements were also normalized to the total protein level.

Evaluating cytotoxic effect of Abisil with MTS assay

MRC5-SV40 cells were seeded at a density of 5×10³ cells/well in a 96-well plate and cultured in 100 μl of DMEM supplemented with 5% FBS at 37° C. 24 hours after seeding cells were treated with Abisil (0; 9; 11; 15; 25; 50; 93; 125, 147 and 186 μg/ml) within 24 hours at 37° C. The level of cell metabolic activity was evaluated using the MTS assay (CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay (MTS), Promega) in accordance with the manufacturer's protocol. The optical density of the stained substance at 590 nm, proportional to the metabolic rate of viable cells, was measured using a plate reader (Tecan). Then, the IC50 values were calculated by the regression analysis, as well as the concentrations at which the most intense cellular metabolism was observed.

Western blotting

MRC5-SV40 cells were cultured on 35 mm Petri dishes with confluency of 50%, treatment with Abisil was carried out in 5% DMEM supplemented with 5% FBS at 37° C overnight. After incubation the cells were washed with cold PBS and lysed in 200 μl of Reporter Lysis Buffer (Promega) containing a Roche protease inhibitor cocktail (# P8349, Sigma-Aldrich), according to the manufacturer's instructions. Lysates were centrifuged for 15 minutes at 12,000 g; total protein content in the protein supernatant was measured by the Bradford assay. Proteins were separated by Laemmli's electrophoresis in a 12% SDS-polyacrylamide gel, electrotransfer to a PVDF membrane (Amersham Hybond P 0.45 μm, Amersham Biosciences, GE Healthcare) was performed in Towbin transfer buffer (25 mM Tris, 192 mM glycine, 20% (v/v) ethanol, pH 8.3). Membranes were blocked in a 4% solution of skim milk in PBST (PBS with 0.05% Tween 20) for 1 hour at room temperature. Incubation with primary and secondary (goat anti-rabbit IgG-HRP, sc-2004, Santa Cruz Biotechnology) antibodies was performed overnight at 4° C and for 1 hour at room temperature, respectively. Immobilon Western Chemiluminescent HRP Substrate (Merck-Millipore, Sigma-Aldrich) was used for detection; the chemiluminescence signal was determined using the Bio-Rad ChemiDoc MP Imaging System (Bio-Rad). Quantification of protein bands was performed using ImageJ software (NIH, Bethesda, MD, USA). The following primary antibodies were used: β-actin (D6A8, #8457, Cell Signaling Technology), LC3 (D11, #3868, Cell Signaling Technology).

Transcriptomic analysis

Sample preparation

MRC5-SV40 cells were plated on 60 mm Petri dishes (3×10⁵ cells per a dish) and cultured overnight at 37° C in DMEM supplemented with 10% FBS. Next day, treatment with Abisil was carried out with concentrations of 5 μg/ml and 50 μg/ml in DMEM with 5% FBS. Control samples were incubated in DMEM with 5% FBS and 0.025% DMSO. After 16 hours, the medium was removed and the cells were washed with PBS, followed by lysis using 500 μl of lysis buffer (MagNA Pure Compact RNA Isolation Kit, Roche). All treatment experiments were performed in triplicate.

RNA isolation, preparation and sequencing of RNA-Seq libraries

Total RNA was extracted using the RNeasy Mini kit (Qiagen, Germany) according to the manufacturer's protocol. The quality and quantity of RNA was determined using an Agilent 2100 bioanalyzer (Agilent Technologies, USA). RNA samples with RINs above 9.0 were used to prepare libraries. Total RNA (2 μg) from each sample was used to prepare the mRNA library using the TruSeq RNA Sample Preparation Kit v2 Low Sample (LS) protocol (Illumina, USA) in accordance with the manufacturer's protocol. The cDNA libraries were sequenced (single end reads, 50 bp) using Illumina NextSeq 500 system in the "Genome" Center (EIMB RAS, Russia, http://www.eimb.ru/rus/ckp/ccu_genome_c.php).

RNA-Seq data processing and differential expression analysis

Raw reads were checked for quality and trimmed using the FastQC and Trimmomatic [18] tools. The trimmed reads were mapped to the human reference genome (GRCh38.80, Ensembl) with splice-aware STAR mapper [19]. Next, 5’-to-3’ transcript read coverage distribution was evaluated using geneBody_coverage.py script from RSeQC package [20] in order to ensure the absence of 3’-tail bias. Read counting per gene was done using featureCounts tool from the Subread package [21]. The further analysis was performed in R environment using edgeR (identification of differentially expressed genes), biomaRt (annotation), topGO and clusterProfiler (GO, KEGG, Reactome enrichment analyses), pathview (visualization) and other packages.

