Journal: Frontiers in Immunology

Article Title: 3-Hydroxystearic acid promotes cholesterol efflux and attenuates atherosclerosis via the ALKBH5/PAX-8/ABCA1 pathway

doi: 10.3389/fimmu.2026.1750021

Figure Lengend Snippet: C18-3OH modulates ABCA1 expression and cholesterol efflux in foam cells via PAX-8 upregulation. (A) RT-qPCR analysis of PAX-8 mRNA expression in foam cells treated with C18-3OH. (B) Western blot analysis of PAX-8 protein expression in foam cells treated with C18-3OH. ** P < 0.01 vs. control, n = 3. (C) Western blot detection of PAX-8 and ABCA1 protein expression in foam cells following PAX-8 knockdown. (D) Quantitative analysis of PAX-8 protein expression (Panel C). (E) RT-qPCR analysis of ABCA1 mRNA expression in foam cells after PAX-8 knockdown. (F) Quantitative analysis of ABCA1 protein expression (Panel C). (G) Effects of C18-3OH and siPAX-8 on cholesterol efflux in foam cells. * P < 0.05, ** P < 0.01 vs. siNC + DMSO; ## P < 0.01 vs. siNC + C18-3OH, n = 3. (H) NBD-cholesterol fluorescence intensity in foam cells; scale bar = 150 μm.

Article Snippet: Primary antibodies diluted in antibody dilution buffer were applied as follows: ABCA1 (Abcam, 1:1000), PAX-8 (Proteintech, 1:3000), ALKBH5 (Proteintech, 1:5000), ELAVL1 (Proteintech, 1:5000); GAPDH (Proteintech, 1:20, 000) and α-Tubulin (Proteintech, 1:20, 000) served as loading controls.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Control, Knockdown, Fluorescence

Journal: Frontiers in Immunology

Article Title: 3-Hydroxystearic acid promotes cholesterol efflux and attenuates atherosclerosis via the ALKBH5/PAX-8/ABCA1 pathway

doi: 10.3389/fimmu.2026.1750021

Figure Lengend Snippet: PAX-8 binds to and regulates ABCA1 expression. (A) Predicted binding sites between PAX-8 and ABCA1 in humans. (B) Predicted binding sites between PAX-8 and ABCA1 in mice. (C) Western blot analysis of PAX-8 and ABCA1 protein expression in foam cells following PAX-8 overexpression. (D) Quantitative analysis of PAX-8 protein expression (Panel C). (E) RT-qPCR analysis of ABCA1 mRNA expression in foam cells after PAX-8 overexpression. (F) Quantitative analysis of ABCA1 protein expression (Panel C). ** P < 0.01 vs. LV-NC, n = 3. (G) ChIP-Seq identification of PAX-8 binding to the ABCA1 locus. Genomic tracks showing PAX-8 binding to the ABCA1 locus in the Input (negative control, top) and ChIP (PAX-8-enriched, bottom) groups. The y-axis indicates relative enrichment of ChIP-Seq signals. The x-axis represents genomic coordinates. (H) ChIP-qPCR validation of PAX-8 targeting regulation of ABCA1. ** P < 0.01 vs. Input, n = 3.

Article Snippet: Primary antibodies diluted in antibody dilution buffer were applied as follows: ABCA1 (Abcam, 1:1000), PAX-8 (Proteintech, 1:3000), ALKBH5 (Proteintech, 1:5000), ELAVL1 (Proteintech, 1:5000); GAPDH (Proteintech, 1:20, 000) and α-Tubulin (Proteintech, 1:20, 000) served as loading controls.

