Journal: Journal of Cellular and Molecular Medicine

Article Title: Lactate regulates pathological cardiac hypertrophy via histone lactylation modification

doi: 10.1111/jcmm.70022

Figure Lengend Snippet: The expression of HKla is upregulated in cardiomyocytes of mice subjected to TAC. (A) The ratios of HW/BW and HW/TL were measured in both the sham and TAC groups ( n = 6 mice/group). (B) Histological analysis of myocardial tissue. (C) Levels of markers for cardiac hypertrophy and fibrosis were measured in mice from the TAC and sham groups. (D) Echocardiography was performed to measure LVEDD, LVESD, LVPWT, heart rate, LVEF and LVFS in both the TAC and sham groups. (E) The levels of LDHA, LDHB and P300 were measured in the hearts of mice from the TAC and sham groups. (F, G) Western blot analysis was conducted to examine the levels of Pan Kla, H3K18la, H3K9la, H4K5la, H4K12la and H3K14la in hearts from mice in the TAC and sham groups. (H, I) The levels of H3K18ac, H4K5ac and pan Kac were measured in cardiomyocytes from both the TAC and sham groups ( n = 3, ** p < 0.01, *** p < 0.001, **** p < 0.0001, compared to the sham group).

Article Snippet: Anti‐histone H3, anti‐H3K18ac, anti‐H4K5ac, anti‐LDHA, anti‐LDHB, anti‐p300, anti‐β‐MHC, anti‐BNP and anti‐GAPDH antibodies were purchased from Abclonal Biotech (Shanghai, China).

Techniques: Expressing, Western Blot

Journal: Journal of Cellular and Molecular Medicine

Article Title: Lactate regulates pathological cardiac hypertrophy via histone lactylation modification

doi: 10.1111/jcmm.70022

Figure Lengend Snippet: Ang II contributes to cardiomyocyte hypertrophy and promotes HKla. (A) After 24 h of Ang II stimulation, the cell surface area of NMCMs increased. (B) Representative western blots of ANP, BNP and β‐MHC in cultured NMCMs treated with Ang II (1 μM) for 24 h. (C) NMCMs were exposed to Ang II (1 μM) for 24 h, and intracellular lactate levels were evaluated using a colorimetric lactate measurement kit. (D) Expression levels of LDHA, LDHB and P300 proteins were detected in NMCMs using western blotting. (E, F) Western blot analysis was performed to measure the expression levels of H3K18la, H3K9la, H4K5la, H4K12la, H3K14la and Pan HKla in NMCMs ( n = 3, * p < 0.05, ** p < 0.01, *** p < 0.001, vs. control group).

Article Snippet: Anti‐histone H3, anti‐H3K18ac, anti‐H4K5ac, anti‐LDHA, anti‐LDHB, anti‐p300, anti‐β‐MHC, anti‐BNP and anti‐GAPDH antibodies were purchased from Abclonal Biotech (Shanghai, China).

Techniques: Western Blot, Cell Culture, Expressing, Control

Journal: Journal of Cellular and Molecular Medicine

Article Title: Lactate regulates pathological cardiac hypertrophy via histone lactylation modification

doi: 10.1111/jcmm.70022

Figure Lengend Snippet: Lactate enhances HKla and exacerbates cardiac hypertrophy. (A) NMCMs were exposed to different concentrations of L‐lactate for 48 h, and intracellular lactate levels were evaluated using a colorimetric lactate measurement kit. (B) Effects of increasing lactate on the surface areas of cardiomyocytes. C‐H NMCMs were cultured with various concentrations of L‐lactate (0, 1, 5 and 10 mM) for 48 h. Afterward, cells at different concentrations were harvested for western blot analysis. (C) Effects of increasing lactate on the levels of cardiac hypertrophy markers. (D) Expression levels of LDHA, LDHB and P300 proteins were detected in NMCMs using western blotting. (E, F) Western blot analysis was performed to measure the expression levels of H3K18la, H3K9la, H4K5la, H4K12la, H3K14la and Pan HKla in NMCMs. (G, H) Expression levels of H3K18ac, H4K5ac and Pan Kac were detected in NMCMs using western blotting ( n = 3, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, vs. indicated group).

