Fig 2: Verification of ADAM10 and ADAM17 knockdown efficiency. Efficient stable, lentiviral ADAM10 (shADAM10), ADAM17 (shADAM17) and ADAM10/17 (shADAM10/17) double knockdown was verified by quantitative real-time PCR (a,d) and western blot analyses in Y79 (b,c) and WERI-Rb1 cells (e,f). Indicated intensity ratios relative to ß-actin, used as a loading control, were calculated using MICRO MANAGER 1.4 software. Values are means of three independent experiments ± SEM. *** p < 0.001 and **** p < 0.0001 statistical differences compared to the control (ctr) group calculated by Student’s t-test.

Fig 3: Effects of ADAM10/17 single and double knockdown on cell viability and proliferation of RB cells. Stable ADAM10 (shADAM10), ADAM17 (shADAM17) and ADAM10/17 (shADAM10/17) knockdown decreases cell viability and proliferation levels of Y79 and WERI-Rb1 cells compared to control cells (ctr) as revealed by WST-1 assays (a), and BrdU stains (b,c). Values are means of three independent experiments ± SEM. ns p > 0.05; ** p < 0.01; *** p < 0.001 and **** p < 0.0001 statistical differences compared to the control (ctr) group calculated by Student’s t-test.

Fig 4: Analysis of L1CAM ectodomain shedding after ADAM10/17 single and double knockdown in Y79 and WERI-Rb1 cell lines. Western blot analysis of ADAM mediated L1CAM ectodomain (L1–200) shedding in cell culture supernatant of Y79 and WERI-Rb1 cells 72 h after ADAM10 (shADAM10), ADAM17 (shADAM17) single and ADAM10/17 (shADAM10/17) double knockdown in comparison to control cells (ctr). Representative western blots of L1 shedding upon ADAM knockdown (a) and quantification of the L1CAM ectodomain expression reveals a significant reduction of L1-200 shedding after ADAM10/17 single and double knockdown in the RB cell lines Y79 and WERI-Rb1 (b). Indicated intensity ratios relative to ß-actin, used as a loading control, were calculated using MICRO MANAGER 1.4 software. Values are means of three independent experiments ± SEM. * p < 0.05 and ** p < 0.01 statistical differences compared to the control (ctr) group calculated by Student’s t-test.

Fig 5: Effects of ADAM10/17 single and double knockdown on apoptosis and anchorage independent growth of RB cell lines. Stable ADAM10 (shADAM10), ADAM17 (shADAM17) and ADAM10/17 (shADAM10/17) knockdown increases caspase mediated apoptosis levels of Y79 and WERI-Rb1 cells compared to control cells (ctr) as revealed by DAPI cell counts (a) and caspase3/7 assays (b). Both RB cell lines show significantly reduced colony formation capacity (c) and colony sizes (d,e) after ADAM10 and ADAM 17 single or double knockdown as revealed by soft agarose assays. Values are means of three independent experiments ± SEM. ns > 0.05; * p < 0.05; ** p < 0.01; *** p < 0.001 and **** p < 0.0001 statistical differences compared to the control (ctr) group calculated by Student’s t-test.