Fig 2: Improved photoreceptor survival after retinal detachment in Pkm2 −/− mice. (a) Representative photomicrographs of TUNEL-stained photoreceptors (green) after 72 hours of retinal detachment. Nuclei of retinal cells are stained with DAPI (blue). INL, inner nuclear layer; ONL, outer nuclear layer. (b) Quantification of TUNEL-positive cells in the ONL. n ≥ 12 sections per group. (c) Caspase 8 activity in detached retinas after 3 days as detected by luminescent assay. N = 3 animals per group; Mean ± SEM; *p < 0.05; ***p < 0.005. (d) Retinal sections of attached and detached retina, 2 months after creation of detachment in Pkm2 +/+ and Pkm2 −/− mice, respectively. (e) Graph summarizing ONL cell viability between attached and detached retina after 2-month detachment. Cell survival was significantly higher in Pkm2 −/− mice as compared to Pkm2 +/+ mice. (f) Graph summarizing ONL area changes between attached and detached retina after 2-month detachment. ONL area was significantly higher in Pkm2 −/− mice as compared to Pkm2 +/+ mice. N = 5 animals per group; Mean ± SEM; *p < 0.05; ***p < 0.005.

Fig 3: Ser227 phosphorylation stabilizes YAP and augments PKM2 transcription upon hypoxia. (A) Representative immunfluorescence images of ULK1 nuclear localization in SW-1990 cells before and after hypoxia stimuli. Scale bar = 20 μm. (B) Representative immunfluorescence images detecting the localization of ULK1 and YAP in YAP knockout (KO) or ULK1 shRNA transfected-SW-1990 cells before and after hypoxia stimuli, respectively. Scale bar = 20 μm. (C) Coimmunoprecipitation assay detecting the abundance of YAP Ser phosphorylation in Flag-tagged wild-type YAP-expressed SW-1990 cells with or without hypoxia stimuli in the presence of CIP treatment. (D) Cellular ubiquitination assays comparing the poly-Ub levels of YAP in Flag-tagged wild-type YAP-expressed SW-1990 cells with or without hypoxia stimuli in the presence of CIP treatment. (E) CHX pulse-chase experiments comparing the turnover of YAP protein in Flag-tagged wild-type YAP (WT)-, mutant YAP Ser227A (S227A)- and Ser227E (S227E)-expressed SW-1990 cells with hypoxia stimuli in the presence or absence of 20 μg/mL CHX treatment for the indicated times, respectively. (F) Cellular ubiquitination assays evaluating the poly-Ub levels of YAP protein in Flag-tagged wild-type YAP (WT)-, mutant YAP Ser227A (S227A)- and Ser227E (S227E)-expressed SW-1990 cells with hypoxia stimuli. (G) Western blotting examining the expression levels of YAP in YAP knockout (KO) SW-1990 cells with Flag-tagged wild-type YAP (WT) or mutant YAP Ser227A (S227A) reconstitution. (H) ChIP analysis for YAP binding to PKM2 gene promoter in YAP knockout (KO) SW-1990 cells with hypoxia stimulation in the presence of Flag-tagged wild-type YAP (WT) or mutant YAP Ser227A (S227A) reconstitution using the indicated antibodies. (I) Western-blotting analyses assessing the levels of PKM2 protein in YAP knockout (KO) SW-1990 cells with hypoxia stimulation in the presence of Flag-tagged wild-type YAP (WT) or mutant YAP Ser227A (S227A) reconstitution. (J) RT-qPCR analyses of PKM2 gene expression in YAP knockout (KO) SW-1990 cells with hypoxia stimulation in the presence of Flag-tagged wild-type YAP (WT) or mutant YAP Ser227A (S227A) reconstitution. Experiments were performed three times, each with quantitative RT-PCR in technical duplicate and real-time values were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Data are expressed as mean ± s.d. **P<0.01 versus YAP KO. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. (K) Luciferase-reporter PKM2 promoter activity analysis of YAP knockout (KO) SW-1990 cells with hypoxia stimulation in the presence of Flag-tagged wild-type YAP (WT) or mutant YAP Ser227A (S227A) reconstitution. Data are expressed as mean ± s.d. of three independent experiments. *P<0.05 versus YAP KO. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value.

Fig 4: PKM2 play a critical role in the keratinocyte glucose metabolism and proliferation. (A) Immunoblotting of HaCaT cell stably expressing Flag-PKM2. Cells were infected with lentivirus containing the empty vector (GFP) or Flag-PKM2 (MOI = 40). The cells were then selected in puromycin (2 μg/ml) for 1 week. Total cell extracts were immunoblotted with antibodies for PKM1, PKM2, Flag, and GAPDH. (B, C) Lactate production and glucose consumption were measured in HaCaT-GFP and HaCaT-Flag-PKM2 cells. (D) Efficiency of PKM2 shRNA knockdown. Immunoblot analyses indicate ∼70% reduction of PKM2 in PKM2 shRNA-expressing cells compared with control shRNA. (E, F) Lactate production and glucose consumption were measured in HaCaT-control shRNA and HaCaT-PKM2 shRNA cells. (G) Proliferation curves of the PKM2 knockdown and control cells. (H) Immunoblotting of NHEK treated with Flag-PKM2 lentivirus or PKM2 shRNA adenovirus. (I, J) Lactate production and glucose consumption were measured in NHEK. NHEK were transfected with Flag-PKM2 lentiviruses or PKM2 shRNA adenovirus. NHEK: normal human epidermal keratinocytes. Data are means ± SEM (n = 6). GAPDH was used as loading control.

Fig 5: Mitochondrial biogenesis is required for PKM2 activation-induced endotoxin tolerance. (A) RAW264.7 cells were treated with control shRNA or shmtTFA lentiviruses for 72 h. Protein expression of mtTFA was measured by Western blot (n=3). (B) NAO signal intensity was used to assess mitochondrial mass in control RAW264.7 cells (con shRNA) and in mtTFA knockdown RAW264.7 cells (shmtTFA) (n=4). (C) mtDNA copy number was determined by measuring MTND1 relative to B2M using RT-PCR (n=3). (D) Supernatant TNF-α and IL-6 levels in RAW264.7 cells stimulated with LPS and/or TEPP-46 were measured using ELISA (n=3). (E) RAW264.7 polarization level was assessed by measuring the relative mRNA expression of Arg1 and CD206 to that of GAPDH using RT-PCR (n=3). *p < 0.05, **p < 0.01, ***p < 0.001.