Fig 2: Hypoxia-induced ER-phagy is BiP dependent.a Immunoblot of UPR proteins in MCF-7 cells transfected with HIF-1a siRNA or control siRNA and subjected to CI-hypoxia (n = 3). b–e Densitometric quantification of UPR proteins; b BiP, c XBP-1s, d ATF4, e CHOP in MCF-7 cells transfected with HIF-1a siRNA or control siRNA and subjected to CI-hypoxia (n = 3). f Western blot analysis of FAM134B in HIF-1a-depleted cells during CI-hypoxia and g HE (1% O2) (n = 3). h Western blot analysis of FAM134B in BiP knockdown cells using siRNA during CI-hypoxia (n = 3). i Confocal immunofluorescence image of MCF-7 cells cultured in HE stained for FAM134B (green), BiP (purple), and nucleus—DAPI (blue); scale bar = 10μm. j Confocal microscopy of MMTV-pyMT breast cancer tissue section stained for FAM134B and BiP; scale bar = 20 μm. k Confocal microscopy of human breast cancer tissue section stained for FAM134B and BiP; scale bar = 100 μm. l Immunoblot illustrating co-immunoprecipitation of BiP with FAM134B during hypoxia compared to normoxia (n = 3). m Crystal violet cell proliferation assay (absorbance is shown) on MCF-7 cells in HE compared to normoxia (n = 6).

Fig 3: CAN inhibited the mTOR-HIF-1 signaling pathway in vivo and in vitro(A–H) HIF-1a, p-mTOR (Ser 2448), and mTOR expression levels in (A) the hearts of the HFD/STZ-induced diabetic mice and in (B) the HL-1 cells were determined using western blot analysis. Relative expression level of (C) Hif-1a mRNA and quantification of (D) HIF-1α protein in the hearts of the HFD/STZ-induced diabetic mice was done using ImageJ software following western blot analysis. Relative expression levels of (E) p-mTOR (Ser 2448)/mTOR protein expression ratio in the hearts of the HFD/STZ-induced diabetic mice was determined using ImageJ following western blot analysis. Relative expression level of (F) Hif-1a mRNA and the quantification of (G) HIF-1α protein in the HL-1 cells was done using RT-qPCR and ImageJ software following western blot analysis. Relative expression levels of (H) p-mTOR (Ser 2448)/mTOR protein expression ratio in the HL-1 cells was determined using ImageJ software following western blot analysis (H).(I) Immunofluorescence analysis of the HL-1 cells using a confocal microscope (I). Magnification, ×600. Green fluorescence indicates p-mTOR (Ser 2448), red fluorescence indicates HIF-1α, blue fluorescence indicates DAPI, and merged indicates merged signal of p-mTOR (Ser 2448), HIF-1α, and DAPI. Data are presented as the mean ± SD. n = 5 or 3. #p < 0.05 and ##p < 0.01 versus normal or blank control; ∗p < 0.05 and ∗∗p < 0.01 versus HFD/STZ or PA. HFD, high-fat diet; STZ, streptozotocin; CAN, canagliflozin; normal, normal control group; HFD/STZ, HFD/STZ-induced diabetic control group; HFD/STZ + CAN, canagliflozin-treated HFD/STZ-induced diabetic group; PA, palmitic acid; Ctrl, untreated normal or blank control; Mod/PA, PA (0.25 mM)-treated control group; 0.625, HL-1 cells were treated with PA (0.25 mM) and CAN (0.625 μg/mL); 1.25, HL-1 cells were treated with PA (0.25 mM) and CAN (1.25 μg/mL); 2.5, HL-1 cells were treated with PA (0.25 mM) and CAN (2.5 μg/mL); PA + CAN, HL-1 cells treated with PA (0.25 mM) and CAN (2.5 μg/mL); p, phosphorylated.

Fig 4: PKM2 Tetramerization Limits HIF1-a, Myc, and mTORC1 Signaling and Engagement of Glycolysis in CD4+ T CellsMurine CD4+ T cells were collected after 72 h of in vitro activation with CD3/CD28 antibodies in the presence of DMSO (CTRL condition) or TEPP-46 100 µM.(A) Results of unbiased Ingenuity Pathway Analysis predicting downregulation of Myc-, Hif-1-a-, and mTOR-regulated pathways by TEPP-46.(B) Heatmaps showing expression of Myc-, Hif-1-a-, and mTOR-regulated genes in resting T cells and T cells activated in the presence of DMSO (Th0 Ctrl) or TEPP-46 (Th0 TEPP).(C) Heatmap showing expression of glycolytic genes in resting, Th0 Ctrl, and Th0 TEPP-46 cells.(D and E) Cells were tested for their glycolytic capacity and oxygen consumption rate (OCR) on a Seahorse XFe96 Analyzer.(D) Quantitative analysis of glycolytic rate and glycolytic capacity of CTRL and TEPP-46-treated cells (n = 4 from 2 independent experiments).(E) Quantitative analysis of basal OCR, maximum respiration and spare respiratory capacity of CTRL and TEPP-46-treated cells (n = 7 from three independent experiments). For (D) and (E), data are the mean ± SD. **p < 0.01 or ***p < 0.001, compared to CTRL condition, by two-tailed unpaired Student’s t test.

Fig 5: (a) Treatment of DRG cultures with bortezomib (1 µM) for 1 h increases HIF1A levels (*P= 0.0115, six wells/group). (b) DRG cultures exposed to PHD inhibitor DMOG (1 mM) for 1 h stabilized HIF1A expression (*P=0.0410, six wells/group). (c) A single intrathecal injection of DMOG (1 µg) produced mechanical allodynia that lasted several days (*P=0.04, **P=0.0027, and ****P<0.0001, five mice/group). IT: intrathecal; IP: intraperitoneal; HIF1A: hypoxia-inducible factor 1 alpha; DMOG: dimethyloxalylglycine.