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 The expression of neutrophils and CD8 + T cells in low- and high-glycolysis clusters, divided by ssGSEA score of glycolysis-related genes, in TCGA and CPTAC PAAD database. Data are presented as box plots showing the median (center line), the first and third quartiles (box bounds), and the whiskers extend to 1.5 times the interquartile range from the box. B The correlation of glycolysis level and neutrophils or CD8 + T cells expression in TCGA ( n = 179 patients) and CPTAC ( n = 140 patients) PAAD database. C Kaplan–Meier plots representing survival probabilities in TCGA-PAAD patients according to the relative level of glycolysis-related or neutrophil-related gene expression. D The diagram illustrating the targets of 2DG and shRNA in the glycolysis process. E CyTOF analysis of tumor-infiltrating immunocytes in subcutaneous tumor models with or without 2DG treatment: tSNE plots showing 12 meta-clusters based on the expression of 41 markers for the immunocytes. F Bar chart of the frequencies of the immune cell subsets in two experimental groups. G A schematic diagram showing the orthotopic PDAC model ( n = 6 mice per group in one experiment) with or without 2DG treatment. H , I Image and weights of the orthotopic tumors in the experimental groups from ( G ) at the end of the experiments. ( n = 6 mice per group). ( J – M ) 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 = 6 mice per group). N A schematic diagram showing the orthotopic tumor model in C57BL/6 J mice ( n = 6 mice per group in one experiment) treated with or without anti-Ly6G/anti-CD8 antibody. O , P Image and weights of the orthotopic tumors in the experimental groups from ( N ) at the end of the experiments ( n = 6 mice per group). Q – S Tumor-infiltrating neutrophils, CD8 + T cells, and GZMB + 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 = 6 mice per group). T , U Representative luminescence images of the mouse model in ( N ) and statistical analysis of the total flux ( n = 6 mice per group). Data represent mean ± SEM. Statistical analysis was conducted using the two-tailed unpaired Student’s t test ( A, I–M, P–S, and U ), two-tailed Pearson’s correlation analysis ( B ), and log-rank test ( C ). Source data are provided as a Source Data file.

Article Snippet: The antibodies used here included anti-L-Lactyl-Histone H3K18 antibody (1:500, PTMBIO, #PTM-1406RM), anti-GRO antibody (1:200, AFFINITY, #AF5403), Ki-67 (1:400, Servicebio, # GB111499 ), anti-Ly6G antibody (1:500, Servicebio, #GB11229), and anti-CD8 antibody (1:200, Cell Signaling Technology, #70306), and anti-PCAF antibody(1:200, Cell Signaling Technology, #3378).

Techniques: Expressing, Gene Expression, shRNA, Isolation, Flow Cytometry, 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 The flowchart illustrates that RNA sequencing was conducted using two PDAC cell lines with or without 2DG treatment. Subsequently, the intersection of detected genes within the chemokine family in both cell lines is identified, and a heatmap is generated to display the log 2 fold change (2DG versus vehicle) of these genes. B , C Relative mRNA levels of Cxcl1 in PDAC cell lines following glycolysis inhibition, either by using 2DG or silencing LDH, were analyzed by qRT-PCR ( n = 3 independent experiments). D , E Relative protein levels of CXCL1 in PDAC cell lines following glycolysis inhibition, either by using 2DG or silencing LDH, were determined using ELISA assay ( n = 3 independent experiments). F Representative IHC images of subcutaneous tumors ( n = 6 mice for 2DG treatment, n = 5 mice for shLDH/shNTC groups) stained by CXCL1 antibody (scale bars = 100 μm). G Relative serum CXCL1 levels in mice treated with or without 2DG were measured by ELISA ( n = 6 mice per group). H Relative serum CXCL1 levels in healthy donors and PDAC patients were measured by ELISA (22 healthy samples and 27 PDAC samples). I Schematic diagram showing the in vitro migration assay: human/mouse neutrophils were co-incubated with the culture medium supernatant from PDAC cells treated with 2DG or LDH-knockdown. J Neutrophils were co-cultured with CD8 + T cells in different proportions, and the proliferation of CD8 + T cells was detected with the CFSE assay ( n = 6 biologically independent samples). K The migratory activity of neutrophils co-incubated with the culture medium supernatant from PDAC cells treated with shLDH and recombinant CXCL1 was analyzed by counting the penetrated cell numbers ( n = 3 biologically independent samples). L The neutrophils were treated with SX-682 or Navarixin for 1.5 h in advance, and the migratory activity of neutrophils co-incubated with the culture medium supernatant from PDAC cell lines treated with shLDH was analyzed by counting the penetrated cell numbers ( n = 3 biologically independent samples). M Representative luminescence images of the orthotopic tumor in mice and statistical analysis of MFI ( n = 5 mice per group). N Tumor-infiltrating neutrophils isolated from the orthotopic tumors were analyzed using flow cytometry ( n = 5 mice per group). Data represent mean ± SEM. Statistical analysis was conducted using the two-tailed unpaired Student’s t test. Source data are provided as a Source Data file.

