Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Circ‐myh8/ KAT7 Affects PANoptosis in Pulmonary Arterial Smooth Muscle Cells: Involvement of Super‐Enhancers in FOSL2 Expression

doi: 10.1161/JAHA.124.040334

Figure Lengend Snippet: A , Gene Set Enrichment Analysis (GSEA) showing that FOSL2 function under hypoxic conditions is enriched in signaling pathways related to apoptosis and inflammatory vesicles. B , Western blot analysis confirming the interference efficiency of siFOSL2. C , RT‐qPCR analysis showing changes in the transcript levels of ZBP1, CASP8, NLRP3, and MLKL, with representative plots (n=4 per group). D , Western blot showing representative graphs and statistical analysis of ZBP1, CASP8, NLRP3, and MLKL expression levels (n=4 per group). E , YP1/PI staining: YP1 (green) stains apoptotic cells, because it is permeable to their cell membranes, whereas PI (red) stains necrotic cells with compromised membranes. Scale bar=50 μm. F , Lactate Dehydrogenase (LDH) assay: measurement of LDH release to assess cell death due to membrane damage (n=6 per group). G , Immunofluorescence showing expression of ASC, RIPK3, and CASP8 in mouse smooth muscle cells. Scale bar=50 μm. Statistical analysis was performed with One‐way Analysis of Variance (ANOVA) analysis followed by Tukey honestly significant difference multiple comparison test for ( C ), ( D ), and ( F ). All values are expressed as mean±SEM. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001. ASC indicates apoptosis‐associated speck‐like protein containing a caspase recruitment domain; CASP8, caspase 8; DAPI, 4',6‐Diamidino‐2‐phenylindole; FOSL2, Fos‐like antigen 2; Hyp, hypoxia; LDH, lactate dehydrogenase; MLKL, RIPK3‐mixed lineage kinase domain‐like pseudokinase; NC, irrelevant nucleotide; NLRP3, nucleotide‐binding oligomerization domain‐like receptor family pyrin domain‐containing 3; Nor, normoxia; PASMCs, pulmonary artery smooth muscle cells; PI, Propidium Iodide; RIPK3, receptor‐interacting serine/threonine‐protein kinase 3; RT‐qPCR, reverse transcriptase‐quantitative polymerase chain reaction; siFOSL2, small interfering RNA targeting FOSL2; YP1, YO‐PRO‐1; ZBP1, Z‐DNA binding protein 1.

Article Snippet: The following primary antibodies were used: FOSL2 (FOS‐like antigen 2) (Millipore, catalog MABS1261, 1:1000; BOSTER, catalog M02615‐2, 1:1000), MAZ (myc‐associated zinc finger protein) (Novus, catalog NB100‐86984, 1:1000), H3K27ac (CST, catalog 8173S, 1:1000), NLRP3 (NOD‐likereceptor protein 3) (CST, catalog 15101S, 1:500; ABclonal, catalog A126694, 1:1000), ASC (apoptosis‐associated speck‐like protein containing a CARD) (CST, catalog 67824S, 1:1000), GSDMD (Gasdermin D; CST, catalog 39754S, 1:500), ZBP1 (BOSTER, catalog A04739‐3, 1:1000), MLKL (mixed lineage kinase domain‐likepseudokinase) (ABclonal, catalog A17312, 1:1000), CASP8 (ABclonal, catalog A0215, 1:1000), RIPK3 (ABclonal, catalog A5431, 1:1000), CASP1 (recombinant caspase 1; BOSTER, catalog BA2220,1:1000), and MYST2 (KAT7, lysine acetyltransferase 7; CST, catalog 58418S, 1:500). β‐actin (ABclonal, catalog AC004, 1:1000) was used as a loading control.

