Research Paper Volume 11, Issue 19 pp 8526—8541

Figure 2. NINJ2 shRNA inhibits human CRC cell survival and proliferation. HT-29 cells (A–K) or the primary human colon cancer cells (“pri-Can-1/-2/-3”, L-N) were infected with lentiviral particles encoding applied NINJ2 shRNA (“Seq1/2/3”) or non-sense control shRNA (“shC”), stable cells were established following puromycin selection; Expression of NINJ2 mRNA (A and L), NINJ1 mRNA (B) and listed proteins (C) were shown; Cell survival was tested by MTT assay (D and M); Cell proliferation was tested by BrdU incorporation assay (E and N), soft agar colony formation assay (F) and EdU staining (G); Cell apoptosis was tested by Annexin V-PI FACS assay (H, results quantified in I), Western blotting of apoptosis-related proteins (J) and TUNEL staining (K). For all the in vitro functional assays, the exact same number of viable cells with different genetic modifications were initially plated into each well/dish (at Day-0, same for all figures). NINJ1 and NINJ2were normalized to the loading control Tubulin (C). “Ctrl” stands for the parental control cells (same for all Figures). For each assay, n=5. * P< 0.05 vs. “shC” cells. Experiments in this figure were repeated three times, and similar results were obtained. Bar= 200 μm (G and K).