Journal: Nature Communications

Article Title: IL-17A is increased in diabetic wounds and impairs keratinocyte function via histone demethylase JMJD3

doi: 10.1038/s41467-025-67456-3

Figure Lengend Snippet: a ChIP analysis of H3K27me3 or IgG at indicated promoters in keratinocytes with (white) or without (blue) IL-17A stimulation. Itga3: n = 5 (unstimulated), n = 4 (IL-17A-stimulated) technical replicates, p = 0.0034, Timp1 : n = 3 technical replicates, p = 0.0046, Ccl20 : n = 3 technical replicates, p = 0.0059, Cxcl1 : n = 3 technical replicates, p = 0.0020, Cxcl3 : n = 3 technical replicates, p = 0.0258, Cxcl5 : n = 3 technical replicates, p = 0.0174. n = 3 independent experiments. b qPCR analysis of keratinocytes treated with DMSO only (white), with IL-17A alone (blue), or with IL-17A and GSK-J4 (1 µM) (red). Itga3 : n = 4 biological replicates, p < 0.0001 (DMSO vs. IL-17A), p = 0.0017 (IL-17A vs. IL-17A and inhibitor), Timp1: n = 6 biological replicates, p < 0.0312 (DMSO vs. IL-17A), p = 0.0029 (IL-17A vs. IL-17A and inhibitor), Ccl20: n = 3 biological replicates, p = 0.0008 (DMSO vs. IL-17A), p = 0.0428 (IL-17A vs. IL-17A and inhibitor), Cxcl1 : n = 3 biological replicates, p = 0.0003 (DMSO vs. IL-17A), p = 0.0294 (IL-17A vs. IL-17A and inhibitor), Cxcl3 : n = 3 biological replicates, p < 0.0001 (DMSO vs. IL-17A), p = 0.0003 (IL-17A vs. IL-17A and inhibitor), Cxcl5 : n = 3 biological replicates, p < 0.0001 (DMSO vs. IL-17A), p = 0.0007 (IL-17A vs. IL-17A and inhibitor), n = 3 independent experiments. c Western blot of ITGA-3 expression in keratinocytes treated with DMSO only (white), with IL-17A alone (blue), or with IL-17A and GSK-J4 (red). Representative densitometry plot is shown. n = 3 independent experiments. d Protein quantification of lysates from keratinocytes treated with DMSO only (white), with IL-17A alone (blue), or with IL-17A and GSK-J4 (red). TIMP-1: n = 6 biological replicates, p = 0.0012 (DMSO vs. IL-17A), p = 0.0068 (IL-17A vs. IL-17A and inhibitor), CCL-20: n = 3 biological replicates, p < 0.0001 (DMSO vs. IL-17A), p = 0.0199 (IL-17A vs. IL-17A and inhibitor), CXCL-1: n = 3 biological replicates, p < 0.0001 (DMSO vs. IL-17A), p = 0.0158 (IL-17A vs. IL-17A and inhibitor), CXCL-3: n = 3 biological replicates, p < 0.0001 (DMSO vs. IL-17A), p = 0.0167 (IL-17A vs. IL-17A and inhibitor), CXCL-5: n = 3 biological replicates, p < 0.0001 (DMSO vs. IL-17A), p = 0.0005 (IL-17A vs. IL-17A and inhibitor), n = 3 independent experiments. e qPCR analysis of keratinocytes treated with a non-targeting control (siNTC) (white) or si Jmjd3 (gray). Jmjd3 : n = 6 (siNTC), n = 4 biological replicates (si Jmjd3 ), p = 0.0192, Itga3: n = 6 (siNTC), n = 4 biological replicates (si Jmjd3 ), p = 0.0019, Timp1: n = 3 (siNTC), n = 4 biological replicates (si Jmjd3 ), p = 0.0205, Ccl20: n = 6 (siNTC), n = 4 biological replicates (si Jmjd3 ), p = 0.0015, Cxcl1: n = 6 (siNTC), n = 4 biological replicates (si Jmjd3 ), p = 0.0019, Cxcl3: n = 6 (siNTC), n = 4 biological replicates (si Jmjd3 ), p = 0.0011, Cxcl5: n = 6 (siNTC), n = 4 biological replicates (si Jmjd3 ), p = 0.0011, n = 3 independent experiments. f qPCR analysis of Jmjd3 fl/fl K14 cre+ (red) and Jmjd3 fl/fl K14 cre- (yellow) keratinocytes. n = 3 biological replicates, Itga3 : p = 0.0184, Timp1 : p = 0.0044, Ccl20 : p = 0.0160, Cxcl1 : p = 0.0371, Cxcl3 : p = 0.0042. n = 3 independent experiments. g , h Scratch assays of primary murine ( n = 3 biological replicates, p < 0.0001 (48 h)) and N/TERT ( n = 3 biological replicates, p = 0.0340 (12 h)) keratinocytes treated with IL-17A alone (blue) or IL-17A and GSK-J4 (red). n = 3 independent experiments. i , j Scratch assays of primary murine ( n = 3 biological replicates, p = 0.0016 (48 h)) and N/TERT ( n = 6 biological replicates (IL-17A alone), n = 4 biological replicates (IL-17A and GSK-J1), p = 0.0223 (8 h), p < 0.0001 (12 h)) keratinocytes treated with IL-17A alone (blue) or IL-17A and GSK-J1 (red). n = 3 independent experiments. k Scratch assay of Jmjd3 fl/fl K14 cre+ (red) and Jmjd3 fl/fl K14 cre- (blue) keratinocytes. n = 3 biological replicates, p = 0.0198 (48 h). n = 3 independent experiments. Data were analyzed for variances, and 2-tailed Student’s t tests for ( a ), ( e ), ( f ) and 1-way ANOVA tests for ( b ), ( d ), ( g – k ) were performed. Data are presented as the mean ± SEM.

