Figure 2Analysis of viability of HCT116 cells assessed 96 h after transfection with independent non-targeting siRNA (two different duplexes used, siCTRL#1 or #2) or targeting VPS4A (duplexes #1 or #2), VPS4B (duplexes #1 or #2) or both VPS4 (various combinations of siVPS4A+siVPS4B duplexes). Data are means of 3 independent experiments ±SEM. All values were normalized, averaged (avrg) viability of siCTRL#1 and #2-transfected cells was set as 100%, the Kruskal-Wallis test followed by Dunn's multiple comparison post hoc test; ns - non-significant (p≥0.05), **p<0.01. Left panel, analysis of clonogenic growth of HCT116 cells assessed 15 days after transfection with non-targeting (siCTRL#1), VPS4A- or/and VPS4B-targeting siRNAs used in various duplex combinations as indicated. Right panel, images of HCT116 clones taken at the day of clonogenic growth assessment. Data are means of 3 independent experiments ±SEM. NT - non-transfected. All values were normalized, clonogenic growth of siCTRL#1-transfected cells was set as 1. One-sample t-test; ns - non-significant (p≥0.05), ****p<0.0001. Viability of RKO and SW480 cells assessed as in (A), growth of DLD-1 cells assessed in BrdU incorporation assay 96 h after siRNA transfection. Various independent non-targeting (siCTRL#1 or #2) and VPS4A- or B-targeting siRNA duplexes were used (#1, #2, #5 or #1, #2, respectively). All values were normalized, averaged (avrg) viability of siCTRL#1 and #2-transfected cells was set as 100%. Data are means of 3 independent experiments ±SEM. The Kruskal-Wallis test followed by Dunn's multiple comparison post hoc; ns - non-significant (p≥0.05), **p<0.01, ***p<0.001. VPS4B copy number and dependency scores of selected cancer cell lines obtained in CRISPR/Cas9 and RNAi screens deposited in the DepMap portal (https://depmap.org/portal/). VPS4B copy number status estimated across different cell lines using TaqMan assay. The whiskers represent the minimal and maximal copy number in a given triplicate readout. The BCL2 gene localized on 18q in the close vicinity to VPS4B was analyzed as a control. VPS4B and VPS4A protein abundance in selected normal and cancer cell lines analyzed by immunoblotting. Vinculin and GAPDH were used as loading controls. Analysis of viability of HOP62 lung cancer cells assessed 144 h after transfection with independent non-targeting siRNA (two different duplexes used, siCTRL#1 or #2) or targeting VPS4A (duplexes #2 or #5), VPS4B (duplexes #1 or #2) or both VPS4 (various combinations of siVPS4A+siVPS4B duplexes). Data are means of 4 independent experiments ±SEM. All values were normalized, cell viability of averaged (avrg) siCTRL#1- and #2-transfected cells was set as 100%. Two-tailed unpaired t-test; ****p<0.0001. Top panel, analysis of clonogenic growth of HOP62 cells assessed 14 days after transfection with various siRNA duplexes as indicated. Bottom panel, images of HOP62 clones taken at the day of clonogenic growth assessment. Data are means of 4 independent experiments ±SEM. All values were normalized, colony area of averaged (avrg) siCTRL#1 and #2-transfected cells was set as 1. The Mann-Whitney U test; **p<0.01. Analysis of viability of SNU410 pancreatic cancer cells assessed 168 h after transfection with independent non-targeting siRNA (three different duplexes used, siCTRL#1, #2 or #3) or targeting VPS4A (duplexes #2, #4 or #5), VPS4B (duplexes #1 or #2) or both VPS4 (various combinations of siVPS4A+siVPS4B duplexes). Data are means of 3 independent experiments ±SEM. All values were normalized, cell viability of averaged (avrg) siCTRL#1-, #2- and #3-transfected cells was set as 100%. Two-tailed unpaired t-test; ****p<0.0001.