Fig 2: Glu supplementation blunts the effects of EAAm on ATF4 induction, mTOR inhibition and cell growth. (A) Western blot analysis of nuclear extracts (ATF4) or total lysates (p70S6K) of M14 untreated (CTRL), incubated with EAAm mixture, or incubated with EAAm mixture plus Glu, Gly, Asp or Ala 10 mM for 3 h. (B) Clonogenic assay in M14 cells left untreated (CTRL), incubated with EAAm mixture (EAAm), or incubated with EAAm mixture plus Glu 10 mM (EAAm + GLU) for 24 h (C) sestrin2 (SESN2) protein expression in HeLa cells transfected with non-targeting (scrambled) or SESN2- specific siRNA. Vinculin is shown as a loading control (D). p-S6 levels immunoblot analysis in Hela cells transfected with control non-targeting siRNA (scrambled), or siRNA against sestrin2 (SESN2), and treated (+) or not (-) with EAAm 1%.for 3 h (E) MTT assay of Hela cells transfected with non-targeting (scrambled) siRNA or siRNA against sestrin 2 (SESN2) and incubated with culture medium (CTRL) or supplemented with 1% EAAm for 24 h (EAAm). Mean (n = 4) ± SEM, **P < 0.01 vs. CTRL. Two-way ANOVA followed by Tukey's post hoc test.

Fig 3: SESN2 interacts with ULK1 on CCCP-induced mitophagy and assists migration of Parkin. (A) HEK293T cells were co-transfected with empty FLAG tagged plasmid and plasmid expressing FLAG tagged SESN2 and subsequently treated with DMSO and CCCP for 3 h. Total cell lysates were subjected to co-immunoprecipitation using anti-FLAG M2 magnetic beads. Western blot analysis shows that SESN2 interacts with ULK1 on CCCP treatment but not with Beclin1. (B) Wild-type and SESN2 knockdown cells were treated with 10 µM CCCP and 500 nM Rapamycin for 3 h and ULK1 mediated serine-14 phosphorylation of Beclin1 was analysed by immunoblotting. (C) HeLa cells stably expressing YFP-tagged Parkin, which were transfected with scrambled siRNA and siRNA targeting SESN2 and treated with CCCP and rapamycin as per mentioned above. Mitochondria was isolated and western blot analysis was done to determine the amount of YFP-Parkin in mitochondria. Note: reduction in Parkin mitochondrial accumulation was observed only in CCCP treated SESN2 knockdown cells. VDAC1, mitochondrial marker and ACTB/Actin, cytosolic marker. (D) HeLa cells stably expressing YFP-Parkin were co-transfected with siRNA targeting Beclin1, HA-tagged phospho-mutant Beclin1 S14A, HA-tagged phospho-mimetic S14D and subsequently treated with 10 µM CCCP (3 h). YFP-Parkin co-localization with TOMM20 in HeLa cells stably expressing YFP-Parkin was scored after counting > 100 cells per sample in three independent experiments. The error bars in graph represents mean ± SEM. Parkin translocation is inhibited in cells with phospho-deficient mutation (S14A) while translocation occurs only on CCCP treatment with the phosphomimetic mutant (S14D). S14A, phospho-deficient mutation; S14D, phosphomimetic mutant. *p < 0.01, # NS. (E) HeLa cells stably expressing YFP-Parkin were co-transfected with scrambled siRNA, siRNA targeting SESN2 and HA-tagged phospho-mimetic S14D of Beclin1. Western blot analysis of mitochondrial fraction and whole cell lysates was done. VDAC1, mitochondrial marker and Actin, cytosolic marker. 10 µM CCCP for 3 h. Immunoblots were converted to gray and white scale using Adobe Photoshop CS6, with linear adjustment of exposure or contrast. Images of immunoblots were cropped for clarity, expanded view of Fig. 5A and E is available in supplementary figures.

Fig 4: Sestrins protect cells from CCCP-induced mitochondrial damage. (A) HEK293T cells were treated with DMSO (vehicle control) and 10 µM CCCP for indicated time points and immunoblot analysis was done using indicated antibodies. Actin blot was used as a loading control. (B) SH-SY5Y cells were treated with DMSO (vehicle control) and 10 µM CCCP for indicated time points and immunoblot analysis was done using indicated antibodies. Actin blot was used as a loading control. (C) HEK293T cells were transfected with scrambled siRNA, siRNA targeting SESN1 and 2 followed by treatment with DMSO (vehicle control) and CCCP for 3 h and 12 h. Cell death analysis was done using PI assay. siSESN1 #3 and siSEN2 #3, si RNA to SESN1 and 2. (D) With similar experimental set up as performed in Fig. 1C, MTT assay was performed to measure the viability of cells. Error bars in the graph represent mean ± SEM from four independent experiments (n = 4). *p < 0.005, #p < 0.01. (E) Cell viability was measured using MTT assay, cells were transfected using scrambled siRNA, siRNA targeting SESN2 and plasmid overexpressing SESN2 as shown, followed by treatment with DMSO and CCCP (12 h). Error bars in the graph represent mean ± SEM from four independent experiments (n = 4). OE-SESN2, cells with overexpressed SESN2. *p < 0.001, #p < 0.05. Immunoblots were converted to gray and white scale using Adobe Photoshop CS6, with linear adjustment of exposure or contrast was done as per required. Images of immunoblots were cropped for clarity.

Fig 5: Overexpression of SESN2 rescue from CCCP-induced damage in PINK1 downregulated cells but not Parkin mitochondrial translocation. (A) HeLa cells stably expressing YFP-Parkin were transfected with scrambled siRNA, siRNA PINK1 along with plasmid expressing kinase dead domain mutant of PINK1 and treated with 10 µM CCCP for 3 h. To investigate the mitochondrial translocation of Parkin, cells were immunostained using antibody against TOMM 20. To evaluate if SESN2 can rescue the effect of PINK1 knockdown, plasmid expressing SESN2 was transfected. Scale bars, 10 µm. (B) Cells were transfected with siRNA targeting SESN2 and PINK1, plasmids expressing PINK1 kinase dead domain mutant and SESN2. Cell viability was determined by MTT assay on DMSO or CCCP treatment (12 h) as indicated. The error bars represents mean ± SEM from three independent experiments (n = 3). It is notable that overexpression of SESN2 was able to protect cells from CCCP induced damage in PINK1 knockdown cells. *p < 0.01, #p < 0.05 C. Evaluation of mitochondria generated superoxide was done by mitoSOX assay in cells which were transfected with siRNA targeting PINK1 and overexpression plasmid of SESN2 followed by treatment with DMSO and CCCP (12 h). The error bars represent mean ± SEM (n = 3). The figure shows overexpression of SESN2 was able to decline increase in CCCP induced generation of mitochondrial ROS in PINK1 knockdown cells. *p < 0.01, #p < 0.05.