Article Snippet: After stimulation, cell free supernatant was collected and analyzed by the University of Michigan Immune Monitoring Shared Resource Core for CCL-20, CXCL-1, CXCL-5 or specific enzyme immunoassay kits for CXCL-3 (Boster Bio) and TIMP-1 (R&D Systems) according to the manufacturer’s instructions.

Techniques: Western Blot, Expressing, Control, Wound Healing Assay

Journal: Autophagy

Article Title: Neutrophil-derived serine proteases induce FOXA2-mediated autophagy dysfunction and exacerbate colitis-associated carcinogenesis via protease activated receptor 2

doi: 10.1080/15548627.2025.2489335

Figure Lengend Snippet: F2RL1/PAR2 regulates lysosome acidity through ATP6V0E1 . (A) A schematic illustration of lysosomal proton pump and mRNA levels in F2RL1 -knockout HT29 cells (generated using BioRender). (B) Relative mRNA levels of V-ATPase subunits ( ATP6V0C , ATP6V0E1 , ATP6V1C2 , and ATP6V1F ) after F2RL1 knockdown in HT29, RKO, and SW620 cells as well as CaCO2 cells treated with F2RL1/PAR2 activator (100 μM, 12 h). (C) Representative western blots of V-ATPase subunits ( ATP6V0C , ATP6V0E1 , ATP6V1C2 , and ATP6V1F ) in HT29, RKO, and SW620 cells following F2RL1 knockdown as well as in CaCO2 cells treated with a F2RL1/PAR2 activator (100 μM, 12 h). (D) Representative images showing lysosome pH detected based on LysoSensor green (upper panel) and CTSB activity (lower panel) following ATP6V0E1 siRNA-mediated silencing or overexpression in F2RL1 -knockdown HT29 cells. Scale bar: 5 µm. (E) Representative western blots of autophagy-related proteins (MAP1LC3/LC3-II/I and SQSTM1/p62) following ATP6V0E1 silencing or overexpression in F2RL1 -knockout HT29 and RKO cells. (F) Representative images of autophagosome-lysosome fusion based on the colocalization of MAP1LC3/LC3-positive autophagosomes (green) with LAMP1-positive lysosomes (red) in HT29 cells after ATP6V0E1 silencing or overexpression after F2RL1 knockdown. Scale bar: 5 µm. Results are presented as mean ± SD. Statistical significance was determined using a two-tailed unpaired Student’s t -test or a two-way ANOVA with Tukey’s post-hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: For western blot, MAP1LC3/LC3 (Cell Signaling Technology 12741), SQSTM1/p62 (Cell Signaling Technology 23214), γH2AX (Abcam, ab81299), F2RL1/PAR2 (Abcam, ab180923), ATP6V0C (Abcam, ab220323), ATP6V1C2 (Abcam, ab176771), ATP6V0E1 (Aviva Systems Biology, OACA00864), ATP6V1F (Abcam, ab190789), FOXA2 (Abcam, ab108422), histone H3 (Cell Signaling Technology, 9715), ZNF600 (Proteintech 20100–1-AP), RXRB (Proteintech 14684–1-AP), GATAD2A (Proteintech 12294–1-AP), TUBB/Beta Tubulin (Proteintech 10068–1-AP), Anti-rabbit IgG, HRP-linked Antibody (Cell Signaling Technology, 7074).