Techniques: Expressing, Binding Assay, Western Blot, Over Expression, Quantitative RT-PCR, ChIP-sequencing, Negative Control, ChIP-qPCR, Biomarker Discovery

Journal: Frontiers in Immunology

Article Title: 3-Hydroxystearic acid promotes cholesterol efflux and attenuates atherosclerosis via the ALKBH5/PAX-8/ABCA1 pathway

doi: 10.3389/fimmu.2026.1750021

Figure Lengend Snippet: Effects of C18-3OH on m 6 A modification of PAX-8 mRNA in foam cells. (A) Predicted score distribution of the human PAX-8 gene query sequence. (B) Predicted score distribution of the murine PAX-8 gene query sequence. (C) Partitioned statistical plot of predicted scores for the human PAX-8 gene query sequence. (D) Partitioned statistical plot of predicted scores for the murine PAX-8 gene query sequence. (E) MeRIP-qPCR analysis of the effect of C18-3OH on m 6 A modification of PAX-8 mRNA in foam cells. ** P < 0.01 vs. control, n = 3.

Article Snippet: Primary antibodies diluted in antibody dilution buffer were applied as follows: ABCA1 (Abcam, 1:1000), PAX-8 (Proteintech, 1:3000), ALKBH5 (Proteintech, 1:5000), ELAVL1 (Proteintech, 1:5000); GAPDH (Proteintech, 1:20, 000) and α-Tubulin (Proteintech, 1:20, 000) served as loading controls.

Techniques: Modification, Sequencing, Control

Journal: Frontiers in Immunology

Article Title: 3-Hydroxystearic acid promotes cholesterol efflux and attenuates atherosclerosis via the ALKBH5/PAX-8/ABCA1 pathway

doi: 10.3389/fimmu.2026.1750021

Figure Lengend Snippet: Effects of C18-3OH on ALKBH5 expression in foam cells. (A) Bioinformatic analysis of methylation-associated proteins regulating PAX-8 in THP-1 cells. (B) RT-qPCR analysis of ALKBH5 mRNA expression in foam cells treated with C18-3OH. (C) Western blot analysis of ALKBH5 protein expression in foam cells treated with C18-3OH. (D) RT-qPCR analysis of ELAVL1 mRNA expression in foam cells treated with C18-3OH. (E) Western blot analysis of ELAVL1 protein expression in foam cells treated with C18-3OH. * P < 0.05, ** P < 0.01 vs. control, n = 3.

Article Snippet: Primary antibodies diluted in antibody dilution buffer were applied as follows: ABCA1 (Abcam, 1:1000), PAX-8 (Proteintech, 1:3000), ALKBH5 (Proteintech, 1:5000), ELAVL1 (Proteintech, 1:5000); GAPDH (Proteintech, 1:20, 000) and α-Tubulin (Proteintech, 1:20, 000) served as loading controls.

Techniques: Expressing, Methylation, Quantitative RT-PCR, Western Blot, Control

Journal: Frontiers in Immunology

Article Title: 3-Hydroxystearic acid promotes cholesterol efflux and attenuates atherosclerosis via the ALKBH5/PAX-8/ABCA1 pathway

doi: 10.3389/fimmu.2026.1750021

Figure Lengend Snippet: ALKBH5 mediates C18-3OH regulation of PAX-8 and ABCA1 expression, PAX-8 mRNA m 6 A modification, and cholesterol efflux. (A) Western blot analysis of ALKBH5 expression in foam cells following lentiviral transfection for ALKBH5 overexpression. ** P < 0.01 vs. control, n = 3. (B) Western blot analysis of PAX-8 and ABCA1 protein expression in foam cells following ALKBH5 overexpression. (C) RT-qPCR analysis of ABCA1 mRNA expression in foam cells after ALKBH5 overexpression. (D) Quantitative analysis of ABCA1 protein expression (Panel B). (E) RT-qPCR analysis of PAX-8 mRNA expression in foam cells after ALKBH5 overexpression. (F) Quantitative analysis of PAX-8 protein expression (Panel B). (G) MeRIP-qPCR analysis of PAX-8 mRNA m6A modification in foam cells following ALKBH5 overexpression. (H) Effect of C18-3OH and LV-ALKBH5 on cholesterol efflux in foam cells. (I) NBD-cholesterol fluorescence intensity in foam cells; scale bar = 150 μm. * P < 0.05, ** P < 0.01 vs. LV-NC + DMSO; ## P < 0.01 vs. LV-NC + C18-3OH, n = 3.

