Journal: Advanced Science

Article Title: MeCP2 Lactylation Protects against Ischemic Brain Injury by Transcriptionally Regulating Neuronal Apoptosis

doi: 10.1002/advs.202415309

Figure Lengend Snippet: MeCP2 lactylation regulates neuronal apoptosis after stroke. A) Heatmap depicting differential lactylation at specific lysine residues of proteins involved in the regulation of neuronal death after stroke. B) PRM quantification of MeCP2 K210 lactylation levels in the brains of sham and MCAO mice ( n = 3 mice per group). C) MeCP2 was immunoprecipitated from sham and MCAO brain tissues, and pan‐Kla and MeCP2 levels were analyzed by Western blot. D) Representative immunofluorescence images showing the proximity ligation assay used to validate MeCP2 lactylation in neurons within the cortex of MCAO mice. Red, in situ PLA detection of MeCP2 and pan‐Kla; green, NeuN; blue, DAPI. E) Heatmap showing differentially lactylated proteins involved in transcriptional regulation between sham and MCAO groups. F) The binding density of MeCP2 was visualized by deepTools: the heatmap presents the CUT&Tag counts at different MeCP2 binding peaks across different treatment groups, ordered by signal strength. G) Distribution of MeCP2 binding peaks across genomic regions in cortical tissues from sham‐operated mice and MCAO mice treated with saline, 2‐DG or 4‐CIN. H) GO enrichment analysis of downregulated and upregulated genes associated with MeCP2 binding sites in the penumbra from MCAO mice. I) Genome browser tracks of MeCP2 CUT&Tag peaks at Pdcd4 and Pla2g6 loci across treatment groups. The purple rectangles indicate the peak regions of MeCP2 on target‐gene promoters. J) Luciferase reporter assay showing relative activity at Pdcd4 and Pla2g6 peaks in HEK293T cells expressing HA‐MeCP2 or HA‐GFP ( n = 3 per group). K) Western blot showing over‐expression of MeCP2 in HEK293 cells, with β‐actin as a loading control. L) Relative luciferase activity at Pdcd4 and Pla2g6 peaks in wild‐type (WT) and MeCP2 −/− HEK293T cells ( n = 4 per group). M) Motif enrichment analysis of MeCP2 binding sites, highlighting top enriched motifs with corresponding p ‐values. N) EMSA demonstrating MeCP2 binding to the CpG‐rich promoters of the target genes Pdcd4 and Pla2g6 . O) ChIP‐qPCR quantification of MeCP2 enrichment at Pdcd4 and Pla2g6 promoter in sham mice and MCAO mice treated with saline, 2‐DG or 4‐CIN ( n = 4 per group). P) Cortical expression of Pdcd4 and Pla2g6 determined by qPCR in sham mice and MCAO mice treated with saline, 2‐DG or 4‐CIN ( n = 5–6 per group). Data are presented as mean ± SEM. # p < 0.05 versus sham control; * p < 0.05, ** p < 0.01, *** p < 0.001 versus MCAO control; ns, not significant.