Article Snippet: Anti‐histone H3, anti‐H3K18ac, anti‐H4K5ac, anti‐LDHA, anti‐LDHB, anti‐p300, anti‐β‐MHC, anti‐BNP and anti‐GAPDH antibodies were purchased from Abclonal Biotech (Shanghai, China).

Techniques: Cell Culture, Western Blot, Expressing

Journal: Journal of Cellular and Molecular Medicine

Article Title: Lactate regulates pathological cardiac hypertrophy via histone lactylation modification

doi: 10.1111/jcmm.70022

Figure Lengend Snippet: Glucose increases lactate levels, which upregulate HKla expression. (A) Lactate levels in NMCMs cultured with different concentrations of glucose were measured by a lactate colorimetric kit. (B) Effects of glucose increase on cardiomyocyte surface areas. (C‐H) NMCMs were cultured for 48 h with different concentrations of glucose (0, 1, 5 and 25 mM). Cells at varying concentrations were harvested and utilized for western blot analysis. (C) Effects of increasing glucose on the levels of cardiac hypertrophy markers. (D) LDHA, LDHB and P300 protein expression levels in NMCMs were assessed using western blotting. (E, F) Western blot analysis was performed to measure the expression levels of H3K18la, H3K9la, H4K5la, H4K12la, H3K14la and Pan HKla in NMCMs. (G, H) Expression levels of H3K18ac, H4K5ac and Pan HKla were detected in NMCMs using western blotting. (I) The glycolysis ability of cardiomyocytes was analysed by Seahorse ECAR assay ( n = 3, * p < 0.05, ** p < 0.01, *** p < 0.001, vs. indicated group).

Article Snippet: Anti‐histone H3, anti‐H3K18ac, anti‐H4K5ac, anti‐LDHA, anti‐LDHB, anti‐p300, anti‐β‐MHC, anti‐BNP and anti‐GAPDH antibodies were purchased from Abclonal Biotech (Shanghai, China).

Techniques: Expressing, Cell Culture, Western Blot, ECAR Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: Lactate regulates pathological cardiac hypertrophy via histone lactylation modification

doi: 10.1111/jcmm.70022

Figure Lengend Snippet: 2‐DG can decrease HKla levels and inhibits the advancement of cardiac hypertrophy. (A) NMCMs were exposed to different concentrations of 2‐DG for 48 h, and intracellular lactate levels were evaluated using a colorimetric lactate measurement kit. (B) The impact of 2‐DG increase on the surface area of cardiomyocytes. (C–H) NMCMs were cultured with various concentrations of 2‐DG (0, 1, 5 and 10 mM) for 48 h, and cells were then collected for western blot analysis. (C) The impact of increasing 2‐DG on the levels of cardiac hypertrophy marker genes. (D) Western blot analysis detected the expression of LDHA, LDHB and P300 proteins in NMCMs. (E, F) Western blot analysis was conducted to examine the expression of H3K18la, H3K9la, H4K5la, H4K12la, H3K14la and Pan HKla in NMCMs. (G, H) Western blot analysis detected the expression of H3K18ac, H4K5ac and Pan Kac levels in NMCMs. (I) The glycolysis ability of cardiomyocytes was analysed by Seahorse ECAR assay ( n = 3, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, vs. indicated group).

Article Snippet: Anti‐histone H3, anti‐H3K18ac, anti‐H4K5ac, anti‐LDHA, anti‐LDHB, anti‐p300, anti‐β‐MHC, anti‐BNP and anti‐GAPDH antibodies were purchased from Abclonal Biotech (Shanghai, China).

Techniques: Cell Culture, Western Blot, Marker, Expressing, ECAR Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: Lactate regulates pathological cardiac hypertrophy via histone lactylation modification

doi: 10.1111/jcmm.70022

Figure Lengend Snippet: Oxamate can reduce HKla levels and inhibit the progression of cardiac hypertrophy. (A) Lactate levels in NMCM cultured with different concentrations of oxamate were measured by a lactate colorimetric kit. (B) The effect of increased oxamate concentration on the surface area of cardiomyocytes was detected. (C–H) The NMCMs model was cultured for 48 h using different concentrations of oxamate (0, 5, 10 and 20 mM), and cells were then collected for western blot analysis. (C) The impact of an increase in oxalate concentration on the levels of marker genes for cardiac hypertrophy. (D) The protein expression levels of LDHA, LDHB and P300 in NMCMs exposed to different concentrations of oxamate were analysed by western blotting. (E and F) Western blot analysis was performed to assess the expression level of H3K18la, H3K9la, H4K5la, H4K12la, H3K14la and Pan HKla in NMCMs. (G and H) Expression of H3K18ac, H4K5ac and Pan Kac levels were detected in NMCMs by western blotting ( n = 3, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, vs. indicated group).