Proteomic analysis

Sample preparation

The MRC5-SV40 line was also used for panoramic proteomic analysis. 3×10⁵ cells were plated on 60 mm Petri dishes. After a night of incubation in the complete medium 16-hour treatment with Abisil at concentration of 50 μg/ml in DMEM medium with 5% FBS was performed. Control samples were treated with DMEM with 5% FBS and 0.025% DMSO. After 16 hours the medium was removed and the cells were washed twice with PBS, then the cells were removed from the dishes in 800 μl PBS using a scraper.

The sample preparation for proteomic analysis was performed using Pierce Mass Spec Sample Prep Kit (Thermo Fisher Scientific, MA, USA) [22]. In addition, Thermo Scientific Pierce Digestion Indicator from the same kit, was used to assess the digestion reproducibility for multiple samples. For sample preparation 1×10⁶ cells (100 μg of protein) were lysed using modified radioimmunoprecipitation assay buffer (RIPA), which was followed by reduction step using 2M dithiothreitol for 45 min at 50° C. For alkylation step iodoacetamide was added to the sample (final volume 50 mM) and incubated at room temperature for 20 min. Immediately after incubation 4 volumes (460 μl) of pre-chilled (-20° C) 100% acetone was added to samples. Two-step enzymatic protein digestion was performed using Lys-C (enzyme-to-substrate ratio = 1:100, 37° C for 2 hours) and trypsin (enzyme-to-substrate ratio = 1:50, 37° C for 17 hours). To remove the digestion buffer samples were dried at the SpeedVac and resuspended in 0.1% TFA buffer. Protein concentration was measured using bicinchoninic acid assay (BCA assay) [23].

LC-MS/MS

LC-MS/MS was performed using an TIMS TOF Pro mass spectrometer (Bruker Daltonics, USA) combined with an UltiMate 3000 nano LC system (Thermo Fisher Scientific, MA, USA). The amount of loaded sample was 100 ng per injection. The tryptic peptide fraction (injection volume 1 μL) was analyzed in triplicate on a Dionex UltiMate 3000 Nano HPLC System (Thermo Fisher Scientific, MA, USA) coupled to a TiMS TOF mass spectrometer (Bruker Daltonics, Bremen, Germany) using a captive spray ion source (positive ion mode, 1600 V) (Bruker Daltonics, Bremen, Germany). HPLC separation was performed on a C18 capillary column (25cm x 75μm 1.6μm) (Ion Optics, Parkville, Australia) by gradient elution. The mobile phase A was 0.1% formic acid diluted in water and mobile phase B was 0.1% formic acid diluted in acetonitrile. The separation was carried out by a 120-min gradient from 4% to 90% of phase B at a flow rate of 0.4 μL/min. Mass-spectrometric measurements were carried out using Parallel Accumulation Serial Fragmentation (PASEF) acquisition method. The measurements were carried out in m/z range from 100 to 1700. The range of ion mobility include a value from 0.60 – 1.60 Vs/cm2 (1/k0). The number of precursor ions per one PASEF cycle (1.2 sec) was 10. Low abundant precursor ions can be analyzed multiple times during PASEF cycle.

Protein identification and quantification

For label-free analysis the obtained MS spectra were processed with MaxQuant software against the SwissProt 2018 database [24]. Oxidation of methionine and acetylation of N-terminal were selected as variable modification. Minimal length for peptide identification was seven amino acids and at least one peptide should be unique for a protein. False discovery rate (FDR) threshold for proteins and peptides was set 0.01. Additionally, match between the runs option were used.

Identification of altered proteins

Protein quantification was carried out using label free quantitation intensities (LFQ) based on relative protein quantification across all samples and represented by a normalized intensity profile that is generated according to the algorithms described by Cox, J. et al. [25]. Statistical processing of proteomic data was performed in R environment. Two samples Welch's t-test was applied to identify the significantly altered proteins between treated and control groups. Correlation between proteins composition in technical runs was estimated using Pearson’s correlation analysis for log-transformed abundance values (the values of correlation coefficient were about 0.9).

