Fig 1: Establishment of a prognostic hypoxic gene signature in osteosarcoma in the TARGET cohort. (A) The chromosomal locations of 197 hypoxic genes. (B) The relationships between the number of trees and error rates. (C) The rank of relative importance of the top five variables. (D) Univariate cox regression analysis of TES, SDC3, MXI1, CAVIN1, and EGFR in osteosarcoma. (E) Heat map visualizing the up- (red) and down-regulated (green) genes in high- and low-risk groups. (F) The distributions and proportions of survival status (alive: green; dead: red). (G) Kaplan-Meier curves of OS between high- and low-risk specimens. The differences were compared by log-rank tests.
Fig 2: Prognostic values of hypoxic genes from the gene signature in osteosarcoma. Kaplan-Meier curves for overall survival of osteosarcoma patients in the high and low (A) CAVIN1, (B) EGFR, (C) SCD3, (D) TES, and (E) MXI1 expression groups from the TARGET cohort. The survival differences were assessed via log-rank tests.
Fig 3: Hypoxia augments Cavin-1/Caveolin-1 interaction and reduces BMPR2 interaction with CAV1 and localization at plasma membrane.a After hPAECs were transfected with control siRNA or CAV1-specific siRNA, hypoxia stimulation with ischemic buffer was induced for 1 or 2 h. Membrane and cytosol fractions were separated by the gradient of sucrose, and lysate of these fractions were immunoblotted. GAPDH was used as cytosol marker and VE-cadherin was used as membrane marker. *P < 0.05 compared with control siRNA. **P < 0.01 compared with control siRNA. ns, not significant. b hPAECs were immunostained with anti-BMPR2 antibody and anti-CAV1 antibody after hypoxia stimulation for 2 h. Higher magnification images were shown in lower left. Scale bar, 50 μm. c Representative fluorescent images of the PLA for the interaction between CAV1 and Cavin-1 or CAV1 and BMPR2 in normoxia or hypoxia stimulated hPAECs. hPAECs were stimulated with normoxia or hypoxia for 2 hr and PLA were assessed with anti-CAV1 antibody, anti-Cavin-1 antibody and anti-BMPR2 antibody. The number of cell samples were 41 in CAV1/Cavin-1 normoxia, 35 in CAV1/Cavin-1 hypoxia, and 10 each in CAV1/BMPR2 normoxia and hypoxia, respectively. *P < 0.05 compared with normoxia. ****P < 0.0001 compared with normoxia. ns, not significant. Scale bar, 20 μm. Data are expressed as the mean ± standard error.
Fig 4: Cavin-1 knockdown rescue BMPR2 localization at plasma membrane and Smad 1/5/9 phosphorylation decreased by CAV1 knockdown.a hPAECs were transfected with control siRNA and/or CAV1-specific siRNA and/or Cavin-1-specific siRNA. Membrane and cytosol fractions were separated by the gradient of sucrose. Lysate of these fractions were immunoblotted to evaluate BMPR2 localization at plasma membrane. GAPDH was used as cytosol marker and VE-cadherin was used as membrane marker. *P < 0.05 compared with control siRNA, #P < 0.05 compared with CAV1 siRNA, ##P < 0.01 compared with CAV1 siRNA. b hPAECs were transfected with control siRNA and/or CAV1-specific siRNA and/or Cavin-1-specific siRNA, and immunostained with anti-BMPR2 antibody and anti-CAV1 antibody. Higher magnification images were shown in upper right. Scale bar, 50 μm. c Phosphorylation of Smad 1/5/9 was evaluated in hPAECs transfected with control siRNA and/or CAV1-specific siRNA and/or Cavin-1-specific siRNA. *P < 0.05 compared with control siRNA, #P < 0.05 compared with CAV1 siRNA. Data are expressed as the mean ± standard error.
Fig 5: MiR-1197/PTRF axis is associated with the abilities of OS cells to invade and proliferate. The viability (A) and colony numbers (B) of SW1353 and HOS cells carrying OE-PTRF, OE-NC, mimic, mimic-NC, or OE-PTRF + mimic were evaluated through the CCK-8 and colony formation experiments. (C) SW1353 and HOS cell invasion was assessed by means of the transwell experiment. # P < 0.05, # # P < 0.01 vs. mimic-NC. ∗ P < 0.05, ∗∗ P < 0.01 vs. OE-NC. & P < 0.05, & & P < 0.01 vs. OE-PTRF + mimic.
Supplier Page from Abcam for Anti-PTRF antibody