Fig. 3
bCAFs produced IL-6 contributes to their protective effects. a ZR-75-1 cells were treated for 48 h with ABT-737 (1 µM) in presence of non-CM or CAFs-CM with or without IL-6 neutralizing antibody (anti IL-6). b ZR-75-1 or T-47D cells were treated for 48 h with ABT-737 (1 µM) in presence of non-CM, control (empty vector) or IL-6 shRNA CAFs-CM. For rescue, recombinant IL-6 (rIL-6; 500 pg/ml) was added on cancer cells within the CM (IL-6 shRNA + rIL-6) or previously during the media conditioning (IL-6 shRNA in presence of rIL-6) (as schematized in the bottom right panel). c ZR-75-1 or T-47D cells were treated for 48 h with ABT-737 1 µM in presence of non-CM, CAFs-CM (untreated), CAFs-CM co-treated with Stattic 8 µM (Stattic 8 µM) or CAFs-CM pre-incubated with Stattic 8 µM during CAFs media conditioning (Stattic pre-incubated). d Top: MCL-1, P-ERK, and ERK expression levels were evaluated by western-blot in ZR-75-1 cells treated for 24 h with ABT-737 (1 µM) alone or in combination with U0126 (5 µM) in presence of non-conditioned media or CAF-conditioned media. MCL-1, P-STAT-3 and STAT-3 expression levels were evaluated by western-blot in ZR-75-1 cells treated for 24 h with ABT-737 (1 µM) in presence of non-conditioned media or CAF-conditioned media. Bottom: ZR-75-1 cells were treated for 48 h with ABT-737 (1 µM) alone or in combination with U0126 (5 µM) or Stattic (8 µM) in presence of non-conditioned media or fibroblasts-conditioned media (NHLF, NF, CAFs). Percentage of positive Annexin-V-FITC cells was measured by flow cytometry. e In a co-culture model, ZR-75-1 cells (1/3) and CAFs (2/3) were treated with ABT-737 1 µM and U0126 5 µM alone or in combination, in DMEM 0.5% FBS for 48 h. CAFs were stained with anti CD-90 PE (bottom). Percentage of positive Annexin-V-APC apoptotic cells was measured by flow cytometry in each of the two cell populations (ZR-75 or CAF) (top). Data are means ± SEM from three independent experiments. P-value was determined by two-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns: not significant