Pathway enrichment analysis

Differentially regulated proteins were divided into several groups for the pathway enrichment analysis: (1) upregulated: FDR < 0.05 (Welch's t-test), two-fold ore greater fold change; (2) downregulated: FDR < 0.05, two-fold ore greater fold change; (3) a group of marginally altered proteins with FDR < 0.05 and fold change less than two-fold. Each protein group was analyzed separately for GO enrichment. Protein interaction network analysis was done using STRINGdb [26]. The results of GO enrichment analysis was grouped by semantic similarity using the REVIGO tool [27].

Data availability

Transcriptome sequencing data are available at NCBI SRA portal (BioProject identifier PRJNA642179).

Cytotoxicity of different concentrations of Abisil

The effect of 48-hour treatment with Abisil on the metabolic activity of cells was studied using the MTS assay on human embryonic lung fibroblast lines (MRC5-SV40 and LECH-4). Abisil demonstrated significant dose dependence (Figure 1). The calculated IC50 values were 149.3 ± 0.67 for MRC5-SV40 and 152.6 ± 0.52 μg/ml for LECH-4, respectively (Table 2). In addition, both cell lines showed a significant increase in metabolic activity (p-value <0.001) when treated with the low drug concentrations (9—15 μg/ml).

Antioxidant activity of Abisil

After a series of experiments, DHE staining method (described in the materials and methods) was chosen as optimal approach to evaluate the levels of ROS after treatment with Abisil in a wide concentration range. This method demonstrated good reproducibility (range of variation 10-25%).

The diagram on the Figure 2 shows the ROS levels assessment results after a 16-hour treatment with Abisil in the range of concentrations 25-125 μg/ml.

The results demonstrate that the most intense decrease in ROS after induction by rotenone is observed for concentrations of 25 and 50 μg/ml (Figure 2). It is worth noting that there is a significant prooxidant effect at higher concentrations of the drug (125 μg/ml). There is also a tendency towards a decrease in ROS in the absence of inductor.

Abisil causes decrease in mitochondrial potential

As Abisil downregulates genes encoding for subunits of the respiratory complex I (according to RNA-Seq data; described below; Supplementary Figure 2), it was tested if Abisil might cause the decrease of mitochondrial potential. Indeed, Abisil treatment induced more than 2 times-fold drop of mitochondrial potential (p-value < 0.01, Mann-Whitney U-test; Figure 3). As a positive control, we used carbonyl cyanide chlorophenylhydrazone (CCCP). This is a lipid-soluble weak acid, mitochondrial toxin and a potent mitochondrial uncoupling agent. It increases the proton permeability across the mitochondrial inner membrane, thus dissipating the transmembrane potential and depolarizing the mitochondria.

Abisil treatment results in intense autophagy after 24 hours

Autophagy was assessed by the amount of LC3II in MRC5-SV40 cells by western blotting (Figure 4A). Figure 4B shows the results of LC3II densitometric analysis with respect to internal control (beta-actin) which are presented as mean ± standard deviation calculated from three independent experiments. There is a significant increase in the content of LC3II complexes (more than 10-fold) when treated with Abisil at concentration of 50 μg/ml, which shows effective stimulation of autophagy.

To carry out an accurate quantitative assessment of the effectiveness of autophagy, a 3D reconstruction of a set of fields was carried out using a z-stack approach on a confocal microscope. 20 fields were used for each of sample type: control (0.5% DMSO), treated with Abisil 50 μg/ml, treated with chloroquine 50 μM and treated with both Abisil and chloroquine). An example of the images obtained is shown below (Figures 5, 6). Data on the number of large autophagosomes are shown in the diagram (Figure 7). As you can see below, autophagy is induced efficiently in 24 hours after treatment.

Abisil does not increases the level of mitophagy and doesn’t affect the number of mtDNA copies

The decrease of mitochondrial potential caused by Abisil treatment might imply that Abisil induces mitophagy [28–30]. In order to verify this hypothesis, MRC5-SV40 cells were treated with Abisil and tested whether Mitotracker Red colocalized with Lysotracker Green (Figures 8, 9). There was an increase of the number of phagolysosomes, but they did not colocalize with Mitotracker Red indicating that Abisil promotes autophagy but does not specifically induce mitophagy in SV40 transformed MRC5. The same treatment was conducted on LECH-4 cells with the same result (data not shown).

Changes in mitochondrial potential detected in previous experiments suggested to test also the copy number of mitochondria DNA. As can be seen from the diagram in Figure 10, the amount of mitochondrial DNA did not change at all.