Techniques: Knock-Out, Generated, Knockdown, Western Blot, Activity Assay, Over Expression, Two Tailed Test

Journal: Autophagy

Article Title: Neutrophil-derived serine proteases induce FOXA2-mediated autophagy dysfunction and exacerbate colitis-associated carcinogenesis via protease activated receptor 2

doi: 10.1080/15548627.2025.2489335

Figure Lengend Snippet: F2RL1/PAR2 regulates the transcription of ATP6V0E1 via YAP1-FOXA2. (A) The overlap between four genes downregulated with F2RL1 knockdown in HT29 cells and transcription factors predicted for ATP6V0E1 . (B) Relative mRNA levels of FOXA2 following F2RL1 knockdown in HT29, RKO, and SW620 cells. (C) Representative western blots of FOXA2 proteins in HT29, RKO, and SW620 cells following F2RL1 knockdown. (D) Representative western blots of FOXA2 in the cytoplasm and nuclei of HT29 cells following F2RL1 knockdown. (E) Representative western blots of autophagy-related proteins (MAP1LC3/LC3-I/II and SQSTM1/p62) and ATP6V0E1 following FOXA2 silencing or overexpression in F2RL1 -knockout HT29 and RKO cells. (F) Representative images of CTSB activity (upper panel) and autophagosome-lysosome fusion based on the colocalization of MAP1LC3/LC3-positive autophagosomes (green) with LAMP1-positive lysosomes (red) in HT29 cells after FOXA2 silencing or overexpression along with F2RL1 knockdown. Scale bar: 5 µm. (G) The bioinformatics analysis of ATP6V0E1 promoter region binding to FOXA2. (H) The luciferase activity of vectors containing ATP6V0E1 or a mutant ATP6V0E1 promoter, which were transfected into HEK-293T cells expressing FOXA2 , showing a dose-dependent response. (I) FOXA2 enrichment in promoter regions of ATP6V0E1 (compared with control IgG binding). (J) Representative western blots of FOXA2, ATP6V0E1, and autophagy-related proteins in HT29 cells treated with ELANE (1 μg/mL, 24 h), CTSG (2 μg/mL, 24 h), and PRTN3 (1 μg/mL, 24 h) with or without overexpression of YAP1 when F2RL1 was knocked down. Results are presented as mean ± SD. Statistical significance was determined using a two-tailed unpaired Student’s t -test or a two-way ANOVA with Tukey’s post-hoc test. * p < 0.05, ** p < 0.01, **** p < 0.0001.