Techniques: Protein-Protein interactions, Western Blot, Quantitative RT-PCR, Expressing, Staining, Lactate Dehydrogenase Assay, Membrane, Immunofluorescence, Comparison, Binding Assay, Reverse Transcription, Real-time Polymerase Chain Reaction, Small Interfering RNA

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Circ‐myh8/ KAT7 Affects PANoptosis in Pulmonary Arterial Smooth Muscle Cells: Involvement of Super‐Enhancers in FOSL2 Expression

doi: 10.1161/JAHA.124.040334

Figure Lengend Snippet: A , Schematic diagram of CRISPR‐Cas9‐mediated knockdown of FOSL2‐SE. B , Representative graph of RT‐qPCR products showing the efficiency of Fosl2‐SE knockdown. C , RT‐qPCR analysis of Fosl2 transcript levels following knockdown with dCas‐SE (CRISPR‐Cas9‐Fosl2‐SE2) (n=4 per group). D , Western blot analysis showing representative graphs and statistical data for FOSL2 protein expression after dCas‐SE knockdown (n=6 per group). E , Prediction of target genes downstream of transcription factors using databases including Global Transcriptional Regulatory Database (GTRD), Find Individual Motif Occurrences (FIMO) JASPAR, PWM Enrichment Analysis (PWMEnrich), Encyclopedia of DNA Elements (ENCODE), and Human Transcription Factor Targets (hTFtarget). F , ChIP‐qPCR analysis: ChIP with FOSL2 antibody followed by qPCR to assess promoter activity of ZBP1, CASP8, NLRP3, and MLKL (n=3 per group). G , RT‐qPCR analysis showing transcript levels of ZBP1, CASP8, NLRP3, and MLKL following dCas‐SE knockdown (n=4 per group). H , Western blot showing representative plots and statistical analysis of ZBP1, CASP8, NLRP3, and MLKL protein expression following dCas‐SE knockdown (n=4, 5, 7 per group). I , LDH assay: measurement of cell death following dCas‐SE knockdown by detecting LDH released from damaged cell membranes (n=6 per group). J , YP1/PI staining: YP1 (green) stains apoptotic cells (permeable to their cell membranes), whereas PI (red) stains necrotic cells with compromised membranes. Scale bar=50 μm. Statistical analysis was performed with 1‐way ANOVA analysis followed by Tukey honestly significant difference multiple comparison test for ( C ), ( D ), ( G ), ( H ), ( I ) and 2‐way ANOVA test for ( F ). All values are presented as mean±SEM. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001. CASP8 indicates caspase 8; ChIP, chromatin immunoprecipitation; CRISPR‐Cas9, clustered regularly interspaced short palindromic repeats‐associated protein 9; FOSL2, Fos‐like antigen 2; LDH, lactate dehydrogenase; Hyp, hypoxia; MLKL, RIPK3‐mixed lineage kinase domain‐like pseudokinase; NLRP3, nucleotide‐binding oligomerization domain‐like receptor family pyrin domain‐containing 3; Nor, normoxia; PI, Propidium Iodide; qPCR, quantitative polymerase chain reaction; RT‐qPCR, reverse transcriptase‐quantitative polymerase chain reaction; SE, super‐enhancer; YP1, YO‐PRO‐1; and ZBP1, Z‐DNA binding protein 1.

Article Snippet: The following primary antibodies were used: FOSL2 (FOS‐like antigen 2) (Millipore, catalog MABS1261, 1:1000; BOSTER, catalog M02615‐2, 1:1000), MAZ (myc‐associated zinc finger protein) (Novus, catalog NB100‐86984, 1:1000), H3K27ac (CST, catalog 8173S, 1:1000), NLRP3 (NOD‐likereceptor protein 3) (CST, catalog 15101S, 1:500; ABclonal, catalog A126694, 1:1000), ASC (apoptosis‐associated speck‐like protein containing a CARD) (CST, catalog 67824S, 1:1000), GSDMD (Gasdermin D; CST, catalog 39754S, 1:500), ZBP1 (BOSTER, catalog A04739‐3, 1:1000), MLKL (mixed lineage kinase domain‐likepseudokinase) (ABclonal, catalog A17312, 1:1000), CASP8 (ABclonal, catalog A0215, 1:1000), RIPK3 (ABclonal, catalog A5431, 1:1000), CASP1 (recombinant caspase 1; BOSTER, catalog BA2220,1:1000), and MYST2 (KAT7, lysine acetyltransferase 7; CST, catalog 58418S, 1:500). β‐actin (ABclonal, catalog AC004, 1:1000) was used as a loading control.

