Fig. 1: Therapy induces senescence (TIS) increase MERCS, but decreases the ER-mitochondria Ca²⁺ flux, which becomes essential for TIS cell survival.

A Schematic diagram of induction and establishment of therapy-induced senescence. B Representative Western blot of p16 and p21 in growing and senescence cells induce with doxorubicin (Sen Doxo) and etoposide (Sen Eto). Bar graph: p16/actin and p21/actin expressed as average fold change over basal levels (growing cells). N = 5, Mean ± S.E., *p < 0.05 (ANOVA Test). C Representative images of SA-β-gal staining in growing, Sen Doxo and Sen Eto and quantitative analysis of stained cells (100 cells per N, N = 3; ****p ≤ 0.0001, ANOVA test). Bar: 50 μm. D Concentration of IL6, IL-1α, IL-1β, MMP3, CXCL1, determined by Luminex assay in supernatant of growing, Sen Doxo and Sen Eto cells. (N = 3, ****p ≤ 0.0001, ANOVA Test). E Representative images of growing, Sen Doxo and Sen Eto labeled with KDEL-BFP to visualize ER and 100 nM TMRE to visualize mitochondria. Bar: 10 µm. Bar graph: Manders index as an indicator of colocalization (20 – 30 cells per N; N = 3; **p ≤ 0.01 *** p ≤ 0.005, ANOVA test). F Representative TEM images and quantification of contact surface area between ER and mitochondria and the distance between ER and mitochondria in growing sen Doxo and Sen Eto. Arrow heads show MERCS (N = 3, 50–70 mitochondria per N, *p < 0.05, ANOVA test). G Representative traces of mitochondrial Ca²⁺ responds in growing, Sen Doxo and Sen Eto cells challenge with histamine using Rhod-2AM and MitoGCamp5. Bar graphs: Quantification of peak Rhod-2AM (N = 3. Mean ± SEM. 50–80 cells were analyzed in each experiment) and MitoGCamp5 (N = 3. mean ± SEM. 20 cells were analyzed in each experiment) fluorescence. ****p ≤ 0.0001, ANOVA test). H Representative traces of cytoplasmic Ca²⁺ signals in growing and Sen Doxo cells challenge with FCCP using Fluo-4AM. Bar graph: Quantification of peak Fluo-4AM fluorescence (N = 3. Mean ± SEM. 20 cells were analyzed in each experiment. *p < 0.05, t test). I Confocal image of GEM-GECO1_mito fluorescence from growing and Sen Doxo IMR90 cells captured at wavelengths 470–500 nm, at wavelengths >520 nm and their overlay. The bar graph compares mean resting [Ca²⁺]_mito (estimated as F470–500/F > 520) in growing and Sen Doxo IMR90 cells (N = 3. mean ± SEM. 12 cells were analyzed, ***P < 0.001, t test). J Representative traces of cytoplasmic Ca²⁺ signals in growing, Sen Doxo and Sen Eto cells challenge with histamine using Fluo-4AM. Bar graph: Quantification of peak Fluo-4AM fluorescence (N = 3. Mean ± SEM. 50–80 cells were analyzed in each experiment. ****p ≤ 0.0001 ANOVA test). K Representative Western blot of MERCS proteins in growing, Sen Doxo, and Sen Eto. Bar graphs are expressed as average fold change over basal levels (growing cells). N = 3–6, mean ± S.E. (ANOVA test). L Representative images of Proximity Ligation Assay (PLA) of the 3 IP3R isoforms with VDAC1, and ER marker PDI with VDAC1 in growing, Sen Doxo and Sen Eto cells. Bar graphs: Quantitative analysis of number of dots per cells (N = 3. mean ± SEM. 20–50 cells were analyzed in each experiment. ****p ≤ 0.0001, ANOVA test). M Representative traces of cytoplasmic Ca²⁺ signals using Fluo-4AM in growing, Sen Doxo and Sen Eto cells treated with 5 µM dmXeB for 1 h and challenge with histamine. (N = 3. Mean ± SEM. 10–15 cells were analyzed in each experiment). N LIVE/DEAD assay to evaluate cellular viability (N = 4; ****p ≤ 0.0001, *** p ≤ 0.005. ANOVA test). O Representative fluorescence images of p16-3MR mice liver treated with two rounds of Doxo (30 mg/k) followed by four rounds of either dmXeB (30 mg/k) or ganciclovir. Bar graph: Quantitative analysis of fluorescence intensity (N = 4. Means ± SEM. Control: 404.2 ± 19.6, Doxo: 444.6 ± 11.6, Doxo + dmXeB: 379.5 ± 9.09 and Doxo + GCV: 407.5 ± 32.0 A.U. ANOVA test). P Upper panel. Representative images of SA-β-gal staining of p16-3MR mice subcutaneous fat treated with two rounds of Doxo (30 mg/k) followed by four rounds of either DmXeB (30 mg/k) or ganciclovir. Bottom panel. Subcutaneous fat with an optical mask used for analysis. Bar graph: Quantitative analysis of relative intensity (N = 4. Means ± SEM. Control: 39.24 ± 13.54, Doxo: 177.5 ± 12.98, Doxo + dmXeB: 54.79 ± 10.83 and Doxo + GCV: 62.74 ± 23.22 R.U. ANOVA test).