Article Snippet: For western blot, MAP1LC3/LC3 (Cell Signaling Technology 12741), SQSTM1/p62 (Cell Signaling Technology 23214), γH2AX (Abcam, ab81299), F2RL1/PAR2 (Abcam, ab180923), ATP6V0C (Abcam, ab220323), ATP6V1C2 (Abcam, ab176771), ATP6V0E1 (Aviva Systems Biology, OACA00864), ATP6V1F (Abcam, ab190789), FOXA2 (Abcam, ab108422), histone H3 (Cell Signaling Technology, 9715), ZNF600 (Proteintech 20100–1-AP), RXRB (Proteintech 14684–1-AP), GATAD2A (Proteintech 12294–1-AP), TUBB/Beta Tubulin (Proteintech 10068–1-AP), Anti-rabbit IgG, HRP-linked Antibody (Cell Signaling Technology, 7074).

Techniques: Knockdown, Western Blot, Over Expression, Knock-Out, Activity Assay, Binding Assay, Luciferase, Mutagenesis, Transfection, Expressing, Control, Two Tailed Test

Journal: Autophagy

Article Title: Neutrophil-derived serine proteases induce FOXA2-mediated autophagy dysfunction and exacerbate colitis-associated carcinogenesis via protease activated receptor 2

doi: 10.1080/15548627.2025.2489335

Figure Lengend Snippet: F2RL1 deficiency promotes DNA damage. (A) Representative images of mitoROS fluorescence staining in HT29 cells after F2RL1 knockdown and pretreatment with BafA1 (100 nM, 12 h) and/or NAC (10 μM, 12 h). Scale bar: 10 µm. (B) Representative western blots of γH2AX and MAP1LC3/LC3-I/II proteins in F2RL1 -knockout HT29 cells following pretreatment with BafA1 (100 nM, 12 h) or NAC (10 μM, 12 h). (C) Representative images of mitoROS fluorescence staining in HT29 cells with FOXA2 and ATP6V0E1 overexpression and stable sh F2RL1 plasmid expression. Scale bar: 10 µm. (D) Representative western blots of γH2AX, FOXA2, and ATP6V0E1 proteins in HT29 cells overexpressing FOXA2 and ATP6V0E1 and stably expressing the sh F2RL1 plasmid. (E) Representative images of γH2AX fluorescence staining in HT29 cells overexpressing FOXA2 and ATP6V0E1 and stably expressing the sh F2RL1 plasmid. Scale bar: 10 µm. (F) Relative mRNA levels of Foxa2 and Atp6v0e in the colon tissues of AOM-induced CRC mice ( n = 5). (G) Representative western blots of FOXA2, ATP6V0E1, and γH2AX in the colon tissues of AOM-induced CRC mice ( n = 3). (H) Gene set enrichment analysis of DEGs between F2rl1 f/f and f2rl1[ ∆IEC] model mice. (I) Representative IHC staining images of γH2AX proteins derived from the colons of F2rl1 f/f and f2rl1[ ∆IEC] mice after the nine-week AOM-DSS challenge. Scale bar: 50 μm. (J) Representative immunofluorescence images of γH2AX (red) in the colons of F2rl1 f/f and f2rl1[ ∆IEC] model mice. EPCAM (green) is an epithelium cell marker. Scale bar: 50 μm. Results are presented as mean ± SD. Statistical significance was determined using a two-way ANOVA with Tukey’s post-hoc test. * p < 0.05, ** p < 0.01.

Article Snippet: For western blot, MAP1LC3/LC3 (Cell Signaling Technology 12741), SQSTM1/p62 (Cell Signaling Technology 23214), γH2AX (Abcam, ab81299), F2RL1/PAR2 (Abcam, ab180923), ATP6V0C (Abcam, ab220323), ATP6V1C2 (Abcam, ab176771), ATP6V0E1 (Aviva Systems Biology, OACA00864), ATP6V1F (Abcam, ab190789), FOXA2 (Abcam, ab108422), histone H3 (Cell Signaling Technology, 9715), ZNF600 (Proteintech 20100–1-AP), RXRB (Proteintech 14684–1-AP), GATAD2A (Proteintech 12294–1-AP), TUBB/Beta Tubulin (Proteintech 10068–1-AP), Anti-rabbit IgG, HRP-linked Antibody (Cell Signaling Technology, 7074).