Article Snippet: Anti‐histone H3, anti‐H3K18ac, anti‐H4K5ac, anti‐LDHA, anti‐LDHB, anti‐p300, anti‐β‐MHC, anti‐BNP and anti‐GAPDH antibodies were purchased from Abclonal Biotech (Shanghai, China).

Techniques: Cell Culture, Concentration Assay, Western Blot, Marker, Expressing

Journal: Journal of Cellular and Molecular Medicine

Article Title: Lactate regulates pathological cardiac hypertrophy via histone lactylation modification

doi: 10.1111/jcmm.70022

Figure Lengend Snippet: GNE‐140 may reduce HKla levels and inhibit the progression of cardiac hypertrophy. (A) Comparison of intracellular lactate levels between the GNE‐140 group and the blank group. (B) The effects of GNE‐140 on the surface areas of NMCMs compared to the blank group. (C–H) NMCMs were treated with GNE‐140 (10 μM) for 48 h. For western blot analysis, cells were collected from both the GNE‐140 and blank groups. (C) The levels of cardiac hypertrophy markers between the GNE‐140 group and the control group. (D) The expression levels of LDHA, LDHB and P300 proteins were detected in the control and GNE‐140 groups by western blotting. (E, F) Western blot analysis was performed to assess the expression levels of H3K18la, H3K9la, H4K5la, H4K12la, H3K14la and Pan Hkla in the control and GNE‐140 groups. (G, H) Western blotting was used to measure the levels of H3K18ac, H4K5ac and Pan Kac in the control and GNE‐140 groups ( n = 3, * p < 0.05, ** p < 0.01, *** p < 0.001, vs. blank group).

Article Snippet: Anti‐histone H3, anti‐H3K18ac, anti‐H4K5ac, anti‐LDHA, anti‐LDHB, anti‐p300, anti‐β‐MHC, anti‐BNP and anti‐GAPDH antibodies were purchased from Abclonal Biotech (Shanghai, China).

Techniques: Comparison, Western Blot, Control, Expressing

Journal: BMC Cancer

Article Title: Acetylation of PGK1 at lysine 323 promotes glycolysis, cell proliferation, and metastasis in luminal A breast cancer cells

doi: 10.1186/s12885-024-12792-8

Figure Lengend Snippet: Identifying and Validating Regulators of PGK1 Acetylation. A Overview of acetyltransferases and deacetylases identified in MCF-10A and T47D cells via proteomic analysis. B Comparison of acetyltransferase expression levels in T47D cells relative to MCF-10A cells, with MCF-10A expression levels set as the baseline (1.0). C Comparison of deacetylase expression levels in T47D cells relative to MCF-10A cells, with MCF-10A expression levels standardized to 1.0. D Western blot analysis illustrating the impact of Nicotinamide (NAM) at concentrations of 25 mM and 50 mM on PGK1 acetylation in T47D cells expressing WT-PGK1. E Quantitative analysis of PGK1 acetylation changes depicted in Fig. 4D. F Western blot showing expression levels of HAT1, p300, and Sirtuin3 in MCF-10A versus T47D cells, with β-actin serving as the loading control. G Co-immunoprecipitation (Co-IP) assays examining interactions between PGK1 and the enzymes p300, Sirtuin3, and HAT1, highlighting the specific regulatory connections

Article Snippet: Related antibodies and dilution ratio were as follows: PGK1 (Santa Cruz, sc-130335, 1:1000), Pan-acetylation (Cell Signaling Technology, 9441, 1:1000), β-actin (Cell Signaling Technology, 4970, 1:1000), FLAG (Sigma, F7425, 1:1000), PCNA(Cell Signaling Technology, 13,110, 1:1000), MMP2 (Cell Signaling Technology, 40,994, 1:1000), MMP9 (Cell Signaling Technology, 13,667, 1:1000), E-cadherin (Proteintech, 20,874–1-AP, 1:2000), N-cadherin (Proteintech, 22,018–1-AP), Vimentin (Proteintech, 10,366–1-AP), HAT1 (PTM Biotechnology, PTM-5195, 1:1000), p300 (Cell Signaling Technology, 70088S, 1:1000), Sirtuin3 (Cell Signaling Technology, 5490S, 1:1000).