Article Snippet: Primary antibodies diluted in antibody dilution buffer were applied as follows: ABCA1 (Abcam, 1:1000), PAX-8 (Proteintech, 1:3000), ALKBH5 (Proteintech, 1:5000), ELAVL1 (Proteintech, 1:5000); GAPDH (Proteintech, 1:20, 000) and α-Tubulin (Proteintech, 1:20, 000) served as loading controls.

Techniques: Expressing, Modification, Western Blot, Transfection, Over Expression, Control, Quantitative RT-PCR, Fluorescence

Journal: Frontiers in Immunology

Article Title: 3-Hydroxystearic acid promotes cholesterol efflux and attenuates atherosclerosis via the ALKBH5/PAX-8/ABCA1 pathway

doi: 10.3389/fimmu.2026.1750021

Figure Lengend Snippet: Effects of C18-3OH on ABCA1, PAX-8, ALKBH5 expression, and PAX-8 mRNA m 6 A methylation in Aortas of apoE −/− Mice. (A) Western blot analysis of ABCA1, PAX-8, and ALKBH5 protein expression in aortas of HFD-fed apoE −/− mice treated with C18-3OH and/or ALKBH5 overexpression. (B) RT-qPCR analysis of ABCA1 mRNA expression in aortas of apoE −/− mice. (C) RT-qPCR analysis of PAX-8 mRNA expression in aortas of apoE −/− mice. (D) RT-qPCR analysis of ALKBH5 mRNA expression in aortas of apoE −/− mice. (E) Quantitative analysis of ABCA1 protein expression (Panel A). (F) Quantitative analysis of PAX-8 protein expression (Panel A). (G) Quantitative analysis of ALKBH5 protein expression (Panel A). (H) MeRIP-qPCR analysis of PAX-8 mRNA m 6 A methylation levels in aortas of apoE −/− mice across experimental groups. ** P < 0.01 vs. Control; # P < 0.05, ## P < 0.01 vs. C18-3OH + AAV-NC. n=6.

Article Snippet: Primary antibodies diluted in antibody dilution buffer were applied as follows: ABCA1 (Abcam, 1:1000), PAX-8 (Proteintech, 1:3000), ALKBH5 (Proteintech, 1:5000), ELAVL1 (Proteintech, 1:5000); GAPDH (Proteintech, 1:20, 000) and α-Tubulin (Proteintech, 1:20, 000) served as loading controls.

Techniques: Expressing, Methylation, Western Blot, Over Expression, Quantitative RT-PCR, Control

Journal: Stem cells (Dayton, Ohio)

Article Title: Dual-specificity phosphatases 13 and 27 as key switches in muscle stem cell transition from proliferation to differentiation.

doi: 10.1093/stmcls/sxae045

Figure Lengend Snippet: Figure 1. Dusp13 is a novel target regulated by MyoD. (A) Clustering heatmap showing differentially expressed genes in RNA-seq data from Pax7- YFP (Quiescent MuSCs; QSCs), MyoD-low (MyoD-tdTomato low), and MyoD-high (MyoD-tdTomato high) (n = 3 per group). (B) Clustering heatmap of differentially expressed genes in RNA-seq between wild-type (WT) growth medium (GM) (WT_GM), MyoD knock-out GM (MKO_GM), WT differentiation medium (WT_DM), and MKO_DM (n = 3 per group). (C) Venn diagrams of indicated clusters from (A, B) and ChIP atlas top 100 genes potentially regulated by MyoD (Left). Venn diagrams of overlapped 35 genes (left) and 80 genes upregulated by MyoD overexpression in MEFs (Right). (D) List of 6 genes from (C). (E, F) Gene expression of MyoD (E) or Dusp13 (F) analyzed by RNA-seq in the Pax7-YFP, MyoD-low, and MyoD-high MuSCs (n = 3 per group). (G, H) Gene expression of MyoD (G) or Dusp13 (H) analyzed by RNA-seq in the WT_GM, WT_DM, MKO_GM, and MKO_DM groups (n = 3 per group). (I) E-box-like sequences in the promoter region of Dusp13 in mammalian species. (J) Luciferase reporter constructs of Dusp13 upstream region (−150 to +50) containing an E-box-like sequence (Top). Relative Dusp13-luciferase reporter activities in HEK293T cells overexpressing MyoD (Bottom) (n = 3 per group). All data are presented as mean ± SEM P values calculated using edge R (E, F) (FDR-corrected), DEseq2 (G, H) (FDR- corrected), or Tukey’s test (J); **P < .01, ***P < .001, n.s., not significant.