Article Snippet: The antibodies used here included anti-L-Lactyl-Histone H3K18 antibody (1:500, PTMBIO, #PTM-1406RM), anti-GRO antibody (1:200, AFFINITY, #AF5403), Ki-67 (1:400, Servicebio, # GB111499 ), anti-Ly6G antibody (1:500, Servicebio, #GB11229), and anti-CD8 antibody (1:200, Cell Signaling Technology, #70306), and anti-PCAF antibody(1:200, Cell Signaling Technology, #3378).

Techniques: RNA Sequencing, Generated, Inhibition, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Staining, In Vitro, Migration, Incubation, Knockdown, Cell Culture, CFSE Assay, Activity Assay, Recombinant, Isolation, Flow Cytometry, 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 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 antibodies used here included anti-L-Lactyl-Histone H3K18 antibody (1:500, PTMBIO, #PTM-1406RM), anti-GRO antibody (1:200, AFFINITY, #AF5403), Ki-67 (1:400, Servicebio, # GB111499 ), anti-Ly6G antibody (1:500, Servicebio, #GB11229), and anti-CD8 antibody (1:200, Cell Signaling Technology, #70306), and anti-PCAF antibody(1:200, Cell Signaling Technology, #3378).

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 antibodies used here included anti-L-Lactyl-Histone H3K18 antibody (1:500, PTMBIO, #PTM-1406RM), anti-GRO antibody (1:200, AFFINITY, #AF5403), Ki-67 (1:400, Servicebio, # GB111499 ), anti-Ly6G antibody (1:500, Servicebio, #GB11229), and anti-CD8 antibody (1:200, Cell Signaling Technology, #70306), and anti-PCAF antibody(1:200, Cell Signaling Technology, #3378).

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

Journal: Cell death & disease

Article Title: A sphingolipid rheostat controls apoptosis versus apical cell extrusion as alternative tumour-suppressive mechanisms.

doi: 10.1038/s41419-024-07134-2

Figure Lengend Snippet: Fig. 5 Manipulation of the sphingolipid rheostat enzymes or ceramide de novo synthesis alters cell death in the hai1afr26 mutant. a Schematic of sphingolipid rheostat showing inhibition of ceramidases with ceranib-2, inhibition of sphingosine kinases with MPA08 or SKI-II, inhibition of S1P phosphatase with a MO targeting sgpp1, inhibition of serine palmitoyltransferase with myriocin or SPT-IN-1 and sphingomyelinases with desipramine, and MOs targeting cers5 and cers6 at the level of de novo synthesis and salvage from sphingosine. b Quantification of aCasp3-positive cells in the tail fins of 4 dpf embryos upon DMSO or ceranib-2 treatment, normalised to fin area. c Quantification of aCasp3-positive cells in the tail fins of control or sgpp1 MO-injected 4 dpf embryos, normalised to fin area. d Quantification of aCasp3-positive cells in the tail fins of 4 dpf embryos upon DMSO, myriocin, or desipramine treatment, normalised to fin area. e Quantification of aCasp3-positive cells in the tail fins of control or cers5 alone, cers6 alone, or both cers5 and cers6 MO-injected 4 dpf embryos, normalised to fin area. For all graphs, means were compared using one-way ANOVA with post-hoc Tukey’s multiple comparison test. N = number of biological replicates, n = number of fish per condition.