Techniques: Comparison, Expressing, Histone Deacetylase Assay, Western Blot, Control, Immunoprecipitation, Co-Immunoprecipitation Assay

Journal: Cell Communication and Signaling : CCS

Article Title: ΔNp63 bookmarks and creates an accessible epigenetic environment for TGFβ-induced cancer cell stemness and invasiveness

doi: 10.1186/s12964-024-01794-5

Figure Lengend Snippet: TGFβ-induced gene expression correlates with changes in histone modification marks orchestrated by p63. (A-B) ChIP-qPCR showing the changes in H3K27ac (A) and H3K27me3 (B) histone marks of the indicated gene loci in MCF10A MII cells, incubated in starvation medium overnight and stimulated or not with TGFβ for the indicated time-periods. (C) ChIP-qPCR showing the effect of TGFβ treatment on p300 binding to the indicated gene loci in MCF10A MII cell. (D-E) ChIP-qPCR experiments showing the effect of p63 depletion on H3K27ac (D) and H3K27me3 (E) marks. MCF10A MII cells transfected with non-targeting control (sictrl) siRNA or with siRNA specific against all p63 isoforms were incubated overnight in starvation medium and treated or not with TGFβ for 24 h. (F) Effect of p63 depletion on p300 recruitment to chromatin. MCF10A MII cells transfected with siRNAs and treated or not with TGFβ as in panels D and E, were subjected to ChIP with p300 antibody and subsequent qPCR analysis. (G) Effect of p63 depletion on p300 expression. MCF10A MII cells transfected with non-targeting control (sictrl) siRNA or with siRNAs specific against all isoforms of p63 were treated or not with TGFβ for 6–24 h and subjected to IB analysis with the indicated antibodies. Data are representative of three independent experiments with similar results. Graphs presented in panels A-F show results of three independent experiments as mean ± SD; * P < 0.05, ** P < 0.01, *** P < 0.001, N.S: not significant difference. The dots represent the individual values from each of the three independent experiments

Article Snippet: The antibodies used for ChIP were raised against p63 (ab124762, Abcam, Cambridge, UK), H3K27ac (39685, Active motif, Carlsbad, CA and ab177178, Abcam, Cambridge, UK), H3K27me3 (61017, Active motif, Carlsbad, CA), p300 (61401, Active motif, Carlsbad, CA, and ab14984, Abcam, Cambridge, UK) and SUZ12 (39357, Active motif, Carlsbad, CA).

Techniques: Expressing, Modification, Incubation, Binding Assay, Transfection, Control

Journal: Cell Communication and Signaling : CCS

Article Title: ΔNp63 bookmarks and creates an accessible epigenetic environment for TGFβ-induced cancer cell stemness and invasiveness

doi: 10.1186/s12964-024-01794-5

Figure Lengend Snippet: H3K27me3 inhibition increases p63 and p300 recruitment to chromatin only in the presence of active TGFβ signaling. (A) ChIP-qPCR showing the recruitment of p63 to the indicated gene loci in MCF10A MII cells treated or not with an EZH2 inhibitor (GSK343) for 48 h in the presence or not of TGFβ stimulation for 24 h. (B) ChIP-qPCR showing the effect of SUZ12 depletion in combination with treatment with a GSK343 inhibitor on the TGFβ-induced p63 binding to the indicated gene loci. (C) qRT-PCR analysis of the effect of TGFβ stimulation and GSK343 inhibition on the expression of LAMB3 , ITGA2 and SERPINE1 genes. MCF10A MII cells were incubated overnight in starvation medium and subsequently treated with 5 µΜ GSK343 inhibitor for 24 h before the stimulation or not with TGFβ for an additional 24 h. (D, E) Effect of p63 silencing on the H3K27ac mark and the recruitment of p300 in MCF10A MII cells treated or untreated with TGFβ and GSK343. Lysates of MCF10A MII cells were subjected to ChIP with H3K27ac (D) of p300 (E) antibodies and subsequent qPCR analysis. Graphs presented in panels A-E show results of three independent experiments as mean ± SD; * P < 0.05, ** P < 0.01, *** P < 0.001, N.S: not significant difference. The dots represent the individual values from each of the three independent experiments

