Fig 2: Inactivation of GSK3ß by p38 MAPK promotes accumulation of the prosurvival factor Mcl-1.(a,b) WT and GSK3ß-KI B cells were activated for two days and the expression of ß-catenin (a), Mcl-1, P-S389 GSK3ß and total GSK3ß (b) were examined by western blotting. GAPDH is shown as a loading control. (c) WT and GSK3ß-KI B cells were activated as in a and Bclx, Bcl2, Bid, Bax and Bad levels were examined by western blotting. (d) Western blot analysis for PUMA in irradiated WT thymocytes (5 Gy) and activated WT and GSK3ß-KI B cells. (e,f) B cells activated as in a were stained with MitoTracker (e) or TMRE (f) and analysed by flow cytometry. The number represents the percentage of cells within the gate. (g) WT and GSK3ß-KI B cells were examined by western blotting for the expression of cleaved caspase-3 (activated caspase-3), full length RIPK1 (RIPK1) and cleaved RIPK1 (cleaved RIPK1). (h) WT and GSK3ß-KI B cells were activated, after 2 days Nectrostatin-1s (Nec-1s) was added and cell viability was determined by cell counting 24 h later (n=3). (i) WT and GSK3ß-KI B cells were activated for 3 days and phospho-MLKL was examined by western blotting. (j) WT and GSK3ß-KI B cells in the presence or absence of the GSK3 inhibitor (GSK3-Inh) were activated and examined by immunostaining and confocal microscopy for Mcl-1 (red) and TOPRO nuclear stain (blue). Scale bar, 3 µm. (k) Mcl-1 levels in nuclear (Nuc) and mitochondrial (Mito) extracts from activated WT and GSK3ß-KI B cells were determined by western blot analysis. Histone and CoxIV (Complex IV) were used as loading controls. (l) WT and GSK3ß-KI B cells were transduced with either an empty retrovirus (E), a retrovirus expressing wildtype Mcl-1 (Mcl) or a retrovirus expressing a Ser140Ala mutant of Mcl-1 (mMcl). Three days after activation cell viability was determined by cell counting. (n=3) and *P value<0.05 as determined by t- test (h) or one-way ANOVA (l). Data are representative of three or more independent experiments.

Fig 3: ML264 treatment causes inactivation of AKT and induces caspase-dependent apoptosis signaling cascade. (A) ML264 treatment causes inactivation of AKT and Bad proteins in EOC cells. EOC cells were treated with 5 and 10 µM ML264 for 48 h. Following treatment, cells were lysed and immunoblotted with antibodies against p-AKT, AKT, p-Bad and GAPDH. (B) ML264 treatment downregulates the expression of anti-apoptotic proteins and inhibitors of apoptosis in EOC cells. EOC cells were treated with indicated doses of ML264 for 48 h. After cell lysis, equal amounts of proteins were separated by SDS-PAGE, transferred to immobilon membrane, and immuno-blotted with antibodies against Bcl-2, Bcl-xl, XIAP, CIAP1 and GAPDH as indicated. (C) Estimation of mitochondrial membrane potential by flow cytometry. EOC cells were treated with 5 and 10 µM ML264 for 48 h. Following treatment, cells were stained with 10 µM JC1 and analysed by flow cytometry. (n = 3) (D) ML264-induced release of cytochrome c. EOC cells were treated with and without 5 and 10 µM ML264 for 48 h. Mitochondrial free, cytosolic fractions and mitochondrial extracts were isolated. Cell extracts were separated on SDS-PAGE, transferred to PVDF membrane, and immunoblotted with an antibody against cytochrome c, Cox IV and GAPDH. (E) Activation of caspases and cleavage of PARP induced by ML264 treatment in EOC cells. EOC cells were treated with and without 5 and 10 µM ML264 for 48 h. Cells were lysed, equal amount of proteins were separated on SDS-PAGE and immuno-blotted with antibodies against caspase-9, caspase-3, cleaved caspase-3, PARP and GAPDH, All the experiments were repeated twice with similar results. All the western blot experiments were repeated at least two times with the same results.

Fig 4: Parkin knockdown reduced the ability of HHT to inhibit cell viability and promote mitophagy. All cells were transfected with the scrambled shRNA or Parkin-shRNA, and treated with or without 25ng/mL HHT. (A) Knockdown of Parkin reversed the substantial increase in cell death induced by HHT. (B) The fluorescence intensity of LC3 was evaluated; the green color indicates LC3, and DAPI was used to stain the nuclei. (C) The MMP was assessed using a mitochondrial membrane potential detection kit. The scramble-HHT group exhibited the lowest level of MMP. (D) Mitochondrial ultrastructure was detected using TEM. The scramble-HHT group showed the most severe mitochondrial damage and massive mitophagic vesicles. (E) WB analysis of levels of LC3-II/LC3-I, Bcl-2, Bax, Tim 23, CoxIV, Parkin and actin (or tubulin) was performed. Each experiment was repeated three times. The data are presented as means±SD. Every experiment was repeated in triplicate. One-way ANOVA analysis was used to assess statistical significance. *P<0.05, **P<0.01, and NS P>0.05 between the indicated pairs of groups.

Fig 5: Formation of MAVS aggregates during Rotavirus infection. A) Crude mitochondrial extracts were prepared from HEK293 cells infected with A5-16 strain (3 M.O.I.) at increasing time points (4, 8 and 12). Extracts were analyzed by SDD-AGE to assess the MAVS aggregate formation. Results revealed MAVS aggregate formation from 4 hpi. B) Role of NSP1 on MAVS aggregation and ubiquitinylation was observed by overexpressing FLAG-MAVS in absence or presence of NSP1. Infection of A5-16 was used for inducing MAVS aggregation, as overexpression of MAVS alone is insufficient for inducing aggregate formation. Results show inhibition of MAVS aggregates in presence of NSP1, which gets restored following MG132 (20 µM) treatment. A fraction of SDD-AGE lysate was analyzed by SDS-PAGE followed by immunoblotting of MAVS, p-IRF3 and COX IV.