Fig. 3: Non-cell autonomous upregulation of anti-apoptotic BCL-2 proteins requires MEK-ERK signalling.

a HeLa tBID2A cells were untreated or treated with 500 nM venetoclax in combination with the indicated inhibitors for 48 h, harvested and protein expression was analysed by western blot (representative blot of two independent repeats). b HeLa tBID2A cells were untreated or treated with 500 nM venetoclax in combination with 500 nM trametinib for 48 h, harvested and protein expression was analysed by western blot (representative blot of three independent repeats). c HeLa tBID2A cells were untreated or treated with 500 nM venetoclax in combination with 500 nM trametinib, harvested and RNA expression was analysed by RT-qPCR. n = 3 independent experiments; mean values ± s.e.m.; Tukey corrected one-way ANOVA. d Supernatant from untreated or venetoclax treated HeLa tBID2A cells was supplemented with 500 nM trametinib before addition onto recipient cells. After 48 h of incubation, recipient cells were harvested and protein expression analysed by western blot (representative blot of three independent repeats). e Supernatant from untreated or venetoclax treated HeLa tBID2A cells was added onto recipient cells. After the indicated times, recipient cells were harvested and protein expression analysed by western blot. Treatment with EGF served as a positive control (representative blot of two independent repeats). f Control or ERK1/2^CRISPR HeLa tBID2A cells were treated directly with venetoclax or with supernatant from control or ERK1/2^CRISPR HeLa tBID2A cells as indicated before harvesting and western blot analysis for protein expression (representative blot of three independent repeats). g Supernatant from control or venetoclax treated HeLa tBID2A cells was supplemented with 500 nM trametinib as indicated before addition onto recipient cells. After 48 h of incubation, the recipient cells were treated with 500 nM venetoclax and 500 nM S63845 and survival was monitored by Sytox green exclusion and livecell imaging. n = 3 independent experiments; mean values ± s.e.m.; Tukey corrected one-way ANOVA. h HeLa cells were treated with the indicated drugs (Etoposide 50 µM, Doxorubicin 2 µM, Paclitaxel 1 µM) for 3 h, followed by 45 h incubation in regular medium. Then the supernatant was harvested, filtered and supplemented or not with trametinib (500 nM) before addition onto recipient cells. After 48 h, cells were harvested and protein expression analysed by western blot. Fold change normalised to loading control is stated below (representative blot of three independent repeats).