Techniques: Fluorescence, Staining, Knockdown, Western Blot, Knock-Out, Over Expression, Plasmid Preparation, Expressing, Stable Transfection, Immunohistochemistry, Derivative Assay, Immunofluorescence, Marker

Journal: Autophagy

Article Title: Neutrophil-derived serine proteases induce FOXA2-mediated autophagy dysfunction and exacerbate colitis-associated carcinogenesis via protease activated receptor 2

doi: 10.1080/15548627.2025.2489335

Figure Lengend Snippet: Neutrophil-derived serine proteases impede autophagy through F2RL1/PAR2. (A) A schematic of F2RL1/PAR2 sites cleaved by different serine proteases. (B) Representative western blots of FOXA2, ATP6V0E1, and autophagy-related proteins in NCM460 glutamine-deprived condition and HT29 cells treated with ELANE (1 μg/mL, 24 h), CTSG (2 μg/mL, 24 h), and PRTN3 (1 μg/mL, 24 h) with or without FOXA2 overexpression. (C) Representative fluorescence images of FOXA2 in NCM460 and HT29 cells treated with ELANE (1 μg/mL, 24 h), CTSG (2 μg/mL, 24 h), PRTN3 (1 μg/mL, 24 h), F2RL1/PAR2-AP (100 μM, 12 h), and trypsin (50 μg/mL, 24 h). Scale bar: 10 µm. (D) Representative images of autophagosome-lysosome fusion based on the colocalization of MAP1LC3/LC3-positive autophagosomes (green) with LAMP1-positive lysosomes (red) in glutamine-deprived NCM460 and HT29 cells treated with ELANE (1 μg/mL, 24 h), CTSG (2 μg/mL, 24 h), and PRTN3 (1 μg/mL, 24 h) with or without FOXA2 overexpression. Scale bar: 5 µm. (E) Representative western blots of FOXA2, ATP6V0E1, and autophagy-related proteins in IEC6 cells treated with F2RL1/PAR2-AP (100 μM, 12 h) and trypsin (50 μg/mL, 24 h) with or without glutamine deprivation or BafA1 (25 nM, 24 h). (F) Representative fluorescence images of FOXA2 in NCM460 and IEC6 cells treated with F2RL1/PAR2-AP (100 μM, 12 h) and trypsin (50 μg/mL, 24 h). Scale bar: 10 µm. (G) Representative images of autophagosome-lysosome fusion based on the colocalization of MAP1LC3/LC3-positive autophagosomes (green) with LAMP1-positive lysosomes (red) in glutamine-deprived IEC6 cells treated with F2RL1/PAR2-AP (100 μM, 12 h) and trypsin (50 μg/mL, 24 h) with or without BafA1 (25 nM, 24 h). Scale bar: 5 µm. (H) The sequencing and western blot-based identification of CRISPR/Cas9-knockout cell lines. (I) Relative mRNA levels of FOXA2 and ATP6V0E1 in HT29 cells treated with ELANE (1 μg/mL, 24 h), CTSG (2 μg/mL, 24 h), and PRTN3 (1 μg/mL, 24 h) with or without overexpression of F2RL1 when F2RL1 was knocked out. (J) Representative western blots of FOXA2, ATP6V0E1, and autophagy-related proteins in HT29 cells treated with ELANE (1 μg/mL, 24 h), CTSG (2 μg/mL, 24 h), and PRTN3 (1 μg/mL, 24 h) with or without overexpression of F2RL1 when F2RL1 was knocked out. (K) The fluorescence intensities detected via FACS, and lysosome pH detected using LysoSensor green in HT29 cells treated with combined ECP (neutrophil-derived serine proteases), including ELANE (1 μg/mL), CTSG (2 μg/mL), and PRTN3 (1 μg/mL) for 24 h with or without overexpression of F2RL1 when F2RL1 was knocked out. Results are presented as mean ± SD. Statistical significance was determined using a two-way ANOVA with Tukey’s post-hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: For western blot, MAP1LC3/LC3 (Cell Signaling Technology 12741), SQSTM1/p62 (Cell Signaling Technology 23214), γH2AX (Abcam, ab81299), F2RL1/PAR2 (Abcam, ab180923), ATP6V0C (Abcam, ab220323), ATP6V1C2 (Abcam, ab176771), ATP6V0E1 (Aviva Systems Biology, OACA00864), ATP6V1F (Abcam, ab190789), FOXA2 (Abcam, ab108422), histone H3 (Cell Signaling Technology, 9715), ZNF600 (Proteintech 20100–1-AP), RXRB (Proteintech 14684–1-AP), GATAD2A (Proteintech 12294–1-AP), TUBB/Beta Tubulin (Proteintech 10068–1-AP), Anti-rabbit IgG, HRP-linked Antibody (Cell Signaling Technology, 7074).