Article Snippet: Final concentrations were as follows: ZDEVD-FMK 10 μM (BD Biosciences, Cat# 550378), MPA08 50 μM (Tocris, Wiesbaden, Germany, Cat# 5803), C2 ceramide 10 μM (Enzo Life Sciences, Lörrach, Germany, Cat# BML-SL1000005), Huzzah-S1P 10 μl/ ml (Avanti Polar Lipids, Birmingham, AL, USA, Cat# 360492 P), C8 ceramide 117 μM (Avanti Polar Lipids, Cat# 860508 P; complexed 1:1 with fatty acid-free bovine serum albumin 1mM, Carl Roth, Karlsruhe, Germany, Cat#0052.1), sphinganine 0.5 μg/ ml (Avanti Polar Lipids, Cat# 860498 P), ceranib-2 1.4 μM (Merck Cat# SML0607), myriocin 25 μM (Merck, Darmstadt, Germany, Cat# M1177), desipramine hydrochloride 7.5 μM (Merck Cat# D3900), SKI II 25 μM (Merck Cat# S5696), SPTIN-1 3.4 μM (MedChemExpress, Sollentuna, Sweden, Cat# HY-125351).

Techniques: Mutagenesis, Inhibition, Control, Injection, Comparison

Journal: Cell death & disease

Article Title: A sphingolipid rheostat controls apoptosis versus apical cell extrusion as alternative tumour-suppressive mechanisms.

doi: 10.1038/s41419-024-07134-2

Figure Lengend Snippet: Fig. 6 Early inhibition of ceramide synthesis in wild-type embryos recapitulates ACE and apoptosis phenotype. a Loss of Matriptase inhibition leads to parallel pro-oncogenic and tumour-suppressive pathways. Sphingolipid rheostat-mediated tumour-suppressive methods include S1P-dependent apical cell extrusion, which preserves epithelial integrity, and C16 ceramide-dependent apoptotic cell death, which may lead to loss of epithelial barrier function. When levels of C16 ceramide are above a certain threshold, a negative feedback loop prevents further de novo ceramide synthesis. In hai1afr26 mutants by 2 dpf, ceramide levels drop due to sustained SphK activity catalysing S1P production, repression of de novo synthesis is lost, and resultant C16 ceramide levels trigger apoptosis. b-c Epidermal aggregates and apoptotic peridermal cells in the caudal fin fold of 4 dpf WT fish, transiently treated with inhibitor of de novo ceramide synthesis myriocin at 2 dpf, with orthogonal views (b’, c’) showing extruding live (yellow arrowhead) or apoptotic (yellow arrow) peridermal cells. Apoptotic cells are labelled with aCasp3 (red), peridermal cells with krt4:GFP (green), basal keratinocytes with p63 (white), and nuclei using DAPI (blue). Scale bar = 50 μm. d Quantification of the numbers of extruded cells collected per fish at 4 dpf upon inhibition of de novo sphingolipid synthesis by myriocin treatment at 2 dpf. White bars show the numbers of live cells, blue bars show the numbers of dead cells, with the proportion of dead cells indicated with blue text. e Quantification of aCasp3-positive cells in the tail fins of 4 dpf embryos upon myriocin treatment, normalised to fin area. f Quantification of proliferating cells in the tail fins of 4 dpf embryos upon myriocin treatment, normalised to fin area. For all graphs, means were compared via two-tailed, unpaired Student’s t-test. N = number of biological replicates, n = number of fish per condition.

Article Snippet: Final concentrations were as follows: ZDEVD-FMK 10 μM (BD Biosciences, Cat# 550378), MPA08 50 μM (Tocris, Wiesbaden, Germany, Cat# 5803), C2 ceramide 10 μM (Enzo Life Sciences, Lörrach, Germany, Cat# BML-SL1000005), Huzzah-S1P 10 μl/ ml (Avanti Polar Lipids, Birmingham, AL, USA, Cat# 360492 P), C8 ceramide 117 μM (Avanti Polar Lipids, Cat# 860508 P; complexed 1:1 with fatty acid-free bovine serum albumin 1mM, Carl Roth, Karlsruhe, Germany, Cat#0052.1), sphinganine 0.5 μg/ ml (Avanti Polar Lipids, Cat# 860498 P), ceranib-2 1.4 μM (Merck Cat# SML0607), myriocin 25 μM (Merck, Darmstadt, Germany, Cat# M1177), desipramine hydrochloride 7.5 μM (Merck Cat# D3900), SKI II 25 μM (Merck Cat# S5696), SPTIN-1 3.4 μM (MedChemExpress, Sollentuna, Sweden, Cat# HY-125351).