Article Snippet: The following primary antibodies were used: Anti‐L‐Lactyl Lysine Rabbit mAb (Cat. PTM‐1401, PTM BIO, China), Anti‐lacty‐MeCP2(Lys210) rabbit pAb (PTM BIO, China), Anti‐lacty‐MeCP2(Lys249) rabbit pAb (PTM BIO, China), Rabbit monoclonal anti‐MeCP2 (Cat. 3456, Cell Signaling Technology, USA), Rabbit monoclonal anti‐PDCD4 (Cat. 9535, Cell Signaling Technology, USA), Rabbit monoclonal anti‐HA‐Tag (Cat. 3724, Cell Signaling Technology, USA), Rabbit polyclonal anti‐Cleaved Caspase‐3 (Cat. 9661, Cell Signaling Technology, USA), Rabbit monoclonal anti‐HDAC1 (Cat. 34589, Cell Signaling Technology, USA), Mouse monoclonal anti‐HDAC2 (Cat. 5113, Cell Signaling Technology, USA), Mouse monoclonal anti‐HDAC3 (Cat. 3949, Cell Signaling Technology, USA), Rabbit monoclonal anti‐CHOP (Cat. 5554, Cell Signaling Technology, USA), Rabbit monoclonal anti‐Caspase‐1 (Cat. 24232, Cell Signaling Technology, USA), Rabbit monoclonal anti‐Phospho‐RIP3 (Thr231/Ser232) (Cat. 91702, Cell Signaling Technology, USA), Mouse monoclonal anti‐AlaRS (Cat. sc‐165990, Santa Cruz Biotechnology, USA), Mouse monoclonal anti‐p300 (Cat. sc‐48343, Santa Cruz Biotechnology, USA), Mouse monoclonal anti‐CBP (Cat. sc‐7300, Santa Cruz Biotechnology, USA), Mouse monoclonal anti‐group VI iPLA2 (Cat. sc‐376563, Santa Cruz Biotechnology, USA), Mouse monoclonal anti‐PCAF (Cat. sc‐13124, Santa Cruz Biotechnology, USA), Mouse monoclonal anti‐KAT2A/GCN5(Cat. 66575‐1‐lg, Proteintech, China), Rabbit polyclonal anti‐RIP3 (Cat. 17563‐1‐lg, Proteintech, China), Mouse monoclonal anti‐b‐actin (Cat. 66009‐1‐lg, Proteintech, China), Mouse GAPDH Monoclonal antibodies (Cat. 60004‐1‐Ig, Protsintech, China).

Techniques: Immunoprecipitation, Western Blot, Immunofluorescence, Proximity Ligation Assay, In Situ, Binding Assay, Saline, Luciferase, Reporter Assay, Activity Assay, Expressing, Over Expression, Control, ChIP-qPCR

Journal: Advanced Science

Article Title: MeCP2 Lactylation Protects against Ischemic Brain Injury by Transcriptionally Regulating Neuronal Apoptosis

doi: 10.1002/advs.202415309

Figure Lengend Snippet: Lactylation of MeCP2 at K210 and K249 regulates apoptotic gene expression in stroke. A) Sequence alignment of the transcriptional repression domain of MeCP2 across species, highlighting conserved MeCP2 lactylation sites. B) Co‐immunoprecipitation and Western blot analysis showing reduced MeCP2 lactylation levels upon K210R or K249R mutation. Luciferase reporter assays showing increased transcriptional activity at the C) Pdcd4 and D) Pla2g6 promoters in HEK293T cells expressing K210R or K249R MeCP2 mutants compared to wild‐type MeCP2 ( n = 3 per group). E) MeCP2 was immunoprecipitated with anti‐HA from HEK293T cells expressing WT or K210R/K249R mutants, and pan‐Kla and HA‐MeCP2 levels were detected by Western blot. Luciferase reporter assays showing transcriptional activity at the F) Pdcd4 and G) Pla2g6 promoters in HEK293T cells expressing WT MeCP2 or K210/K249 mutants ( n = 4 per group). H) EMSA analysis showing recombinant wild‐type and mutant MeCP2 binding to the CpG‐rich promoters of Pdcd4 and Pla2g6 . I,J) Western blot analysis of cleaved caspase‐3 levels in primary neurons expressing wild‐type MeCP2, or MeCP2 mutants under OGD/R conditions ( n = 4–7 per group). K–M) Western blot analysis of GVI PLA2 and PDCD4 protein levels in neurons expressing wild‐type MeCP2, or MeCP2 mutants under OGD/R conditions ( n = 7–8 per group). Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001; ns, not significant.