Article Snippet: Primary antibodies were against PAX7 (sc-81648; SantaCruz), MYOD (sc-377460; SantaCruz), MYOD (ab133627, Abcam), RFP (600-401-379, Rockland), LAMININ (sc-59854; SantaCruz), MYOGENIN (124800; Abcam), MYOGENIN (MA5-11486; Thermo Fisher Scientific), (MyHC; DSHB; MF20), and KI67 (MA514520; Thermo Fisher Scientific).

Techniques: RNA Sequencing, Knock-Out, Over Expression, Gene Expression, Luciferase, Construct, Sequencing

Journal: Stem cells (Dayton, Ohio)

Article Title: Dual-specificity phosphatases 13 and 27 as key switches in muscle stem cell transition from proliferation to differentiation.

doi: 10.1093/stmcls/sxae045

Figure Lengend Snippet: Figure 2. Absence of skeletal muscle phenotype in Dusp13 knock-out (13 KO) mice during development and regeneration. (A) Hematoxylin and eosin (H&E) staining of Soleus (SOL) and Plantaris (PLA) muscle cross-sections of male Dusp13 knock-out (13 KO) and WT mice at 10 weeks. Scale bar: 100 μm. (B, C) Cross-sectional area (CSA) of SOL (B) or PLA (C) (n = 3 per group). (D) Immunofluorescence of PAX7 and LAMININ in intact TA muscles. Scale bar: 100 µm. The area in the white dotted boxes is shown at a higher magnification. Scale bar: 25 μm. (E) Average number of PAX7 (+) nuclei in DAPI (+) in (D). (F) H&E staining of tibialis anterior (TA) muscle cross-sections of male 13 KO and WT mice at 12 weeks, 7 days post cardiotoxin (CTX) injury. Scale bar: 100 µm. (G) CSA of damaged TA muscles (n = 3 per group). (H) Immunofluorescence of PAX7 with LAMININ on sections of TA muscles from male WT and Dusp13 KO mice, 7 days post-CTX injury. Nuclei are stained with DAPI. Scale bar: 100 µm. The area in the white dotted boxes is shown at a higher magnification. Scale bar: 25 μm. (I) Average number of PAX7 (+) nuclei in (H) are quantified (n = 3–4 independent experiments). (J) Immunofluorescence of MYOGENIN with LAMININ on sections of TA muscle from male WT and 13 KO mice, 7 days post-CTX injury. Nuclei are stained with DAPI. Scale bar: 100 µm. The area in the white dotted boxes is shown at a higher magnification. Scale bar: 25 μm. (K) Average number of MYOGENIN (+) nuclei in (J) are quantified (n = 3–4 independent experiments). All data are presented as mean ± SEM P values calculated using Student’s t-test; n.s., not significant.

Article Snippet: Primary antibodies were against PAX7 (sc-81648; SantaCruz), MYOD (sc-377460; SantaCruz), MYOD (ab133627, Abcam), RFP (600-401-379, Rockland), LAMININ (sc-59854; SantaCruz), MYOGENIN (124800; Abcam), MYOGENIN (MA5-11486; Thermo Fisher Scientific), (MyHC; DSHB; MF20), and KI67 (MA514520; Thermo Fisher Scientific).

