kat2b sgrna1  (Addgene inc)

Journal: Nature Communications

Article Title: Histone lactylation increases CXCL1 expression for neutrophil infiltration and immune escape in pancreatic cancer

doi: 10.1038/s41467-026-69311-5

Figure Lengend Snippet: A Molecular docking simulated the binding affinities of PCAF for acetyl-CoA and lactyl-CoA. B Co-IP was performed to confirm the interaction between H3K18la and PCAF in PANC1 and KPC cells. PCAF and H3K18la were detected on the same gel. C, D Western blot analysis demonstrates the level of H3K18la in PDAC cells with or without the treatment of PCAF inhibitors (bromosporine or Embelin). Histone H3 and H3K18la blots are from parallel-processed separate gels (size conflict) with samples from the same experiment. E Western blot analysis demonstrates the level of H3K18la in PDAC cells following the knockdown of Pcaf . Histone H3, PCAF, β-actin and H3K18la blots are from parallel-processed separate gels with samples from the same experiment. F Western blots of in vitro histone acetylation or lactylation assay, the samples as indicated. Blots are from parallel-processed separate gels with samples from the same experiment. G , H Relative Cxcl1 RNA and protein levels with the vehicle or bromosporine treatment were analyzed by qRT-PCR and ELISA ( n = 3 independent experiments), respectively. I Relative Cxcl1 RNA levels after Pcaf knockdown were analyzed by qRT-PCR ( n = 3 independent experiments). J A schematic diagram showing the orthotopic tumor construction in C57BL/6 J mice ( n = 5 mice per group in one experiment, 2 × 10 6 KPC-luc cells per mouse) with or without bromosporine treatment. K , L Image and weights of the orthotopic tumors in the experimental groups from ( J ) at the end of the experiments ( n = 5 mice per group). M Lactylation levels of H3K18 and PanK in orthotopic tumors with or without bromosporine treatment were detected by western blot ( n = 4 biologically independent samples per group). Histone H3, H3K18la, and PanKla blots are from parallel-processed separate gels (size conflict) with samples from the same experiment. N Serum CXCL1 levels in mice with or without bromosporine treatment were measured by ELISA ( n = 5 mice per group). O – R Tumor-infiltrating neutrophils, CD8 + T cells, GZMB + CD8 + T cells, and PD-1 + CD8 + T cells isolated from the orthotopic tumors were analyzed using flow cytometry. Representative graphs of flow cytometry (left panel) and statistical analysis of the cell ratio (right panel) ( n = 5 mice per group). S The migratory abilities of neutrophils co-incubated with the culture medium supernatant from shNTC/ Pcaf PDAC cells treated with recombinant CXCL1 were analyzed by counting the penetrated cell numbers ( n = 3 biologically independent samples). T Representative IHC images stained by CXCL1 or H3K18la antibody (scale bar = 100 μm, left panel), correlation analysis of H-scores of CXCL1 and H3K18la in PDAC samples ( n = 21 patients, right panel). Data represent mean ± SEM. Statistical analysis was conducted using the two-tailed unpaired Student’s t test ( G – I , L , and N – S ) and two-tailed Pearson’s correlation analysis ( T ). Unless otherwise indicated, all western blots had three independent experimental repetitions with consistent results. Source data are provided as a Source Data file.

Article Snippet: The primary antibodies used here included anti-LDHA antibody (Proteintech, #19987-1-AP, 1:3000), anti-LDHB antibody (Proteintech, #14824-1-AP, 1:5000), anti-H3 antibody (Proteintech, #17168-1-Ap, 1:3000), anti-PCAF antibody (Cell Signaling Technology, #3378S, 1:1000), anti-P300 antibody (Proteintech, #20695-1-AP, 1:500), anti-GCN5 antibody (Proteintech, #66575-1-Ig, 1:3000), anti-beta Actin antibody (PTMBIO, #PTM-5028, 1:1000), anti-human CXCL1 Polyclonal antibody (Proteintech, #12335-1-AP), anti-mouse CXCL1 Polyclonal antibody (Proteintech, #30322-1-AP), anti-L-Lactyl Lysine antibody (PTMBIO, #PTM-1401RM, 1:1000), anti-L-Lactyl-Histone H3K18 antibody (PTMBIO, #PTM-1406RM, 1:1000), anti-L-Lactyl-Histone H3K9 antibody (PTMBIO, #PTM-1419RM, 1:1000), anti-L-Lactyl-Histone H3K14 antibody (PTMBIO, #PTM-1414RM, 1:1000), anti-H4 antibody (PTMBIO, #PTM-1009, 1:1000), anti-L-Lactyl-Histone H4K12 antibody (PTMBIO, #PTM-1411RM, 1:1000), anti-L-Lactyl-Histone H4K8 antibody (PTMBIO, #PTM-1415RM, 1:1000), anti-Acetyl-Histone H3 (Lys18) antibody (PTMBIO, #PTM-114RM, 1:1000), anti-Acetyllysine antibody (PTMBIO, #PTM-101, 1:1000), and anti-Acetyl-Histone H4 (Lys8) antibody (PTMBIO, #PTM-190, 1:1000).

