Fig 1: Mfn1 silencing reduces galectin-9 expression and secretion in senescent cells. (a–c) B16-F1 cells were treated with DMSO, TMZ or 223Ra: (a) representative Western-Blot (WB) of intracellular levels of galectin-9. (b) Quantification of the WB in (a), galectin-9 was normalized using tubulin as loading control and results were expressed relative to control condition. Unpaired t-test, two tails, *P = 0.014 (n = 4). (c) Lgals9 mRNA reported relative to control value. One-way ANOVA (P = 0.0003), Tukey post-hoc for multiple comparisons, *P = 0.005, **P = 0.0003 vs control (n = 3–6). (d–g) ShScr and shMfn1 cells were treated with TMZ or DMSO: (d) Galectin-9 secretion assessed by ELISA. Two-way ANOVA, main effects of treatment (P < 0.0001), shRNA (P = 0.0002) and their interaction (P = 0.0002). Tukey post-hoc for multiple comparisons, different letters are significantly different (P ≤ 0.0002). (e) Lgals9 mRNA, reported relative to control value. Two-way ANOVA, main effects of treatment (P = 0.0015), shRNA (P = 0.002) and their interaction (P = 0.002). Tukey post-hoc for multiple comparisons, different letters are significantly different (P ≤ 0.0009). (f) Representative western-blot of intracellular levels of galectin-9. (g) Quantification of protein levels in (f). Galectin-9 was normalized using tubulin as loading control and expressed relative to control condition. Two-way ANOVA, main effects of treatment (P < 0.0001), shRNA (P < 0.0001) and their interaction (P < 0.0001). Tukey post-hoc for multiple comparisons, different letters are significantly different (P < 0.0001). Results are the mean ± SD (n = 3–5). The uncropped images of the blots, with different degrees of exposure, can be found in Supplementary Figs. S10 and S11.
Fig 2: Mfn1 silencing improves the response to chemotherapy with a DNA damaging agent in a mice model of melanoma. shScr or shMfn1 B16-F1 cells were injected subcutaneously in the right hind leg of the mice, and when tumors became palpable mice were treated with three doses of DTIC (arrows). (a) Tumor size was measured at different time points p.t.i.. Two-way ANOVA with Tukey post-hoc analysis with respect to control (shScr) * P ≤ 0.01. Results are the mean ± SEM (n = 10). (b) Day 12 p.t.i., tumor volume before DTIC treatment. Unpaired t-test, two tails ***P < 0.0001 (n = 23). (c) Day 25 p.t.i., tumor volume after treatment. Two-way ANOVA, main effects of treatment (P = 0.0005), shRNA (P = 0.3) and their interaction (P = 0.6). Tukey post-hoc for multiple comparisons, different letters are significantly different (P ≤ 0.02). (d–f) mRNA levels of senescence marker or SASP components in the tumor. Two-way ANOVA (main effects of treatment, shRNA and their interaction) and Tukey post-hoc for multiple comparisons, different letters are significantly different: (d) Cdkn1a (p21) in the tumors (P < 0.0001, P = 0.004, P = 0.03), Tukey post-hoc P ≤ 0.0008. (e) Ccl5 (P < 0.0001, P = 0.3, P = 0.2), Tukey post-hoc P ≤ 0.0036. (f) Lgals9 (P < 0.0001, P = 0.5, P = 0.5), Tukey post-hoc P ≤ 0.009. (b–f) Results are the mean ± SD (n = 10).
Supplier Page from Abcam for Mouse galectin 9/Gal-9 ELISA Kit