Techniques: Derivative Assay, Western Blot, Over Expression, Fluorescence, Sequencing, CRISPR, Knock-Out

Journal: Autophagy

Article Title: Neutrophil-derived serine proteases induce FOXA2-mediated autophagy dysfunction and exacerbate colitis-associated carcinogenesis via protease activated receptor 2

doi: 10.1080/15548627.2025.2489335

Figure Lengend Snippet: Neutrophil levels correlate with F2RL1/ PAR2-FOXA2 expression in colitis and CRC. (A) FOXA2 expression between healthy individuals and patients with UC in the GSE4183 ( N = 15), GSE2443625 ( N = 15), GSE224758 ( N = 16), GSE117993 ( N = 43), and GSE109142 ( N = 206) datasets. (B) Relative FOXA2 expression in the GSE220063 dataset for CRC ( N = 6) and CAC ( N = 5). (C) TCGA dataset analysis of FOXA2 expression between patients with low and high neutrophil index ( N = 128). (D) The correlation between FOXA2 and ATP6V0E1 expression in UC in the GSE128682 ( N = 44) and GSE174159 ( N = 39) datasets (left panel); correlation between F2RL1 , FOXA2 , and ATP6V0E1 expressions in COAD and READ in TCGA dataset (right panel). (E) The multicolor immunofluorescence of FOXA2 (green), MPO (yellow), and PANCK (purple) in active and inactive UC ( N = 8). Bar: 25 μm. (F) PANCK + FOXA2 + cells/mm 2 in active and inactive UC ( N = 8). (G) FOXA2 average nuclear intensity of total and ≤25 μm in active UC ( N = 8). (H) The correlation between PANCK + FOXA2 + and MPO + cells/mm 2 ( N = 8). (I) The multicolor immunofluorescence of FOXA2 (green), MPO (yellow), and PANCK (purple) in CRC ( N = 3). Bar: 25 μm. (J) FOXA2 average nuclear intensity of total and ≤25 μm ( N = 3). (K) PANCK + FOXA2 + cell ratios in MPO + cells in CRC ( N = 3). Results are presented as mean ± SD. Statistical significance was determined using a two-tailed unpaired Student’s t -test or a one-way ANOVA with Tukey’s post-hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: For western blot, MAP1LC3/LC3 (Cell Signaling Technology 12741), SQSTM1/p62 (Cell Signaling Technology 23214), γH2AX (Abcam, ab81299), F2RL1/PAR2 (Abcam, ab180923), ATP6V0C (Abcam, ab220323), ATP6V1C2 (Abcam, ab176771), ATP6V0E1 (Aviva Systems Biology, OACA00864), ATP6V1F (Abcam, ab190789), FOXA2 (Abcam, ab108422), histone H3 (Cell Signaling Technology, 9715), ZNF600 (Proteintech 20100–1-AP), RXRB (Proteintech 14684–1-AP), GATAD2A (Proteintech 12294–1-AP), TUBB/Beta Tubulin (Proteintech 10068–1-AP), Anti-rabbit IgG, HRP-linked Antibody (Cell Signaling Technology, 7074).

Techniques: Expressing, Immunofluorescence, Two Tailed Test