Extended Data Fig. 6: GAS5 functions as a tumour suppressor by controlling TCA flux.
From: Mitochondrial long non-coding RNA GAS5 tunes TCA metabolism in response to nutrient stress
(a) RT-qPCR detection of GAS5 expression level in empty vector control (EV) or GAS5 overexpressed MDA-MB-231. Data are presented as mean values ± S.D., n = 3 biologically independent experiments, Two-sided Student’s t-test, ***P < 0.001. (b) Cellular NADH/NAD+ ratio was determined in empty vector control (EV) or GAS5 overexpressed MDA-MB-231 cells under 8-hour glucose starvation. Data are presented as mean values ± S.D., n = 3 biologically independent experiments, Two-sided Student’s t-test, **P < 0.01. (c-d) Mitochondria were isolated from 8-hour glucose starved (d) or normal (c) MDA-MB-231 cells and the mitochondrial NADH/NAD+ ratio level was measured. Data are presented as mean values ± S.D., n = 3 biologically independent experiments, Two-sided Student’s t-test, ***P < 0.001. (e) Mitochondria were isolated from MDA-MB-231 cells and the mitochondrial ATP generation was measured. Data are presented as mean values ± S.D., n = 3 biologically independent experiments, Two-sided Student’s t-test, *P < 0.05. (f) Oxygen consumption rate (OCR) profile was monitored in empty vector control (EV) or GAS5 overexpressed MDA-MB-231 cells with a Seahorse XF24 analyzer for 100 min. The indicated metabolic inhibitors were injected sequentially at different time points as indicated. Data are presented as mean values ± S.D., n = 3 biologically independent experiments. (g) RT-qPCR detection of GAS5 expression level in empty vector control (EV) or GAS5 overexpressed MDA-MB-468 cells. Data are presented as mean values ± S.D., n = 3 biologically independent experiments, Two-sided Student’s t-test, ***P < 0.001. (h) Cellular NADH/NAD+ ratio was determined in empty vector control (EV) or GAS5 overexpressed MDA-MB-468 cells. Data are presented as mean values ± S.D., n = 3 biologically independent experiments, Two-sided Student’s t-test, **P < 0.01. (i) Mitochondria were isolated from MDA-MB-468 cells and the mitochondrial NADH/NAD+ ratio level was measured. Data are presented as mean values ± S.D., n = 3 biologically independent experiments, Two-sided Student’s t-test, ***P < 0.001. (j) Cellular ATP was determined in empty vector control (EV) or GAS5 overexpressed MDA-MB-468 cells. Data are presented as mean values ± S.D., n = 3 biologically independent experiments, Two-sided Student’s t-test, *P < 0.05. (k) Mitochondria were isolated from MDA-MB-468 cells and the mitochondrial ATP level was measured. Data are presented as mean values ± S.D., n = 3 biologically independent experiments, Two-sided Student’s t-test, *P < 0.05. (l) Cellular malate level was determined in empty vector control (EV) or GAS5 overexpressed MDA-MB-468 cells. Data are presented as mean values ± S.D., n = 3 biologically independent experiments, Two-sided Student’s t-test, *P < 0.05. (m) Cellular citrate level was determined in empty vector control (EV) or GAS5 overexpressed MDA-MB-468 cells. Data are presented as mean values ± S.D., n = 3 biologically independent experiments, Two-sided Student’s t-test, **P < 0.01. (n) Cell proliferation viability was assessed by cell counting in empty vector control (EV) or GAS5 overexpressed MDA-MB-231 cells. Data are presented as mean values ± S.D., n = 3 biologically independent experiments, two-way ANOVA analysis, **P < 0.01. (o) Colony formation assay in empty vector (EV) control or GAS5 overexpressed MDA-MB-231 cells. Data are presented as mean values ± S.D., n = 3 biologically independent experiments, Two-sided Student’s t-test, ***P < 0.001. (p) Cell proliferation viability of GAS5 overexpressed MDA-MB-231 cells with 0.2 mM citrate rescue or not was analyzed by CCK8 assay in the indicated time points. Data are presented as mean values ± S.D., n = 3 biologically independent experiments, two-way ANOVA analysis, **P < 0.01. (q) In vivo analyses of tumors’ weight in xenograft mouse model were shown. Data are presented as mean values ± S.D., n = 5 biologically independent samples of xenograft tumors per group. Two-sided Student’s t-test, **P < 0.01, ***P < 0.001. (r) RT-qPCR detection of CCND1 (gene of cyclin D1) expression level in the indicated xenograft tumors. Data are presented as mean values ± S.D., n = 5 biologically independent samples of xenograft tumors per group, Two-sided Student’s t-test, *P < 0.05, ***P < 0.001. (s) RT-qPCR detection of SIRT3 expression in adjacent normal tissues (N) and malignant breast cancer (T) (SYSUCC Cohorts. n = 48 biologically independent samples of tumors with matched adjacent normal tissues. Two-sided Student’s t-test, **P < 0.01. (t) Analysis of SIRT3 expression in adjacent normal tissues (N) and malignant breast cancer (T), in other cohorts’ microarray data sets from Oncomine database. Data are presented as box plots with median (horizontal line) and 25%/75% quartiles with whiskers to the last point; Two-sided Student’s t-test, **P < 0.01. (u-w) Immunohistochemical (IHC) detection of MDH2 (u), FH (v) or CS (w) was performed in adjacent normal tissues and matched malignant breast cancer tissues (Bio MAX, n = 100 biologically independent samples of patients, Gehan-Breslow test, **P < 0.01). Scale bar, 100 µm.