Article Snippet: The following primary antibodies were used: Anti‐L‐Lactyl Lysine Rabbit mAb (Cat. PTM‐1401, PTM BIO, China), Anti‐lacty‐MeCP2(Lys210) rabbit pAb (PTM BIO, China), Anti‐lacty‐MeCP2(Lys249) rabbit pAb (PTM BIO, China), Rabbit monoclonal anti‐MeCP2 (Cat. 3456, Cell Signaling Technology, USA), Rabbit monoclonal anti‐PDCD4 (Cat. 9535, Cell Signaling Technology, USA), Rabbit monoclonal anti‐HA‐Tag (Cat. 3724, Cell Signaling Technology, USA), Rabbit polyclonal anti‐Cleaved Caspase‐3 (Cat. 9661, Cell Signaling Technology, USA), Rabbit monoclonal anti‐HDAC1 (Cat. 34589, Cell Signaling Technology, USA), Mouse monoclonal anti‐HDAC2 (Cat. 5113, Cell Signaling Technology, USA), Mouse monoclonal anti‐HDAC3 (Cat. 3949, Cell Signaling Technology, USA), Rabbit monoclonal anti‐CHOP (Cat. 5554, Cell Signaling Technology, USA), Rabbit monoclonal anti‐Caspase‐1 (Cat. 24232, Cell Signaling Technology, USA), Rabbit monoclonal anti‐Phospho‐RIP3 (Thr231/Ser232) (Cat. 91702, Cell Signaling Technology, USA), Mouse monoclonal anti‐AlaRS (Cat. sc‐165990, Santa Cruz Biotechnology, USA), Mouse monoclonal anti‐p300 (Cat. sc‐48343, Santa Cruz Biotechnology, USA), Mouse monoclonal anti‐CBP (Cat. sc‐7300, Santa Cruz Biotechnology, USA), Mouse monoclonal anti‐group VI iPLA2 (Cat. sc‐376563, Santa Cruz Biotechnology, USA), Mouse monoclonal anti‐PCAF (Cat. sc‐13124, Santa Cruz Biotechnology, USA), Mouse monoclonal anti‐KAT2A/GCN5(Cat. 66575‐1‐lg, Proteintech, China), Rabbit polyclonal anti‐RIP3 (Cat. 17563‐1‐lg, Proteintech, China), Mouse monoclonal anti‐b‐actin (Cat. 66009‐1‐lg, Proteintech, China), Mouse GAPDH Monoclonal antibodies (Cat. 60004‐1‐Ig, Protsintech, China).

Techniques: Gene Expression, Sequencing, Immunoprecipitation, Western Blot, Mutagenesis, Luciferase, Activity Assay, Expressing, Recombinant, Binding Assay

Journal: Biochemical Pharmacology

Article Title: Tylvalosin exhibits anti-inflammatory property and attenuates acute lung injury in different models possibly through suppression of NF-κB activation

doi: 10.1016/j.bcp.2014.04.015

Figure Lengend Snippet: Tylvalosin inhibits PLA2 expression and activation. (A) iPLA2 VI, (B) cPLA2-IVA, (C) p-cPLA2-IVA and (D) sPLA2-IVE were analyzed by immunohistochemistry in sections of lungs from ctrl, LPS and LPS + tylvalosin (25, 50 and 100 mg/kg, i.g.) treatment mice. Representative photomicrographs of staining are shown with arrows. Immunohistochemical analyses for PLA2 show positive staining for PLA2 mainly localized in the epithelial bronchial cells (see arrows a1), in inflammatory cells in subbronchial epithelial (see arrows, a2), and in leukocytes (a3). Score analysis of positive cells, performed on Ctrl, LPS and LPS + tylvalosin (25, 50 and 100 mg/kg, i.g.) treatment mice. Strong immunostaining was observed in the epithelial bronchial cells (see arrows a1), in inflammatory cells in subbronchial epithelial (see arrows, a2), and in leukocytes (a3) from LPS-treated mice. The immunohistochemical images were analyzed quantitatively using Image Pro-Plus v6.0. Mean optical density (MOD) are expressed as mean ± SD. Original magnification 200×.

Article Snippet: Mouse mAb iPLA2, rabbit mAb cPLA2 and goat mAb sPLA2 were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Activation Assay, Immunohistochemistry, Staining, Immunohistochemical staining, Immunostaining