Techniques: Inhibition, Activity Assay, Two Tailed Test

Journal: Journal of Biological Chemistry

Article Title: Targeting of Functional Antibody-Decay-accelerating Factor Fusion Proteins to a Cell Surface

doi: 10.1074/jbc.m100436200

Figure Lengend Snippet: FIG. 6. Inhibition of cell surface C3 deposition by IgG-DAF fusion proteins. Control unlabeled or dansyl-labeled CHO cells were incubated with 100 nM CH1-DAF or CH3-DAF (15 min/37 °C) and then sensitized to complement with anti-CHO cell membrane antiserum. Human serum depleted of C8 was then added to a final concentration of 10%. Following incubation (45 min, 37°C), cells were washed, and C3 deposition was measured by flow cytometry using FITC-conjugated anti-C3 antibody. Histograms show relative fluorescence, with mean relative fluorescence indicated next to each peak. The data are repre- sentative of four experiments.

Article Snippet: The cells were then sensitized with anti-CHO cell membrane antiserum (10%), and NHS depleted of complement protein C8 (Quidel, San Diego, CA) added to a final concentration of 10%.

Techniques: Inhibition, Control, Labeling, Incubation, Membrane, Concentration Assay, Flow Cytometry, Fluorescence

Journal: Nature Communications

Article Title: Methionine restriction alleviates kidney fibrosis through epigenetic repression of the TGF-β-Smad3-Hoxc8/P-TEFb axis

doi: 10.1038/s41467-025-68061-0

Figure Lengend Snippet: A Motif analysis of transcription factor (TF) binding sites in TGF-β-upregulated genes in NRK-49F cells. B Heatmap showing TFs sensitive to MetR treatment in NRK-49F cells. C Quantitative RT-PCR analysis of mRNA levels for Hoxc8 , Hoxc9 , Prrx2 , and Hoxb9 in the indicated groups. From left to right: **** P = < 0.0001, ns P = 0.1239, ** P = 0.0047, *** P = 0.0006, respectively, by two-tailed unpaired Student’s t -test. Da t a are presented as mean ± SEM. D Quantitative RT-PCR analysis of mRNA levels for fibrosis marker genes ( Acta2 , Col1a1 , and Col3a1 ) in NRK-49F cells transfected with control siRNA or siRNAs targeting Hoxc8 , Prrx2 , and Hoxb9 . **** P = < 0.0001. by two-tailed unpaired Student’s t -test. Data are presen t ed as mean ± SEM. E Western blot analysis of Hoxc8 in NRK-49F cells treated with TGF-β and MetR. F Western blot analysis of Hoxc8, α-SMA, Col1a1, and Fibronectin expression in NRK-49F cells treated with TGF-β and siHoxc8. G Heatmap showing gene expression values in NRK-49F cells treated with or without TGF-β and siHoxc8. Rows represent Z-scores calculated for each group. H Venn diagram illustrating the overlap between MetR-suppressed and siHoxc8-suppressed genes (left panel). GO analysis of the overlapping genes (right panel). I Quantitative RT-PCR analysis of mRNA levels for fibrosis marker genes, Vim and Acta2 , in NRK-49F cells with or without Hoxc8 overexpression under the indicated conditions. From left to right: * P = < 0.0417, ns P = 0.6504, ** P = 0.0016, ns P = 0.2471, respectively, by two-tailed unpaired Student’s t -test. Data are presented as mean ± SEM. J Western blot analysis of α-SMA and Hoxc8 expression in NRK-49F cells with or without Hoxc8 overexpression and MetR treatment. Independent experiments = 3. Source data are provided as a Source Data file.

Article Snippet: Immunohistochemistry was performed on formalin-fixed, paraffin-embedded kidney sections from both humans and mice using antibodies against Hoxc8 (15448-1-AP, Proteintech, 1:200 dilution) and α-SMA (ab5694, Abcam, 1:100 dilution).

Techniques: Binding Assay, Quantitative RT-PCR, Two Tailed Test, Marker, Transfection, Control, Western Blot, Expressing, Gene Expression, Over Expression

Journal: Nature Communications

Article Title: Methionine restriction alleviates kidney fibrosis through epigenetic repression of the TGF-β-Smad3-Hoxc8/P-TEFb axis

doi: 10.1038/s41467-025-68061-0

Figure Lengend Snippet: A Western blot analysis of histone methylations (H3K4me3, H3K36me3, H3K9me3, H3K27me3) and total H3 in NRK-49F cells under the indicated conditions. Independent experiments = 3. B Heatmap of normalized H3K4me3 ChIP-seq signals in NRK-49F cells (left). Average H3K4me3 signal intensity around transcription start sites (TSS) of TGF-β-upregulated genes (right). C Heatmap of normalized H3K36me3 ChIP-seq signals in NRK-49F cells (left). Average H3K36me3 ChIP-seq signal intensity around TSS and transcription end sites (TES) of TGF-β-upregulated genes (right). D Gene tracks displaying H3K4me3 and H3K36me3 ChIP-seq profiles for Acta2 , Col1a1 , and Col3a1 under the indicated conditions. E Gene tracks displaying H3K4me3 and H3K36me3 ChIP-seq profiles for Hoxc8 under the indicated conditions. Source data are provided as a Source Data file.