Techniques: Knock-Out, Staining, Immunofluorescence, Muscles

Journal: Stem cells (Dayton, Ohio)

Article Title: Dual-specificity phosphatases 13 and 27 as key switches in muscle stem cell transition from proliferation to differentiation.

doi: 10.1093/stmcls/sxae045

Figure Lengend Snippet: Figure 3. Dusp27 KO mice exhibit comparable muscle development and regeneration to WT mice. (A) qPCR analysis of Dusp27 mRNA levels in gastrocnemius muscles of male WT and 13 KO mice at 12 weeks (n = 3 per group). (B) Gene expression of Dusp27 in RNA-seq data from Pax7-YFP, MyoD-low, and MyoD-high groups (n = 3 per group). (C) E-box-like sequences in the promoter region of Dusp27 in mammalian species. (D) Luciferase reporter constructs of Dusp27 upstream region (−1000 to +200) containing an E-box-like sequence (Top). Relative Dusp27-luciferase reporter activities in HEK293T cells overexpressing MyoD (Bottom) (n = 3 per group). (E, G) H&E staining of SOL and PLA muscle cross-sections. Scale bar: 100 μm. (F, H) CSA of SOL (F) or PLA (H) muscles of male Dusp27 KO (27 KO) and WT (n = 3–6 independent experiments) mice at 12 weeks. (I) Immunofluorescence of PAX7 and LAMININ in intact TA muscles. Scale bar: 100 µm. The area in the white dotted boxes is shown at a higher magnification. Scale bar: 25 μm. (J) Average number of PAX7 (+) nuclei in DAPI (+) in (I). (K) H&E staining of TA muscle cross-sections of male 27 KO and WT mice at 12 weeks (n = 5–6

Article Snippet: Primary antibodies were against PAX7 (sc-81648; SantaCruz), MYOD (sc-377460; SantaCruz), MYOD (ab133627, Abcam), RFP (600-401-379, Rockland), LAMININ (sc-59854; SantaCruz), MYOGENIN (124800; Abcam), MYOGENIN (MA5-11486; Thermo Fisher Scientific), (MyHC; DSHB; MF20), and KI67 (MA514520; Thermo Fisher Scientific).

Techniques: Muscles, Gene Expression, RNA Sequencing, Luciferase, Construct, Sequencing, Staining, Immunofluorescence

Journal: Stem cells (Dayton, Ohio)

Article Title: Dual-specificity phosphatases 13 and 27 as key switches in muscle stem cell transition from proliferation to differentiation.

doi: 10.1093/stmcls/sxae045

Figure Lengend Snippet: Figure 4. Loss of both Dusp13 and Dusp27 impairs muscle regeneration after acute muscle injury. (A) H&E staining of tibialis anterior (TA) muscle cross- sections of male Dusp13 and Dusp27 double knock-out (DKO) (n = 3) and WT (n = 3) mice at 12-15 weeks of age, 3, 7, and 28 days post-cardiotoxin (CTX) injury. Scale bar: 100 µm. (B–D) Cross-sectional area (CSA) of damaged TA muscles at indicated time points. (E, F) Immunofluorescence of PAX7 (E) or MYOGENIN (F) with LAMININ on sections of TA muscles from male WT and DKO mice, 7 days post-CTX injury. Nuclei are stained with DAPI. Scale bar: 100 µm. The area in white dotted boxes is shown at a higher magnification. Scale bar: 25 μm. (G) Iimmunofluorescence of KI67 and MYOGENIN with LAMININ on sections of TA muscles from male WT and DKO mice, 7 days post-CTX injury. Nuclei are stained with DAPI. Scale bar: 100 µm. The area in white dotted boxes is shown at a higher magnification. Scale bar: 25 μm. Arrowheads indicate KI67+/MYOGENIN + cells. Arrows indicate KI67-/MYOGENIN + cells. (H, I) Average number of PAX7 (+) or MYOGENIN (+) nuclei in (E) and (F) are quantified (n = 5-8 independent experiments). (J) Frequency of Ki67 (+) nuclei in MYOGENIN (+) in (G) are quantified (n = 5 per group) (K) Immunofluorescence of PAX7 and MYOD on cultured MuSCs from female WT and DKO mice. Nuclei are stained with DAPI. Scale bar: 100 µm. (L, M) Quantification of the proportion (L) and numbers (M) of MuSCs undergoing self-renewal (PAX7 (+), MYOD (−)), activation (PAX7 (+), MYOD (+)), and differentiation [PAX7 (+), MYOD (−)] in (K).

