Fig 2: Gaq deficiency attenuated PKD/PKCµ activation and reduced HDAC 5 phosphorylation level to inhibit NF?BIA expression during decidualization.(A) Western blot analysis of the p-PKD/PKCµ, PKD/PKCµ, p-HDAC5, and HDAC5 expression in whole-cell lysates of proliferative (D0) and decidualized human endometrial stromal cell (HESC) treated with differential culture medium for 2, 4, and 6 days. ß-Tubulin was used as the loading controls. (B) Western blot analysis of the IGFBP-1, p-PKD/PKCµ, PKD/PKCµ, I?Ba, p-HDAC5, and HDAC5 expression in whole-cell lysates after treatment with PKD/PKCµ activator, Phorbol 12-myristate 13-acetate (PMA, 1 nM) in CON and KO HESC treated with differential culture medium for 4 days. GAPDH was used as the loading controls. (C) Western blot analysis of the I?Ba, p-HDAC5, HDAC5, p-PKD/PKCµ, and PKD/PKCµ expression in whole-cell lysates after treatment with PMA (1 nM) and or PKD/PKCµ inhibitor CID755673 for 0, 2, and 4 hr in CON and KO HESC treated with differential culture medium for 4 days. ß-Tubulin was used as the loading controls. (D) Immunofluorescence staining of p-HDAC5 in CON and KO decidualized stromal cells treated with differential culture medium for 4 days. Scale bars, 100 µm. (E) Western blot analysis of the p-HDAC5 expression in cytoplasm and nucleus after treatment with PMA (1 nM) in CON and KO HESC treated with differential culture medium for 4 days. GAPDH and H3 were used as the loading controls. (F) Chromatin immunoprecipitation (ChIP)-qRT-PCR analysis of enrichment of HDAC5 on promoters of NF?BIA in WT HESC treated with differential culture medium for 4 days (n = 3). (G–J) qRT-PCR analysis of NF?BIA, IL-11, IL-1ß, and IGFBP-1 mRNA expression levels after treatment with HDAC5 inhibitor LMK235 (2 µM), in CON and KO HESC treated with differential culture medium for 4 days. Representative data are shown from three independent experiments (n = 3). (F) was analyzed with unpaired Student’s t-test. (G–J) were calculated with one-way analysis of variance (ANOVA) with Bonferroni’s multiple comparison tests. *p < 0.05 and **p < 0.01. Figure 5—source data 1.Original blot images of Figure 5.

Fig 3: FGF2 activates KLF2 through the AMPK-HDAC5 pathway. (A) Skeletal muscle tissues' WB assay for AMPK, p-AMPK, p-HDAC5, and HDAC5. AMPK and p-AMPK protein levels from (A) are quantified in (B), normalised to GAPDH band density. HDAC5 protein level from (A) is evaluated in (C), normalised in respect to Histone 3 band density, coupled with p-HDAC5 level from (A), normalised in relation to GAPDH band density. (D) Using DAPI labelling, skeletal muscle slices labelled with HDAC5 and the EC marker CD31 reveal distinct nuclei. Scale bars indicate 100 μm. Nuclear HDAC5 and CD31 double-positive cells computation (E). (F) Images from a dual luciferase reporter gene test. (G) Relative luciferase activity was plotted as a box plots. (H) Skeletal muscle tissues' WB assay for AMPK, p-AMPK, p-HDAC5, KLF2, HDAC5, and NFE2L2. (I) Quantification of protein level of AMPK, p-AMPK, p-HDAC5, KLF2 from (H) with normalized to GAPDH band density, HDAC5 and NFE2L2 protein levels from (H) with normalized to Histone 3 band density. (J-L) Images of skeletal muscle slices with DAPI-stained nuclei that have had HDAC5, KLF2, NFE2L2, and EC marker CD31 stained. Scale bars: 100 μm. (M-O) Quantification of nuclear HDAC5, KLF2, NFE2L2 and CD31 double-positive cells, the percentages of double positive cells versus total CD31 positive cells are indicated. (P) Skeletal muscle tissues' WB assay for SOD1, SLC7A11, and GPX4. SOD1, SLC7A11, and GPX4 protein levels are evaluated in (Q) and normalised based on GAPDH band density. (R) Photographs of skeletal muscle slices with distinct nuclei shown by DAPI staining and DHE and 4-HNE staining. Scale bars: 100 μm. Immunofluorescence data from (R) are measured in (S), which reveals the average optical density of 4-HNE and the degree of ROS. (T) Content of Fe2+ was plotted as a histogram. (U) A histogram showing the content of MDA. (V) A histogram showing the GSH content. (W) Schematic representation of proposed molecular mechanisms highlighting the roles of AMPK-HDAC5 signaling, KLF2, NFE2L2, oxidative stress, ferroptosis in the pathophysiology of I/R-induced microvascular injury in extremities. Data are presented as the mean ± SD (n = 3-5 per group). Significance: ns, not significant; *P < 0.05.