Techniques: CRISPR, Knockdown, Quantitative RT-PCR, Western Blot, Expressing, ChIP-qPCR, Activity Assay, Lactate Dehydrogenase Assay, Staining, Comparison, Chromatin Immunoprecipitation, Binding Assay, Real-time Polymerase Chain Reaction, Reverse Transcription

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Circ‐myh8/ KAT7 Affects PANoptosis in Pulmonary Arterial Smooth Muscle Cells: Involvement of Super‐Enhancers in FOSL2 Expression

doi: 10.1161/JAHA.124.040334

Figure Lengend Snippet: A and B , Western blot: Representative graphs and statistical data depicting the expression of H3K27ac and FOSL2 proteins in mPASMCs treated with JQ1 and iBET inhibitors under 24‐hour hypoxic conditions (n=4 per group). C and E , RT‐qPCR: Quantification of transcript levels for Fosl2, Zbp1, Casp8, Nlrp3, and Mlkl in mPASMCs subjected to 24‐hour hypoxia in the presence of JQ1 and iBET inhibitors (n=4 per group). D , Enzyme‐Linked Immunosorbent Assay (ELISA): Measurement of NLRP3 protein levels, showing a significant increase following inhibitor treatment (n=4 per group). F , Western blot: Representative graphs and statistical data for the expression of ZBP1, CASP8, NLRP3, and MLKL proteins following the addition of JQ1 and iBET inhibitors (n=4 per group). G , Immunofluorescence: Expression patterns of ASC, RIPK3, and CASP8 following treatment with JQ1 and iBET inhibitors. Scale bar=50 μm. Conditions: Nor and Hyp. Statistical analysis was performed with 1‐way ANOVA analysis followed by Tukey honestly significant difference multiple comparison test for ( A ) through ( F ). All data are presented as mean±SEM. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001. ASC indicates apoptosis‐associated speck‐like protein containing a caspase recruitment domain; CASP8, caspase‐8; DAPI, 4',6‐Diamidino‐2‐phenylindole; FOSL2, Fos‐like antigen 2; H3K27ac, acetylation of lysine 27 of histone H3; Hyp, hypoxia; mPASMCs, mouse pulmonary artery smooth muscle cells; MLKL, RIPK3‐mixed lineage kinase domain‐like pseudokinase; NLRP3, nucleotide‐binding oligomerization domain‐like receptor family pyrin domain‐containing 3; Nor, normoxia; SE, super‐enhancer; and ZBP1, Z‐DNA binding protein 1.

Article Snippet: The following primary antibodies were used: FOSL2 (FOS‐like antigen 2) (Millipore, catalog MABS1261, 1:1000; BOSTER, catalog M02615‐2, 1:1000), MAZ (myc‐associated zinc finger protein) (Novus, catalog NB100‐86984, 1:1000), H3K27ac (CST, catalog 8173S, 1:1000), NLRP3 (NOD‐likereceptor protein 3) (CST, catalog 15101S, 1:500; ABclonal, catalog A126694, 1:1000), ASC (apoptosis‐associated speck‐like protein containing a CARD) (CST, catalog 67824S, 1:1000), GSDMD (Gasdermin D; CST, catalog 39754S, 1:500), ZBP1 (BOSTER, catalog A04739‐3, 1:1000), MLKL (mixed lineage kinase domain‐likepseudokinase) (ABclonal, catalog A17312, 1:1000), CASP8 (ABclonal, catalog A0215, 1:1000), RIPK3 (ABclonal, catalog A5431, 1:1000), CASP1 (recombinant caspase 1; BOSTER, catalog BA2220,1:1000), and MYST2 (KAT7, lysine acetyltransferase 7; CST, catalog 58418S, 1:500). β‐actin (ABclonal, catalog AC004, 1:1000) was used as a loading control.

