Journal: Acta pharmacologica Sinica
Article Title: Circular RNA circDhx32 promotes cardiac inflammatory responses in mouse cardiac ischemia-reperfusion injury via binding to FOXO1 competed with AdipoR1.
doi: 10.1038/s41401-025-01593-9
Figure Lengend Snippet: Fig. 1 CircDhx32 is upregulated in I/R-treated mouse hearts and H/R-treated cardiomyocytes. a Heatmap showing numerous circRNAs with significant differential m6A modifications in I/R-treated mice. b, c RIP revealed the binding of circDhx32 with a m6A antibody in I/R- treated mice or H/R-induced cardiomyocytes. The Y-axis represents the percentage of input for each IP sample according to the formula: % Input =1/10*2Ct [IP] – Ct [input]. n = 3. **P < 0.01 vs. Sham, *P < 0.05 vs.. Ctrl. P values were determined via an unpaired t test. d Sanger sequencing was performed to validate that circDhx32 originated from its host gene. e Expression of linear RNA (Dhx32) and circDhx32 after Act D treatment for 24 h. n = 6. **P < 0.01 vs. Act D-. P values were determined via an unpaired t test. f Relative RNA expression of GAPDH and circDhx32 in cardiomyocytes with or without RNase R incubation. n = 5. **P < 0.01 vs. Mock. P values were determined via an unpaired t test. g An RNA fractionation assay was used to evaluate the distribution of circDhx32 in the nucleus and cytoplasm under H/R conditions. n = 5. *P < 0.05 vs. Ctrl. P values were determined via an unpaired t test. h Subcellular localization of circDhx32 in H/R-induced cardiomyocytes was detected via FISH. The nuclei were stained with DAPI. Cy3 was used to stain for circDhx32. Scale bar = 20 µm. n = 4.
Article Snippet: After blocking, the membranes were incubated with primary antibodies against GAPDH (#TA-08, ZsBio, Beijing, China, 1:1000), lamin B1 (#A11495, ABclonal, Wuhan, China, 1:500), YTHDF2 (24744-1-AP, Proteintech, Wuhan, China, 1:1000), YTHDC1 (14392-1-AP, Proteintech, 1:1000), ALKBH5 (67811-1-AP, Proteintech, 1:1000), FOXO1 (18592-1-AP, Proteintech, 1:1000), the adiponectin receptor (14361-1-AP, Proteintech, 1:1000), p65 (10745-1-AP, Proteintech, 1:1000), p-p65 (#ab76302, Abcam, Cambridge, UK, 1:1000), AMPKα (D5A2) (5831S, Cell Signaling Technology, MA, USA, 1:1000) and p-AMPKα (Thr172) (2535S, Cell Signaling Technology, 1:1000) at 4 °C overnight.
Techniques: Binding Assay, Sequencing, Expressing, RNA Expression, Incubation, Fractionation, Staining
![Fig. 1 CircDhx32 is upregulated in I/R-treated mouse hearts and H/R-treated cardiomyocytes. a Heatmap showing numerous circRNAs with significant differential m6A modifications in I/R-treated mice. b, c RIP revealed the binding of circDhx32 with a m6A antibody in I/R- treated mice or H/R-induced cardiomyocytes. The Y-axis represents the percentage of input for each IP sample according to the formula: % Input =1/10*2Ct [IP] – Ct [input]. n = 3. **P < 0.01 vs. Sham, *P < 0.05 vs.. Ctrl. P values were determined via an unpaired t test. d Sanger sequencing was performed to validate that circDhx32 originated from its host gene. e Expression of linear RNA (Dhx32) and circDhx32 after Act D treatment for 24 h. n = 6. **P < 0.01 vs. Act D-. P values were determined via an unpaired t test. f Relative RNA expression of <t>GAPDH</t> and circDhx32 in cardiomyocytes with or without RNase R incubation. n = 5. **P < 0.01 vs. Mock. P values were determined via an unpaired t test. g An RNA fractionation assay was used to evaluate the distribution of circDhx32 in the nucleus and cytoplasm under H/R conditions. n = 5. *P < 0.05 vs. Ctrl. P values were determined via an unpaired t test. h Subcellular localization of circDhx32 in H/R-induced cardiomyocytes was detected via FISH. The nuclei were stained with DAPI. Cy3 was used to stain for circDhx32. Scale bar = 20 µm. n = 4.](https://pub-med-unpaywalled-images-cdn.bioz.com/pub_med_ids_ending_with_8033/pm40528033/pm40528033__page5_image1.jpg)