Techniques: Binding Assay, Co-Immunoprecipitation Assay, Western Blot, Knockdown, In Vitro, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Isolation, Flow Cytometry, Incubation, Recombinant, Staining, Two Tailed Test

Journal: Nature Communications

Article Title: Histone lactylation increases CXCL1 expression for neutrophil infiltration and immune escape in pancreatic cancer

doi: 10.1038/s41467-026-69311-5

Figure Lengend Snippet: A A schematic diagram showing the subcutaneous tumor model ( n = 6 mice per group in one experiment, 6 × 10 6 PANC02 cells per mouse) treated with bromosporine and anti-PD-1 antibody. B. The tumor volume growth curves of subcutaneous tumors from ( A ). C , D Image and weights of the subcutaneous tumors at the end point of experiments ( n = 6 mice per group). E The statistical analysis of the cell ratio of tumor-infiltrating neutrophils and CD8 + T cells isolated from subcutaneous tumors ( n = 3 mice per group). F Western blot analysis demonstrates H3K18la levels in the subcutaneous tumors ( n = 3 biologically independent samples per group) from ( A ). Histone H3 and H3K18la blots are from parallel-processed separate gels (size conflict) with samples from the same experiment. G Relative serum CXCL1 levels in mice treated with or without bromosporine/anti-PD-1 antibody were measured by ELISA ( n = 6 mice per group). H A schematic diagram showing the combinational treatment schedule for the orthotopic KPC-luc tumor model ( n = 21 mice per group in one experiment, 2 × 10 6 KPC-luc cells per mouse). I , J Image and weights of the orthotopic tumors at the end point of experiments ( n = 6 mice per group). K – N Tumor-infiltrating neutrophils, CD8 + T cells, GZMB + CD8 + T cells, and PD-1 + CD8 + T cells were analyzed using flow cytometry. Representative graphs of flow cytometry (left panel) and statistical analysis of the cell ratio (right panel) ( n = 6 mice per group). O Western blot analysis showing the H3K18la levels in the orthotopic tumors ( n = 3 biologically independent samples per group) from ( H ). Histone H3 and H3K18la blots are from parallel-processed separate gels (size conflict) with samples from the same experiment. P Representative luminescence images and Quantification of radiance intensity of the mouse model in ( H ). Q Survival probability of mice with orthotopically transplanted PDAC ( n = 15 mice per group). R A working model displaying the signaling pathway through which the aerobic glycolysis-mediated Lactate-PCAF-H3K18la-CXCL1 axis modulates the tumor microenvironment in pancreatic cancer, and the scientific basis for the development of a novel therapeutic strategy for PDAC. Data represent mean ± SEM. Statistical analysis was conducted using the two-tailed unpaired Student’s t test ( B , D , E , G , J – N , P ) and the log-rank test ( Q ). Source data are provided as a Source Data file.