Article Snippet: Primary antibodies were against PAX7 (sc-81648; SantaCruz), MYOD (sc-377460; SantaCruz), MYOD (ab133627, Abcam), RFP (600-401-379, Rockland), LAMININ (sc-59854; SantaCruz), MYOGENIN (124800; Abcam), MYOGENIN (MA5-11486; Thermo Fisher Scientific), (MyHC; DSHB; MF20), and KI67 (MA514520; Thermo Fisher Scientific).

Techniques: Staining, Knock-Out, Muscles, Immunofluorescence, Cell Culture, Activation Assay

Journal: Stem cells (Dayton, Ohio)

Article Title: Dual-specificity phosphatases 13 and 27 as key switches in muscle stem cell transition from proliferation to differentiation.

doi: 10.1093/stmcls/sxae045

Figure Lengend Snippet: Figure 6. Overexpression of Dusp13 induces precocious muscle differentiation independent of phosphatase activity. (A) Schematic diagram illustrating adenovirus infection into MuSCs from 15 to 16-week-old male Pax7-YFP mice on day 4 of growth medium (GM) culture. (B) Number of cultured MuSCs after transduction with adenovirus vectors overexpressing GFP (pAd-EGFP) or Dusp13 (pAd-Dusp13-EGFP). (C) Immunofluorescence staining of PAX7 and MYOGENIN in cultured MuSCs after transduction with adenovirus vectors overexpressing GFP (pAd-EGFP) or Dusp13 (pAd-Dusp13-EGFP) on GM day 4. Nuclei are stained with DAPI. Arrows indicate PAX7 (+)/MYOGENIN (+) cells. (n = 6 per group). Scale bar: 100 µm (upper); 25 µm (bottom). (D) The proportion of each population in (C). (E) Schematic diagram illustrating adenovirus infection into MuSCs from 15 to 16-week-old male Pax7-YFP mice on GM day 9. (F) Immunofluorescence staining of MYOGENIN in cultured MuSCs after transduction with adenovirus vectors overexpressing GFP (pAd-EGFP) or Dusp13 (pAd-Dusp13-EGFP) on GM day 9. Nuclei are stained with DAPI. Scale bar: 100 µm (G, H) Myotube width (G) and Fusion index (numbers of myonuclei per myotube) (H) in (F) are quantified (50 myotubes analyzed per each experiment) All data are presented as mean ± SEM. P values calculated using Student’s t-test; *P < .05, **P < .01, ***P < .001, n.s., not significant.

Article Snippet: Primary antibodies were against PAX7 (sc-81648; SantaCruz), MYOD (sc-377460; SantaCruz), MYOD (ab133627, Abcam), RFP (600-401-379, Rockland), LAMININ (sc-59854; SantaCruz), MYOGENIN (124800; Abcam), MYOGENIN (MA5-11486; Thermo Fisher Scientific), (MyHC; DSHB; MF20), and KI67 (MA514520; Thermo Fisher Scientific).

Techniques: Over Expression, Activity Assay, Infection, Cell Culture, Transduction, Immunofluorescence, Staining