Article Snippet: The antibodies used for ChIP were raised against p63 (ab124762, Abcam, Cambridge, UK), H3K27ac (39685, Active motif, Carlsbad, CA and ab177178, Abcam, Cambridge, UK), H3K27me3 (61017, Active motif, Carlsbad, CA), p300 (61401, Active motif, Carlsbad, CA, and ab14984, Abcam, Cambridge, UK) and SUZ12 (39357, Active motif, Carlsbad, CA).

Techniques: Inhibition, Binding Assay, Quantitative RT-PCR, Expressing, Incubation

Journal: Cell Communication and Signaling : CCS

Article Title: ΔNp63 bookmarks and creates an accessible epigenetic environment for TGFβ-induced cancer cell stemness and invasiveness

doi: 10.1186/s12964-024-01794-5

Figure Lengend Snippet: Identification of ΔNp63-interacting proteins. (A) Heatmap showing the peptide intensities derived from the MS/MS spectrum for control samples (Ctrl) and TGFβ-treated samples. Log10 expression represents peptide intensities derived from quantifying area under curve (AUC) of significantly enriched peaks. (B) Illustration of the number of the identified p63 interactors, enriched in both control condition and TGFβ-treated condition (common), uniquely enriched in control condition (control) or uniquely enriched in TGFβ-treated condition (TGFβ). C, D UMAP plots visualizing the most significantly enriched molecular functions derived from the gene ontology database and associated with proteins identified in control samples (C) and TGFβ-treated samples (D) . (E) Heatmap depicting the intensity patterns of proteins involved in R-SMAD binding in the respective samples. Scaled AUC was calculated using Z-score method representing quantified AUC of peptide intensities. (F) p63 interaction with p300 and SMAD2/3. MCF10A MII cells, starved in 0.2% FBS medium and stimulated with TGFβ or not for 6 h, were subjected to nuclear-cytosolic fractionation. The nuclear lysates (input nuclear) were immunoprecipitated (IP) with p63-specific antibody, or IgG control, and analyzed by immunoblotting utilizing specific antibodies, as indicated. One of three independent experiments with similar results, is shown

Article Snippet: The antibodies used for ChIP were raised against p63 (ab124762, Abcam, Cambridge, UK), H3K27ac (39685, Active motif, Carlsbad, CA and ab177178, Abcam, Cambridge, UK), H3K27me3 (61017, Active motif, Carlsbad, CA), p300 (61401, Active motif, Carlsbad, CA, and ab14984, Abcam, Cambridge, UK) and SUZ12 (39357, Active motif, Carlsbad, CA).

Techniques: Derivative Assay, Tandem Mass Spectroscopy, Control, Expressing, Binding Assay, Fractionation, Immunoprecipitation, Western Blot

Journal: Cell Communication and Signaling : CCS

Article Title: ΔNp63 bookmarks and creates an accessible epigenetic environment for TGFβ-induced cancer cell stemness and invasiveness

