Fig 2: Expression and association of OR7E156P and HIF1A under normoxia or hypoxia conditions (A) Glioma tissues samples from the core region, intermediate region, and the peripheral region were collected and examined by Hematoxylin and eosin (H&E) staining for histopathological characteristics and the immunofluorescence (IF) staining for the protein content and distribution of HIF1A. (B) The expression of HIF1A and OR7E156P in glioma tissues samples from the core region, intermediate region, and the peripheral region was examined using real-time PCR. (C) The correlation of HIF1A and OR7E156P expression in tissue samples was analyzed using Pearson’s correlation analysis. (D, E) A human fetal glial cell line SVG p12 and four glioma cell lines, U87-MG, T98-G, U251-MG, and LN229 were exposed to 1% or 20% oxygen and examined for the expression of OR7E156P using real-time PCR and protein levels of HIF1A using Immunoblotting. (F) U251-MG and U87-MG cells were transfected with Lsh-OR7E156P, exposed to 1% or 20% oxygen, and then examined for the protein levels of HIF1A by Immunoblotting. *P < 0.05, **P < 0.01, ## P < 0.01.

Fig 3: Dynamic effects of HIF1A and OR7E156P on glioma in vitro and in vivo U251-MG and U87-MG cells were transduced with Lsh-OR7E156P or Lsh-NC, exposed to 1% O2 or 20% O2, and examined for cell invasion by Transwell (A) and DNA synthesis capacity by EdU (B). (C) Conditioned medium was obtained from Lsh-OR7E156P- or Lsh-NC-infected U251-MG and U87-MG cells and used for human umbilical vein endothelial cell (HUVEC) culturing; the content of VEGF in culture medium was determined by ELISA. (D) The tube formation capacity of HUVEC in different conditional culture medium was determined. (E) Tumor volumes and weights from mice bearing tumors derived from glioma cells infected or co-infected with Adv-HIF1A (Adv-NC as control) and/or Lsh-OR7E156P (Lsh-NC as control) were measured and calculated. HIF-1a protein content and distribution in tumor tissues was examined by IHC staining. *P < 0.05, **P < 0.01, #p < 0.05, ## P < 0.01.

Fig 4: Intense Light Increases Cardiac Adenosine-cAMP and Glycolytic Flux via PER2 in the Uninjured Heart(A) Infarct sizes in C57BL/6J mice that were housed under intense light (IL; 10,000 lux, L:D 14:10 h) for 7 days and subjected to 60 min of in situ myocardial ischemia followed by 2 h reperfusion at ZT3 or ZT15 (mean ± SD; n = 6; Student’s t test).(B–D) C57BL/6J mice exposed to voluntary wheel running for 1 versus 2 weeks. Shown are infarct sizes after 60 min of myocardial ischemia and 2 h reperfusion at ZT3 (B) or circadian amplitude (C) and distance walked measurements in relation to infarct sizes (D, mean ± SD; n = 6; Student’s t test).(E–H) Wheel running measurements during or infarct size studies after 2 weeks of wheel running at ZT3 in C57BL/6J or Per2-/- mice (mean ± SD; n = 5; Student’s t test).(E) Distance walked.(F) Circadian amplitude.(G and H) Infarct size measurements (G) and one representative infarct size staining and one wheel running activity recording are shown (H).(I and J) Adenosine (I) or cAMP (J) levels in heart tissue from C57BL/6J or Per2-/- mice at ZT3 after 7 days of room light (RL; 200 lux, L:D 14:10 h) or intense light (IL; 10,000 lux, L:D 14:10 h) housing (mean ± SD; n = 5; ANOVA with Tukey’s multiple comparison test).(K) Cardiac U-13C-glucose-1,6-bisphosphate levels at ZT3 from C57BL/6J mice that were housed under RL or IL for 7 days (mean ± SD; n = 4; Student’s t test).(L and M) Phosphofructokinase (PFK) activity in both heart tissue (L) and plasma samples (M) from C57BL/6J or Per2-/- mice at ZT3 after 7 days of RL or IL housing (mean ± SD; n = 4–5; ANOVA with Tukey’s multiple comparison test).(N) HIF1A-hypoxia response element (HRE) binding was determined at ZT3, ZT9, ZT15, and ZT21 (mean ± SD; n = 5; *p < 0.05 for ZT21 versus ZT3 in RL- and IL-housed mice via Student’s t test).(O) C57BL/6J or Per2-/- mice housed under IL for 7 days before 60 min myocardial ischemia and 2 h reperfusion at ZT3 (mean ± SD; n = 5; Student’s t test).(P) Representative infarct staining.

Fig 5: miR-143 is involved in the cross-talk between OR7E156P and HIF1A (A) A schematic diagram showing the selection of miRNAs related to glioma and might simultaneously target OR7E156P and HIF1A. Five miRNAs were selected: miR-17, miR-20a, miR-153, miR-519d, and miR-143-5p. (B) U251-MG and U87-MG cells were infected with Lsh-OR7E156P and examined for the expression of miR-17, miR-20a, miR-153, miR-519d, and miR-143-5p by real-time PCR. (C) U251-MG and U87-MG cells were exposed to 1% or 20% oxygen and examined for the expression of miR-17, miR-20a, miR-153, miR-519d, and miR-143-5p by real-time PCR. (E) Wild-type and mutant-type OR7E156P or HIF1A 3’UTR luciferase reporter vectors were constructed as described in the Materials and Methods section. These vectors were co-transfected in 293T cells with miR-143 mimics or miR-143 inhibitor. The luciferase activity was determined. (F) RIP assays were performed to confirm the binding of miR-143 to OR7E156P and the 3’-UTR of HIF1A using AGO2 antibody. The levels of miR-143, OR7E156P, and HIF1A in precipitated AGO2 proteins were examined using real-time PCR. *P < 0.05, **P < 0.01.