Article Snippet: Immunohistochemistry was performed on formalin-fixed, paraffin-embedded kidney sections from both humans and mice using antibodies against Hoxc8 (15448-1-AP, Proteintech, 1:200 dilution) and α-SMA (ab5694, Abcam, 1:100 dilution).

Techniques: Western Blot, ChIP-sequencing

Journal: Nature Communications

Article Title: Methionine restriction alleviates kidney fibrosis through epigenetic repression of the TGF-β-Smad3-Hoxc8/P-TEFb axis

doi: 10.1038/s41467-025-68061-0

Figure Lengend Snippet: A Heatmap of normalized Hoxc8 ChIP-seq signals in NRK-49F cells treated with or without TGF-β. B Gene Set Enrichment Analysis (GSEA) of genes with enhanced Hoxc8 binding comparing TGF-β-treated cells to control cells. The enrichment plot demonstrates enrichment of these genes in TGF-β-upregulated genes. C GSEA of genes with enhanced Hoxc8 binding comparing TGF-β-treated cells to TGF-β+MetR-treated cells. The enrichment plot demonstrates enrichment of these genes in MetR-suppressed genes. D GSEA of genes with enhanced Hoxc8 binding comparing TGF-β-treated cells to TGF-β+siHoxc8-treated cells. The enrichment plot demonstrates enrichment of these genes in siHoxc8-suppressed genes. E Genome browser tracks showing Hoxc8 ChIP-seq signals at the Hoxc8 locus in control and TGF-β-treated NRK-49F cells. F Schematic representation of luciferase reporter constructs containing either wild-type (WT) or mutant (Mut) Hoxc8 binding sites. The WT construct includes four tandem Hoxc8 binding sites with sequences highlighted in red, while the Mut construct contains mutated binding sites. G Luciferase assay showing Hoxc8-mediated activation of luciferase reporters containing WT or mutant Hoxc8 binding sites in NRK-49F cells. From left to right: *** P = 0.0002, ns P = 0.9864, respectively, by two-tailed unpaired Student’s t -test. Data are presented as mean ± SEM. H Quantitative RT-PCR analysis of Hoxc8 mRNA levels in NRK-49F cells transfected with empty vector or human TY1-HOXC8 plasmid. *** P = 0.0006, by two-tailed unpaired Student’s t -test. Data are presented as mean ± SEM. I Western blot analysis showing the expression of endogenous Hoxc8 and exogenous (TY1-tagged) HOXC8 proteins in NRK-49F cells transfected with empty vector or TY1-HOXC8 plasmid. Independent experiments = 3. Source data are provided as a Source Data file.

Article Snippet: Immunohistochemistry was performed on formalin-fixed, paraffin-embedded kidney sections from both humans and mice using antibodies against Hoxc8 (15448-1-AP, Proteintech, 1:200 dilution) and α-SMA (ab5694, Abcam, 1:100 dilution).

Techniques: ChIP-sequencing, Binding Assay, Control, Luciferase, Construct, Mutagenesis, Activation Assay, Two Tailed Test, Quantitative RT-PCR, Transfection, Plasmid Preparation, Western Blot, Expressing

Journal: Nature Communications

Article Title: Methionine restriction alleviates kidney fibrosis through epigenetic repression of the TGF-β-Smad3-Hoxc8/P-TEFb axis