doi: 10.1186/s12964-024-01794-5

Figure Lengend Snippet: Activation of SMAD2 and SMAD3 transcription factors drives the p63 selectivity on histone modulation complexes. (A) Effect of ALK5 inhibition on p63/p300 interaction. Lysates of MCF10A MII cells treated with ALK5 kinase inhibitor (SB505124) or not (control) in the presence of TGFβ stimulation were subjected to IP with p300-specific antibody or IgG control, and analyzed by IB with the indicated antibodies. ( B ) Effect of p38 inhibition on p63/SMAD2/3 interaction. Lysates of MCF10A MII cells treated with p38 kinase inhibitor (SB203580) or not (control) in the presence of TGFβ stimulation were subjected to IP with SMAD2/3 antibody or IgG control, and analyzed by IB with the indicated antibodies. (C) Effect of SMAD2/3 depletion on p63/p300 interaction. MCF10A MII cells transfected with non-targeting control (sictrl) siRNA or with siRNA specific against SMAD2 and SMAD3 were incubated in starvation medium and treated or not with TGFβ for 6 h. Cell lysates were subjected to IP with p300-specific antibody or IgG control, and analyzed by IB with the indicated antibodies. (D) Effect of p63 depletion on SMAD3/p300 interaction. MCF10A MII cells transfected with non-targeting control (sictrl) siRNA or with siRNA specific against all p63 isoforms were incubated in starvation medium overnight and treated or not with TGFβ for 45 min. Cell lysates were subjected to IP with a p300-specific antibody or IgG control, and analyzed by IB with the indicated antibodies. (E , F) Effect of SMAD2/3 depletion on p300 recruitment to chromatin (E) and H3K27ac (F) . MCF10A MII cells transfected with siRNAs and starved as in panel C were treated or not with TGFβ for 24 h. Cell lysates were subjected to ChIP with p300 (E) or H3K27ac (F) antibodies and subsequent qPCR analysis. Graphs presented in panels E-F show results of three independent experiments as mean ± SD; * P < 0.05, ** P < 0.01. The dots represent the individual values from each of the three independent experiments. (G) Effect of SMAD2/3 depletion on p63/NCOR2 interaction. MCF10A MII cells transfected with siRNAs and starved as in panel A were treated or not with TGFβ for 6 h. Cell lysates were subjected to IP with p63 and analyzed by IB with the indicated antibodies. One of three independent experiments with similar results, is shown. ( H ) Graph illustrating the relative IP NCOR2/input as average values from the quantification of three independent experiments performed as described in panel G. The dots represent the individual values from each of the three independent experiments

Article Snippet: The antibodies used for ChIP were raised against p63 (ab124762, Abcam, Cambridge, UK), H3K27ac (39685, Active motif, Carlsbad, CA and ab177178, Abcam, Cambridge, UK), H3K27me3 (61017, Active motif, Carlsbad, CA), p300 (61401, Active motif, Carlsbad, CA, and ab14984, Abcam, Cambridge, UK) and SUZ12 (39357, Active motif, Carlsbad, CA).

Techniques: Activation Assay, Inhibition, Control, Transfection, Incubation

Journal: Cell Communication and Signaling : CCS

Article Title: ΔNp63 bookmarks and creates an accessible epigenetic environment for TGFβ-induced cancer cell stemness and invasiveness

doi: 10.1186/s12964-024-01794-5

Figure Lengend Snippet: Schematic illustration of the effect of active TGFβ signaling on ΔNp63-dependent transcription. (A) In the absence of active TGFβ pathway, ΔNp63 is bound to NURD/PRC2 and NCOR/SMRT/HDAC3 complexes on TGFβ/SMAD target regulatory genomic loci. These regions showing high H3K4me1 are bookmarked for transcription by ΔNp63; however, the presence of the H3K27 tri-methylation mark results in condensed chromatin and inactive transcription. (B) Activation of TGFβ signaling leads to phosphorylation of ΔNp63 at Ser66/68 via p38 MAPK, nuclear translocation of SMAD2/3 transcription factors and complex formation between ΔNp63, SMAD2/3 and p300. p300 catalyzes the acetylation of K27 on H3, which promotes chromatin accessibility and activation of gene transcription favoring cancer cell stemness and invasiveness. Dynamic interactions with chromatin are shown with two anti-parallel arrows. Arrow thickness correlates to the prevalent interaction (association or dissociation). Created with BioRender.com

Article Snippet: The antibodies used for ChIP were raised against p63 (ab124762, Abcam, Cambridge, UK), H3K27ac (39685, Active motif, Carlsbad, CA and ab177178, Abcam, Cambridge, UK), H3K27me3 (61017, Active motif, Carlsbad, CA), p300 (61401, Active motif, Carlsbad, CA, and ab14984, Abcam, Cambridge, UK) and SUZ12 (39357, Active motif, Carlsbad, CA).

Techniques: Methylation, Activation Assay, Translocation Assay