Article Snippet: The primary antibodies used here included anti-LDHA antibody (Proteintech, #19987-1-AP, 1:3000), anti-LDHB antibody (Proteintech, #14824-1-AP, 1:5000), anti-H3 antibody (Proteintech, #17168-1-Ap, 1:3000), anti-PCAF antibody (Cell Signaling Technology, #3378S, 1:1000), anti-P300 antibody (Proteintech, #20695-1-AP, 1:500), anti-GCN5 antibody (Proteintech, #66575-1-Ig, 1:3000), anti-beta Actin antibody (PTMBIO, #PTM-5028, 1:1000), anti-human CXCL1 Polyclonal antibody (Proteintech, #12335-1-AP), anti-mouse CXCL1 Polyclonal antibody (Proteintech, #30322-1-AP), anti-L-Lactyl Lysine antibody (PTMBIO, #PTM-1401RM, 1:1000), anti-L-Lactyl-Histone H3K18 antibody (PTMBIO, #PTM-1406RM, 1:1000), anti-L-Lactyl-Histone H3K9 antibody (PTMBIO, #PTM-1419RM, 1:1000), anti-L-Lactyl-Histone H3K14 antibody (PTMBIO, #PTM-1414RM, 1:1000), anti-H4 antibody (PTMBIO, #PTM-1009, 1:1000), anti-L-Lactyl-Histone H4K12 antibody (PTMBIO, #PTM-1411RM, 1:1000), anti-L-Lactyl-Histone H4K8 antibody (PTMBIO, #PTM-1415RM, 1:1000), anti-Acetyl-Histone H3 (Lys18) antibody (PTMBIO, #PTM-114RM, 1:1000), anti-Acetyllysine antibody (PTMBIO, #PTM-101, 1:1000), and anti-Acetyl-Histone H4 (Lys8) antibody (PTMBIO, #PTM-190, 1:1000).

Techniques: Isolation, Western Blot, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Two Tailed Test

Journal: bioRxiv

Article Title: SAGA/ATAC complexes sustain aberrant chromatin regulation and promote tumorigenesis in diffuse midline glioma

doi: 10.64898/2026.01.22.701194

Figure Lengend Snippet: a, Growth curves of SU-DIPGXIII cells transduced with shRNAs to knockdown both KAT2A and KAT2B, showing significantly reduced growth compared to control shRNA-transduced cells. b, Number of live SU-DIPGXIII+Cas9 cells expressing a control plasmid (HA/flag-GFP) or sgRNAs to KO SAGA-associated acetyltransferase activity ( TADA2B sgRNAs), or de-ubiquitinase activity ( ENY2 sgRNA). c, Schematic illustrating inhibition of histone-modifying modules of SAGA/ATAC complexes using chemical probes to target SAGA/ATAC-dependent histone acetylation (GSK4027, CPTH2, garcinol), SAGA-dependent H2A/H2B de-ubiquitination (USP22si-02), or WDR5-mediated H3K4me3 methylation (OICR-9529, WM856, WDR5-IN-4, and WDR5-IN-6). d, Average normalized cell viability from CellTiter-Glo assays conducted seven days after starting treatment of H3K27M mutant cells (teal curves) or non-transformed, H3 wildtype, control cells (black curves) with increasing doses of the KAT2A/2B bromodomain-targeting acetyltransferase inhibitor, GSK4027. e, Immunoblots confirming that treatment of BT245 cells with GSK4027 reduced H3K9ac in a dose-dependent manner, but not total H3 or SGF29 protein abundance. f-h, Dose response curves showing inhibition of H3K27M mutant DMG cell viability, but no effect on control H3 wild-type cells (black curves) following seven days of treatment with the DUB inhibitor, USP22i-S02 ( f, yellow curves), the WDR5 WIN site inhibitor, WDR5-IN-4 ( g, blue curves), or the WDR5 WBM site inhibitor, WDR5-IN-6 ( h, blue curves). i-j Average number of live SU-DIPGXIIIP*+ZsG/luc cells ( i ), and average BT245 ( j ) cell viability (CellTiter-Glo) following treatment with combinations of GSK4027 and USP22si-02 to simultaneously target both SAGA/ATAC-dependent histone acetyltransferase activity and SAGA-dependent H2A/H2B de-ubiquitination. k-l, BLISS synergy plots showing a combined effect of GSK4027 and WDR5-IN-6 treatment in reducing SU-DIPGXIIIP*+ZsG/luc growth ( k, **p<1.74×10 -3 ), and a combined effect of GSK4027 and WDR5-IN-4 treatment in reducing BT245 cell growth ( l , ****p<2.5×10 -10 ).

Article Snippet: KAT2A sgRNA1 and KAT2B sgRNA1 were obtained from AddGene (cat# 138185 and 138186).

Techniques: Transduction, Knockdown, Control, shRNA, Expressing, Plasmid Preparation, Activity Assay, Inhibition, Ubiquitin Proteomics, Methylation, Mutagenesis, Transformation Assay, Western Blot, Quantitative Proteomics