doi: 10.1038/s41467-025-68061-0

Figure Lengend Snippet: A Co-immunoprecipitation (Co-IP) analysis showing the interaction between FLAG-CDK9 and HA-HOXC8 in 293 T cells. B Co-IP analysis showing the interaction between MYC-CDK9 and FLAG-HOXC8 in 293 T cells. C Co-IP analysis of endogenous Hoxc8, Cdk9, and CycT1 in NRK-49F cells treated with or without TGF-β. D Composite plots showing normalized Hoxc8 ChIP-seq signals across gene bodies (3 kb ± ChIP-seq peak summit) in control, TGF-β-treated, and TGF-β+siHoxc8-treated NRK-49F cells. E Composite plots showing Pol II Ser2P ChIP-seq signals across gene bodies (3 kb ± ChIP-seq peak summit) in control, TGF-β-treated, and TGF-β+siHoxc8-treated NRK-49F cells. F Cumulative distribution plot of Pol II pausing index (PI) for genes overlapped between MetR- and siHoxc8-suppressed gene sets. G Working model of the regulation of Hoxc8 and pro-fibrotic gene expression under TGF-β and MetR treatments. Top panel (red dashed box): TGF-β induces Hoxc8 expression through Smad3 activation. Once upregulated, Hoxc8 undergoes self-activation and interacts with P-TEFb to promote transcriptional elongation of pro-fibrotic genes. Bottom panel (blue dashed box): MetR suppresses TGF-β-induced myofibroblast activation by inhibiting Hoxc8 expression. This disrupts P-TEFb recruitment, thereby reducing transcriptional elongation of pro-fibrotic genes. Independent experiments = 3. Source data are provided as a Source Data file.

Article Snippet: Immunohistochemistry was performed on formalin-fixed, paraffin-embedded kidney sections from both humans and mice using antibodies against Hoxc8 (15448-1-AP, Proteintech, 1:200 dilution) and α-SMA (ab5694, Abcam, 1:100 dilution).

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, ChIP-sequencing, Control, Gene Expression, Expressing, Activation Assay

Journal: Nature Communications

Article Title: Methionine restriction alleviates kidney fibrosis through epigenetic repression of the TGF-β-Smad3-Hoxc8/P-TEFb axis

doi: 10.1038/s41467-025-68061-0

Figure Lengend Snippet: A IHC staining assessing Hoxc8 expression in kidneys, accompanied by corresponding quantitative analysis. From left to right: ** P = 0.0042, * P = 0.0343, respectively, by two-tailed unpaired Student’s t -test. Data are presented as mean ± SEM. n = 6 biologically independent samples. Scale bar = 100 μm. B Schematic view of experimental design in the Hoxc8 conditional knockout mice. C Immunofluorescence staining of Hoxc8 and PDGFR-α in kidney sections from sham or UUO-operated Hoxc8 WT and Hoxc8 FKO mice. Arrows denote cells with co-localization of PDGFR-α and Hoxc8. The experiment shown is representative of 6 independent biological replicates, all yielding similar results. Scale bar = 25 μm. D IHC analysis of α-SMA expression. Quantification of α-SMA-positive staining is shown as the percentage of stained area. **** P < 0.0001, by two-tailed unpaired Student’s t -test. n = 6 biologically independent samples. Data are presented as mean ± SEM. Scale bar = 100 μm. E Masson staining of kidney sections from sham or UUO-operated Hoxc8 WT and Hoxc8 FKO mice to assess fibrosis. Quantification of fibrosis is shown as the percentage of the fibrotic area. **** P < 0.0001, by two-tailed unpaired Student’s t -test. n = 6 biologically independent samples. Data are presented as mean ± SEM. Scale bar = 100 μm. F Quantitative RT-PCR analysis of mRNA expression for Hoxc8 , Acta2 , Col3a1 , and Col1a1 in kidney sections from sham or UUO-operated Hoxc8 WT and Hoxc8 FKO mice. From left to right: * P = 0.0190, * P = 0.0237, * P = 0.0368, * P = 0.0147, respectively, by two-tailed unpaired Student’s t -test. n = 6 biologically independent samples. Data are presented as mean ± SEM. G IHC analysis of HOXC8 expression in human kidney biopsies from control, ADPKD ( n = 23), and IgAN patients ( n = 40). Scale bar = 50 μm. Quantification of HOXC8 expression is shown as H-score. From left to right: ** P = 0.0036, *** P = 0.0010, respectively, by two-tailed unpaired Student’s t -test. Data are presented as mean ± SEM. H Correlation analysis between HOXC8 expression and estimated glomerular filtration rate (eGFR) in kidney biopsies from IgAN patients. Source data are provided as a Source Data file.

Article Snippet: Immunohistochemistry was performed on formalin-fixed, paraffin-embedded kidney sections from both humans and mice using antibodies against Hoxc8 (15448-1-AP, Proteintech, 1:200 dilution) and α-SMA (ab5694, Abcam, 1:100 dilution).

Techniques: Immunohistochemistry, Expressing, Two Tailed Test, Knock-Out, Immunofluorescence, Staining, Quantitative RT-PCR, Control, Filtration