Fig 1: Amylin drives the expression of HIF1a and PFKFB3 in hiPSC-CMs.a, b Effect of pre-treatment of hiPSC-CMs with a human amylin antibody for 24 h on amylin-induced HIF1a (a) and PFKFB3 (b) mRNA levels (n = 6/group). c Effect of pre-treatment of hiPSC-CMs with siRNA for HIF1a (siHIF1a) or for vehicle control (siCTL) on amylin-induced HIF1a (n = 4/group). d Effect of pretreatment of hiPSC-CMs with siHIF1a, siRNA for PFKFB3 (siPFKFB3), or siCTL on amylin-induced PFKFB3 (n = 4/group). e, f Effect of HIF1a inhibitor YC-1 on the upregulation of HIF1a (e) and PFKFB3 (f) induced by amylin (n = 6/group). Data represent mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 by One-way ANOVA with Tukey’s post-test.
Fig 2: Activation of HIF1a, PFKFB3, and PFKFB3 phosphorylation at Ser461 in the myocardium of NHPs with HF.Expression of HIF1a (a–e), PFKFB3 (f–j), and pSer461-PFKFB3 (k–l) in hearts from CTL (n = 7) and HF (n = 6) NHPs. qRT-PCR for HIF1a mRNA (a) and PFKFB3 (h) in hearts from NHPs with HF (n = 6) and CTL (n = 7). Immunohistochemistry of HIF1a (b) and PFKFB3 (f) (brown color) in heart sections from NHPs with HF and CTL as used in panel (a). Quantifications of the relative positive signal intensity for HIF1a (c) and PFKFB3 (g) in a 284 × 214 µm field of view of heart sections from NHPs as used in panels (a). Western blot analysis with anti-HIF1a (d), anti-PFKFB3 (i), and anti-pSer461-PFKFB3 (k) antibodies on heart lysates as used in panels (a). Quantifications of protein expression in western blot were shown in bar graphs (e, j, and l). Scale bar, 100 µm. Data represent mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 by Student’s t test.
Fig 3: Cardiac amylin accumulation activated HIF1a and PFKFB3 in isolated RVCMs.a–j Increased expression of HIF1a (a–d, i) and PFKFB3 (e–h, j) in RVCMs treated with or without H-amy for 2 h. a, b Immunofluorescence staining (a, green color, mouse anti-HIF1a antibody; blue color, Hoechst 33342) in cells treated under the above conditions and b quantification of HIF1a positive signal was shown in bar graph (n = 10/group). c, d Western blot analysis (blot, c and bar graph, d) of HIF1a (mouse anti-HIF1a antibody) in RVCMs treated as in panel (a) (n = 3/group). e, f Immunofluorescence staining (e, red color, rabbit anti-PFKFB3 antibody; blue color, Hoechst 33342) in cells under the above conditions and f quantification of PFKFB3 positive signal was shown in bar graph (n = 10/group). g, h Western blot analysis (blot, g and bar graph, h) of PFKFB3 (rabbit anti-PFKFB3 antibody) in RVCMs treated as in panel (a) (n = 3/group). i, j Increased mRNA levels of HIF1a (i) and PFKFB3 (j) in RVCMs incubated with amylin (n = 10/group). Scale bar, 100 µm. Data represent mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 by Student’s t test.
Fig 4: Cardiac amylin accumulation activated HIF1a and PFKFB3 in hiPSC-CMs.a Immunostaining of a-actinin (a, green color, mouse anti-a-Actinin antibody), positive signal of human amylin (a, red color, rabbit anti-human amylin antibody) and Hoechst 33342 (blue color) in cells treated with H-amy and b quantification of amylin positive signal was shown in bar graph (n = 6/group). c–l Expression of HIF1a (c–g) and PFKFB3 (h–l) in hiPSC-CMs treated with CTL and H-amy. Positive signal of HIF1a (c, green color, mouse anti-HIF1a antibody), PFKFB3 (h, red color, rabbit anti-PFKFB3 antibody) and Hoechst 33342 (c and h, blue color) levels were quantified as shown in bar graph (d, HIF1a; i, PFKFB3, n = 9/group). Western blot analysis of HIF1a (e, f) and PFKFB3 (j, k) in hiPSC-CMs incubated under the two conditions described in panel (c) (n = 3/group). qRT-PCR of HIF1a (g) and PFKFB3 (l) in myocytes incubated with CTL and H-amy (n = 5/group). Scale bar, 100 µm. Data represent mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.01 by Student’s t test.
Supplier Page from Abcam for Anti-PFKFB3 (phospho S461) antibody [EPR19735]