Techniques: Western Blot, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Comparison, Binding Assay

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Circ‐myh8/ KAT7 Affects PANoptosis in Pulmonary Arterial Smooth Muscle Cells: Involvement of Super‐Enhancers in FOSL2 Expression

doi: 10.1161/JAHA.124.040334

Figure Lengend Snippet: A , Schematic representation of the hypoxia (10%) model in mice, incorporating a lentiviral construct carrying shRNA for the knockdown of circ‐myh8. B , Representative RT‐qPCR plots demonstrating the efficiency of circ‐myh8 knockdown (n=4 per group). C and D , Statistical analysis of right ventricular pressure and the right heart hypertrophy index (n=6 per group). E and F , Representative images of HE and Masson trichrome staining of lung tissues from the model mice. G , Immunofluorescence analysis showing FOSL2 expression in the circ‐myh8 knockdown model. Scale bar=25 μm. H , RT‐qPCR analysis indicating changes in FOSL2 transcript levels following circ‐myh8 knockdown (n=4 per group). I and M , Western blot analysis: representative and statistical data on the protein expression of FOSL2, ZBP1, CASP8, MLKL, NLRP3, and CASP1 (n=4,3 per group). J through L , Statistical analysis of the area, mean fluorescence intensity, and integrated fluorescence intensity (n=4 per group). N and O , Colocalization immunofluorescence images of FOSL2 with ZBP1 and CASP8 in the circ‐myh8 knockdown model. Scale bar=25 μm. Statistical analysis was performed with 1‐way ANOVA analysis followed by Tukey honestly significant difference multiple comparison test for ( B ) through ( D ), ( H ) through ( M ). All values are expressed as mean±SEM. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001. α‐SMA indicates smooth muscle actin alpha; CASP1, caspase 1; CASP8, caspase 8; sh‐circ, shRNA denotes lentiviral knockdown of circ‐myh8; FOSL2, Fos‐like antigen 2; KAT7, lysine acetyltransferase 7; MLKL, RIPK3‐mixed lineage kinase domain‐like pseudokinase; NC, negative control; NLRP3, nucleotide‐binding oligomerization domain‐like receptor family pyrin domain‐containing 3; HE, hematoxylin and eosin; and RT‐qPCR, reverse transcriptase‐quantitative polymerase chain reaction; and ZBP1, Z‐DNA binding protein 1.

Article Snippet: The following primary antibodies were used: FOSL2 (FOS‐like antigen 2) (Millipore, catalog MABS1261, 1:1000; BOSTER, catalog M02615‐2, 1:1000), MAZ (myc‐associated zinc finger protein) (Novus, catalog NB100‐86984, 1:1000), H3K27ac (CST, catalog 8173S, 1:1000), NLRP3 (NOD‐likereceptor protein 3) (CST, catalog 15101S, 1:500; ABclonal, catalog A126694, 1:1000), ASC (apoptosis‐associated speck‐like protein containing a CARD) (CST, catalog 67824S, 1:1000), GSDMD (Gasdermin D; CST, catalog 39754S, 1:500), ZBP1 (BOSTER, catalog A04739‐3, 1:1000), MLKL (mixed lineage kinase domain‐likepseudokinase) (ABclonal, catalog A17312, 1:1000), CASP8 (ABclonal, catalog A0215, 1:1000), RIPK3 (ABclonal, catalog A5431, 1:1000), CASP1 (recombinant caspase 1; BOSTER, catalog BA2220,1:1000), and MYST2 (KAT7, lysine acetyltransferase 7; CST, catalog 58418S, 1:500). β‐actin (ABclonal, catalog AC004, 1:1000) was used as a loading control.

Techniques: Construct, shRNA, Knockdown, Quantitative RT-PCR, Staining, Immunofluorescence, Expressing, Western Blot, Fluorescence, Comparison, Negative Control, Binding Assay, Reverse Transcription, Real-time Polymerase Chain Reaction