Fig 2: PHB is indispensable for QCR2-dependent p53 signaling and cell cycle arrest. (a) After transfection of HeLa cells with NC siRNA or QCR2 siRNA-2, the cells were used for immunofluorescence studies for nuclei (DAPI, blue), PHB or QCR2 (green), and mitochondria (MitoTracker, red). Arrows indicate nuclear translocation of PHB (scale bar = 20 µm). (b) Western blots of HeLa extracts fractionated into cytoplasm, nuclear and mitochondrial fractions after transfection with siRNAs for the indicated protein. PCNA as a nuclear marker, PDHA1 as a mitochondria maker and a-tubulin as a cytoplasm marker. (c, d) Intact nucleus from HCT116 cells transfected with Control Ad or QCR2-Flag Ad (c) and NC siRNA or QCR2 siRNA-2 (d) were isolated, and the lysates were immunoprecipitated using an anti-p53 antibody or control IgG. Immunocomplexes were analyzed using anti-p53 and anti-PHB antibodies. (e) HeLa cells were transfected with the indicated siRNAs for 96 h, and cell lysates were analyzed by western blotting for indicated proteins. (f) HeLa cells were transfected with the indicated siRNAs for 96 h, and cells serum-starved for 12 h (starved) and then restimulated with 10% FBS and taxol containing-medium for 24 h were assessed for cell cycle distribution via FACS analysis. (g) The bar graph represents quantification analyses of SA-ß-gal positive HeLa cells transfected with the indicated siRNAs (*p < .05, ***p < .001, student's t-test, n = 3). (h) A549 cells were transfected with the indicated siRNAs, and lysates were immunoprecipitated with an anti-p53 antibody. The resulting immunocomplexes were analyzed by western blotting using anti-p53 and anti-Ub antibodies. (i) A549 cells were transfected with siRNAs, followed by treatment with 1 µg/µL CHX for the indicated time points 72 h later. Endogenous p53 expression was examined by western blotting. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig 3: Changes in the fractionation profiling of proteins from the mitochondrial matrix in UVA-exposed cells compared to controls reflect mitochondrial fragmentation(A) Profile plots obtained for FH and OAT in control and irradiated samples. Lines represent the means of relative abundance, and shadowed intervals represent standard errors.(B) Immunostaining of FH (green) in HaCaT cells exposed to UVA or mock-treated. PDHA1 (red) was immunolabeled as a structural mitochondrial marker. The nucleus was stained with Hoechst (blue). Three independent experiments were performed, and similar results were obtained.(C) Immunostaining of OAT (green) in HaCaT cells exposed to UVA or mock-treated. Similarly, PDHA1 (red) was used as a mitochondrial marker, and the nucleus was stained with Hoechst (blue). Three independent experiments were performed, and similar results were obtained. The bars indicate the 20 µm scale.

Fig 4: Clinical correlation between EMD and PDHA. (A-D) EMD (A), EMD ISGylation (B), global ISGylation (C) and PDHA (D) level in LUAD and adjacent normal tissues (n = 60/group) from cohort #6. (E) Correlation between PDHA and EMD in LUAD tissues (n = 60/group) from cohort #6. (F) EMD expression and ISGylation in LUAD and adjacent normal tissues (n = 6/group) from cohort #6. The EMD level in each co-IP sample was adjusted to the same protein content. (G) Correlation between EMD and its ISGylation in LUAD tissues (n = 60/group) from cohort #6. (H) Correlation between PDHA and EMD ISGylation in LUAD tissues (n = 60/group) from cohort #6. (I-K) EMD (I), EMD ISGylation (J) and PDHA (K) level in stage I, II and III LUAD tissues from cohort #6. (L-N) EMD (L), EMD ISGylation (M) and PDHA (N) level in tumour diameter <3 cm and =3 cm tissues from cohort #6. IB images were selected from three biological replicates. Data in A-D and L-N were analysed by a Student's t-test. Data in E, G, H were analysed by a Spearman rank-correlation analysis. Data in I-K were analysed by a one-way anova test. **, P < 0.01

Fig 5: Effect of siRNA knockdowns on mitophagic flux and evaluation of LC3 involvement by proximity ligation assay (PLA). (A) Representative widefield images of mCherry-EGFP- SYNJ2BP-TM H9c2 cells analyzed 48 h after transfection with scrambled siRNA (siScr) or siRNA against Ulk1, Atg7, Rab9a, Rab7a or Rab5a respectively. (B) Quantification of the effect of 48 h siRNA knockdowns by assessment of number of red-only dots per cell in cells containing red-only dots in control conditions against a 12 h PepA and E64d treatment. The data is presented as mean ± SEM from 3 independent experiments, with more than 100 cells per condition (total number analyzed per experiment was over 1200 cells). (C) Western blots showing the expression levels of the siRNA targeted proteins in control and siRNA treated cells for verification of successful knockdown. (D) Representative confocal images of detected PLA puncta (white) using anti-MAP1LC3B and anti-PDHA1 antibody during normal (GLU) and galactose (GAL) adapted conditions. The enlarged boxes display the PLA puncta on the mitochondria network and small mitochondrial fragments but their absence on red-only dots. (E) Quantification of the number of PLA puncta per cell in 10 images (with more than 50 cells in total) per condition from two independent experiments. The individual datapoints are per frame cell averages. * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001. Scale bar: 10 µm.