RIP1 Maintains DNA Integrity and Cell Proliferation by Regulating PGC-1Α-Mediated Mitochondrial

RIP1 maintains DNA integrity and cell proliferation by regulating PGC-1α-mediated mitochondrial oxidative phosphorylation and glycolysis

Supplementary Information

Supplementary Legends for Figures and Table

Supplementary Figure 1p53-dependent inhibition of proliferation in RIP1 knockdown cells. (a) Proliferation in H460 control (Cont) cells and two clones of RIP1 knockdown (KD) cells. Relative cell number was determined with MTT (20 µg/ml) assay with respective readings from day 1 as 1. Western blot shows the efficiency of RIP1 knockdown. β-Actin was probed as a loading control. The valuesare mean ± S.D. from triplicate samples.*P0.05versus control cells at the identical time points.(b) Representative images of colony formation in A549 control and RIP1 KD cells seeded in soft agar for 10 days.(c)Expression of p53 and p21 ina panel of control (Cont) and RIP1 KDlung cancer cell lines. GAPDH and β-tubulin were detected as loading controls. (d) p53 and p21 expression in H460 RIP1 KD cells transfected with control (si-Cont, 10 nM) or p53 siRNA (si-p53, 10 nM) for two days. GAPDH was probed as a loading control. (e) Proliferation of control (Cont) and RIP1 KD H23, H2009 and H1299 cells. The valuesare mean ± S.D. from triplicate samples.*P0.05versus control cells at the identical time points.Western blotting shows the efficiency of knockdown. GAPDH was used as a loading control. (f) Bax and PARP expression in A549 control (Cont) and RIP KD cells. PARP was deliberately overexposed to detect possible cleavage. GAPDH was used as a loading control. (g) Basal NF-κB activity in control (Cont) and RIP KD cells detected with NF-κB luciferase reporter assay. Luciferase activity was measured 24 hours post-transfection.The valuesare mean ± S.D. from triplicate samples.

Supplementary Figure2RIP1 downregulation impaired DNA repair capacity.(a) Phosphorylation of ATM, CHK2 and γH2AX in H460 control (Cont) and RIP1 KD cells. GAPDH was used as a loading control. (b) γH2AX expression in H1299 control (Cont) and RIP1 knockdown (KD) cells. β-Tubulin was probed as a loading control. Result was obtainedfrom the identicalexperiment in Supplementary Figure1c. (c) ROS levels in control (Cont) and RIP KD cells of A549 and H460 determined by addition of dichlorodihydrofluorescein diacetate (CM-H2DCFDA, 5 μM). Results were normalized to total cellular protein. The valuesare mean ± S.D. from triplicate samples.(d) γH2AX expression in A549 RIP1 KD-1 cells untreated or treated with NAC (630 µM) for 48 hours. GAPDH was probed as a loading control. (e) Cell death in nonconfluent MEFs (WT, RIP1-/- and RIP1-/- reconstituted with RIP1) 16 hours after treatment with 3 μg/ml of adriamycin (Adr) for 1 hour. A group of cells were treated with TNF (20 ng/ml) as a control. Cell death was determined by measuring LDH release in the culture media against total LDH as 100 (%) (Promega cytotoxicity assay kit). The valuesare mean ± S.D. from triplicate samples.(f) Response of H460 control (Cont) and RIP1 KD cells to treatment with adriamycin (Adr, 1 μg/ml), camptothecin (CPT, 10 μM) or BPDE (0.4 μM) for 30 hours. The valuesare mean ± S.D. from triplicate samples.*P0.05. Supplementary Figure 3RIP1 down-regulation enhanced glycolysis.(a) Lactate production, LDH activity and glucose consumption in H460 control (Cont) and RIP1 KD cells. Results were normalized to total cellular protein. *P0.05versus control cells. (b) LDH activities in H1299 and WT and RIP-/- MEFs. *P0.05versusrespective control cells. (cand d) Cell viability of H460 control (Cont) and RIP1 KD cells and WT and RIP-/- MEFs treated with various concentrations of 2-DG or oxamate for 30 hours. Cell viability was determined with MTT assay.*P0.05. (e) Lactate production in A549 control and RIP1 KD-1 cell treated with various concentrations of 2-DG for 24 hours. *P0.05versusrespective untreated cells (0 mM of 2-DG). (f) Proliferation of A549 control and RIP1 KD-1 cells treated with 0.5 mM oxamate for4days, and production of lactate from the cells treated with various concentrations of oxamate for 24 hours. *P0.05versusrespective untreated cells. Allvaluesare mean ± S.D. from triplicate samples.

Supplementary Figure 4Enhanced glycolysis was associated with DNA damage.(a, b and c) Expression of phosphorylation of ATM, CHK2 and γH2AX in A549 control, RIP1 KD-1 and H460 RIP1 KD-8 cells treated with low concentrations of 2-DG or oxamate for 48 hours. GAPDH and β-actin were probed as loading controls. (dand e) Relative NAD+ levels in cytoplasmic, cytosolic and nuclear fractions from H460 control (Cont) and RIP1 KD cells (d), and in cytosolic fraction from WT and RIP1-/- MEFs (e). The values are mean ± S.D. from triplicate samples.*P0.05versusrespective control cells.

Supplementary Figure 5RIP1 knockdown suppressed PGC-1α expression.(a) PGC-1α protein expression in H460 control (Cont) and RIP1 KD cells. GAPDH was used as loading control. (b) Expression of WT RIP1 and its mutants. GAPDH was used as a loading control. (c) PGC-1α and p53 expression in A549 RIP1 KD-1 cells transfected with control (Cont, 10 nM) or p53 siRNA (10 nM) for 48 hours. GAPDH was probed as a loading control.

Supplementary Figure 6Down-regulation of PGC-1α impaired OXPHOS.(aand b) Expression of cyt c, COXIV and MnSOD (a) and relative complex IV enzyme activity (b)in control (Cont) and RIP1 KD H460 cells. β-Tubulin was probed as a loading control in Western blot.The valuesare mean ± S.D. from triplicate samples.*P0.05versus control.(c) The mitochondrial mass in A549 and H460 control (Cont) and RIP1 KD cells determined by MitoTrackerTM(50 nM) staining and flowcytometry analysis. A reference line was provided. (d) mtDNA content in A549 control (Cont) and RIP1 KD cells. The COXI and 18S were amplified with qPCR in total DNA extracts to calculate the ratio. The valuesare mean ± S.D. from triplicate samples.(e) Survival of H460 control (cont) and RIP1 KD cells cultured in normal, glucose-free media [Glu(-)], or glucose-free media supplemented with 10 mMof glucose (Glu) or galactose(Gal) for 24 hours.The valuesare mean ± S.D. from triplicate samples.*P0.05.

Supplementary Table 1 Reduced expression of genes in mitochondrial respiratory system components in A549RIP1 knockdown cells. Microarray analysis was carried out as described in Materials and Methods. Gene expression in A549 RIP1 KD-1 cells with≥ 2 fold reduction comparedwithcontrol cells waslisted.