Abisil induce changes in cell energy metabolism

Using the Seahorse XF-24 extracellular flux analyzer, we analyzed changes in mitochondrial respiration and glycolysis rates after between Abisil-treated and control cells. The results are presented at the Figure 11. While the rate of oxygen consumption drops slightly, the glycolytic activity is significantly reduced. Thus, we see that both the current activity of glycolysis and its maximum rate, possible for the cell (glycolytic capacity), decrease proportionally.

Transcriptomic changes induced by Abisil treatment

We performed RNA-Seq analysis for MRC5-SV40 cells treated with Abisil at concentrations 5 and 50 μg/ml as well as control cells (Supplementary Tables 1–4). Taken at both concentrations, Abisil treatment induced vast transcriptomic shift spanning several thousand genes. The effect of treatment with both concentrations was generally similar (see the Supplementary Figure 3). Surprisingly, at a concentration of 5 μg/ml, the effect was even more pronounced. In this case, 2075 genes passed FDR < 0.05 threshold and had at least 1.5-fold expression level changes. We noted a predominant decrease in expression. So, among the top 200 differentially expressed genes (DEGs), 189 were downregulated. The lists of DEGs between 5 and 50 μg/ml significantly overlapped. The spectra of enriched Gene Ontology terms also overlapped. At both concentrations of Abisil, the lists of downregulated DEGs were strongly enriched with genes involved in cell cycle and related processes. In addition, we noted overrepresentation of genes involved in mRNA processing, protein folding, fatty acid elongation, NIK/NF-κβ and WNT signaling, antigen processing and presentation, cellular response to hypoxia.

Among biological processes, participants of which were overrepresented in upregulated genes, we noticed Notch signaling pathway, positive and negative regulation of apoptotic process, heterocycle catabolic process and other processes. We noticed bidirectional expressional changes of genes encoding for ribosomal proteins, but it is hard to say about the prevalence of any direction.

Interestingly, KEGG enrichment analysis revealed significant overrepresentation of downregulated DEGs among participants of oxidative phosphorylation, DNA replication process, basal transcription factors, spliceosome subunits, proteasome subunits (for both Abisil concentrations). Figure 12 shows the transcriptomic effect of Abisil treatment on genes involved in several mostly affected KEGG pathways. For a more detailed info, see the Supplementary Table 9.

The comparison of samples treated with different Abisil concentrations (5 and 50 μg/ml) revealed 1281 genes that passed FDR < 0.05 threshold, but only 305 of them had 1.5-fold or greater expression level changes. The lists of DEGs demonstrated enrichment in genes involved in the regulation cell proliferation and growth, cytoskeleton organization, RNA splicing, cell metabolism, including biosynthesis of amino acids, chemotaxis and cell adhesion, extracellular matrix organization, ion transport. The complete results of GO enrichment analyses are presented in the Supplementary Tables 5–8.

Mainly, the effect of Abisil on biological processes and cellular pathways can be described as: 1) suppressing genes participating in oxidative phosphorylation, DNA replication (especially at low Abisil concentrations), mRNA transcription and splicing, RNA transport, fatty acid biosynthesis, protein export, proteolysis; 2) activating phosphatidylinositol signaling, cytosolic DNA-sensing pathway, PPAR signaling pathway, various pathways involved in the immune response; 3) bidirectional impact on cell cycle (predominantly, downregulation) and endocytosis.

Two pathways, cell cycle and oxidative phosphorylation, require special attention (Figure 13 and Supplementary Figure 2). The pattern of transcriptomic impact of Abisil on genes participating in cell cycle is ambiguous, although in general downregulation strongly prevails. This is especially evident when cells were treated with Abisil in low concentration (Figure 13). One can see a decrease in the expression levels of cyclins CycA, CycB, CycH, as well as cyclin-dependent kinases CDK4, CDK6, CDK7 and other genes that contribute to cell cycle progression. However, against this background, a decrease in the expression levels of several genes involved in the negative regulation of the cell cycle (Kip1, Kip2, PCNA, Rb, etc.) is also noted.

Regarding the oxidative phosphorylation pathway, it is remarkably, that Abisil induces not only reducing mitochondrial potential itself (see the Figure 3), but also reduces the expression of genes participating oxidative phosphorylation. (Supplementary Figure 2) Taken at both concentrations, it causes a decrease in the expression mainly for genes of the mitochondrial genome: NADH dehydrogenase (ND1-6) and cytochrome C oxidase (COX1-3). Expression levels of genes from the cell nuclear genome, whose products are involved in oxidative phosphorylation (NDUFA, NDUFB, succinate dehydrogenase, ATPase), are altered to a much lesser extent. Both up- and downregulations are noted mainly for treatment with high concentration of Abisil (Supplementary Figure 2).

Worth noting, Abisil treatment results in predominant downregulation of many genes encoding for kinases and kinase-associated proteins (KKPs; containing “kinase” keyword in a gene name). Among top 50 differentially expressed KKPs after treatment with Abisil at 5 μg/ml, 35 are downregulated, and the average log2(fold change) for all these 50 genes is -0.45. The concentration 50 μg/ml gives less pronounced result.

Thus, such effect is more pronounced for low concentration of the drug (5 μg/ml). Moreover, we noticed that 27 of 30 (90%) KKPs downregulated after 50 μg/ml treatment (FDR < 0.05) lies within the list of 70 downregulated KKPs after 5 μg/ml treatment (FDR < 0.05). These 27 KKPs are: BUB1, MAP2K4, MNAT1, LYN, AXL, JAK1, CSNK2A2, AK6, TAB2, SNRK, AK3, CLK1, DYRK1A, CDKN3, CDK6, FASTKD3, SH3KBP1, PRKACB, MOB1B, TTK, PXK, MAP3K20, PRKRA, RPS6KC1, PRKD3, NRK, PIK3R1.

Figure 14 illustrates the results of the protein-protein interaction network analysis for top 200 genes that are downregulated after Abisil treatment (5 μg/ml). We can see several net modules, that are responsible for translation, mRNA processing, regulation of cell cycle and other activities. Even though Abisil stimulates autophagy (as demonstrated above), however, at the transcriptome level, we do not see a consistent and unidirectional change in the expression of genes involved in autophagy, mTOR and AMPK signaling.

Analysis of panoramic proteomic sequencing results

Large-scale quantitative proteome profiling was performed to reveal differentially expressed proteins in MRC5-SV40 cells treated with Abisil (50 μg/ml) compared to non-treated ones. Total 4906 proteins have been detected either for treated or non-treated cells. As it was noticed for transcriptome profiling, we observed that Abisil treatment mainly induces downregulation of expression. 4002 proteins were detected in non-treated MRC5-SV40 cells and only 3068 in Abisil-treated (2164 proteins were detected in both conditions). The detailed data on the proteome profiling is presented in the Supplementary Table 10.

Gene Ontology, KEGG and Reactome overrepresentation analysis revealed that the list of top 300 down-regulated proteins is enriched with participants of cell cycle regulation, exocytosis, secretion. Both lists, top 300 down- and top 300 up-regulated proteins were highly enriched with participants of RNA processing, translation, mitochondrial organization, oxidative phosphorylation, cell metabolism, transport, gene expression regulation, histone modification, membrane trafficking, ER to Golgi anterograde transport, and many other biological processes.

Protein-protein interaction (PPI) networks for down- and up-regulated proteins is presented in the Supplementary Figures 4, 5. Both networks are highly enriched with PPIs (p < 10^-16) according to the STRING database analysis results. We can see that proteins, participating in translation, mitochondrial organization, RNA processing, form compact subnets (marked with colors), whereas proteins involved in cell metabolism and intracellular transport are spread over the whole network.

To sum up the analysis, it is possible to conclude that the content of many proteins in the cell decreases (possibly, because of autophagy) after Abisil treatment. The expression of genes/proteins, involved in translation, mitochondrial organization, gene expression regulation, cell metabolism and transport, is significantly altered (either down- or upregulated).

It is remarkably that the content of such proteins as mTOR, RICTOR (Rapamycin-insensitive companion of mTOR), Cytochrome c, RBL1 (Retinoblastoma-like protein 1), Ras-related protein Rab-13, TGF-beta receptor type-1 (TGFBR1), Tumor necrosis factor receptor superfamily member 12A (TNFRSF12A), Cyclin-dependent kinase 9 (CDK9), Mitogen-activated protein kinases (MAPK14, MAP3K6, MAP3K2, MAPK8, MAPK3, MAP4K5, MAPK14) and Renin receptor (ATP6AP2) was significantly reduced (or even not detected by MS) after treatment with Abisil. However, the expression of some MAPKs (MAP4K4, MAP3K7, MAPK9, ZAK, MAPK1) was upregulated (or was detected only in non-treated cells).