Journal: Journal of Biochemical and Molecular Toxicology

Article Title: Renal Tubular Epithelial Cells Senescence Induced by 1‐Aminopyrene Activates Fibroblast via BRD4 Driven Senescence‐Associated Secretory Phenotype Signaling

doi: 10.1002/jbt.71124

Figure Lengend Snippet: 1 ‐ AP induces tubular epithelial senescence and promotes paracrine fibroblast activation via a SASP‐mediated mechanism. (A) Representative images and quantification of SA‐β‐gal positive RTECs following exposure to 1‐AP after 72 h. Scale bars, 5 µm. (B) DAPI‐stained nuclei (blue) and quantification showing a significant reduction in cell number following exposure to 1‐AP after 72 h. Scale bars, 2 µm. (C) Representative images and quantification of immunofluorescence analysis of Ki67 in RTECs following 1‐AP treatment after 72 h. Scale bars, 5 µm. (D) RT‐qPCR analysis of p16, p21, and p53 mRNA expression showing upregulation of senescence‐associated cell cycle regulators in RTECs following exposure to 1‐AP after 48 h. (E) Representative images and quantification of immunofluorescence analysis of p21 in RTECs following 1‐AP treatment after 72 h. Scale bars, 5 µm. (F) Heatmap for RT‐qPCR analysis of senescence‐associated secretory phenotype (SASP) factors (IL‐1β, IL‐6, IL‐8, MCP‐1, TGF‐β1, and CXCL1) in RTECs following 1‐AP treatment after 48 h. (G) Schematic illustration of the experimental design showing 1‐AP induced senescence in RTECs and paracrine activation of renal fibroblasts via conditioned media may leads to TIF or renal fibrosis. (H) Heatmap for RT‐qPCR analysis of fibrotic markers ( α‐SMA, POSTN, Col1a2, TIMP1 , and CTGF ) in NRK52F renal fibroblasts exposed to conditioned media from 1‐AP‐treated TECs for 48 h. (I–K) Representative images and quantification of immunofluorescence analysis of α‐SMA (I), POSTN (J), and COL1A1 (K) in NRK52F fibroblasts, demonstrating significant fibroblast activation and extracellular matrix induction following exposure to TEC‐derived conditioned media after 48 h. Scale bars, 20 µm. Data are presented as mean ± SD, n = 3. Statistical analysis was performed using two‐tailed unpaired Student's t‐test. Exact p ‐values are indicated in the graphs; ns, not significant. DAPI used as nuclear counterstain.

Article Snippet: Primary antibodies for AhR antibody (182705‐10 G, Meck), Phospho‐Histone H2A.X (Ser139) Antibody (20E3, cell signaling), BRD4 antibody (ER1901‐02, Thermo‐Fischer), Ki‐67 antibody (27309‐1‐AP, proteintech), p21 (2947, cell signaling), α‐SMA antibody (14395‐1‐AP, proteintech), periostin (19899‐1‐AP, proteintech), COL1A1 antibody (55131‐1‐AP, proteintech), Histone H3K9/14ac (Acetyl H3K9/14) Antibody (A‐4021‐025, epigentek), Histone H3K27me3 (H3K27 Trimethyl) Antibody (A‐4039‐025, epigentek), Histone H3K9me3 (H3K9 Trimethyl) Antibody (A‐4036‐025, epigentek), IL‐6 (21865‐1‐AP, proteintech), TGF‐beta1 (21898‐1‐AP, proteintech) and Secondary antibody anti‐Mouse Alexa‐Fluro 555 ( A31570 , Thermo‐Fischer) and anti‐Rabbit Alexa‐Fluro 488 (A11008, Thermo‐Fischer), TUNEL Assay Kit (25879, cell signaling) and Senescence β‐Galactosidase (SA‐β‐gal) Staining Kit (9860, cell signaling) were purchased.

Techniques: Activation Assay, Staining, Immunofluorescence, Analysis, Quantitative RT-PCR, Expressing, Derivative Assay, Two Tailed Test

Journal: Journal of Biochemical and Molecular Toxicology

Article Title: Renal Tubular Epithelial Cells Senescence Induced by 1‐Aminopyrene Activates Fibroblast via BRD4 Driven Senescence‐Associated Secretory Phenotype Signaling

doi: 10.1002/jbt.71124

Figure Lengend Snippet: AhR signaling is activated in RTECs by 1‐AP but is insufficient for SASP‐mediated fibroblast activation. (A) Representative images and quantification of immunofluorescence analysis of AHR expression in RTECs following 1‐AP treatment ± AhR inhibitor (10 μM) after 48 h. Scale bars, 20 µm. (B) Heatmap for RT‐qPCR analysis of AhR target genes ( CYP1A1, CYP1A2 ) and senescence markers ( p16, p21, p53 ) following 1‐AP treatment ± AhR inhibitor after 48 h. (C) Heatmap for RT‐qPCR analysis of SASP genes ( IL‐1β, IL‐6, IL‐8, MCP‐1, TGF‐β1, CXCL1 ) following 1‐AP treatment ± AhR inhibitor after 48 h. Conditioned media (CM) transfer from NRK‐52E/RTECs treated with 1‐AP ± AhR inhibitor to NRK‐52F fibroblasts after 48 h showing (D) Heatmap for RT‐qPCR analysis of fibroblast activation and extracellular matrix genes ( α‐SMA, POSTN, Col1a2, TIMP‐1, CTGF ), (E–G) Quantification of immunofluorescence analysis of α‐SMA, POSTN and COL1A1 in NRK52F fibroblasts. Data are presented as mean ± SD, n = 3. Statistical analysis was performed using two‐tailed unpaired Student's t‐test. Exact p ‐values are indicated in the graphs; ns, not significant. DAPI used as nuclear counterstain.

Article Snippet: Primary antibodies for AhR antibody (182705‐10 G, Meck), Phospho‐Histone H2A.X (Ser139) Antibody (20E3, cell signaling), BRD4 antibody (ER1901‐02, Thermo‐Fischer), Ki‐67 antibody (27309‐1‐AP, proteintech), p21 (2947, cell signaling), α‐SMA antibody (14395‐1‐AP, proteintech), periostin (19899‐1‐AP, proteintech), COL1A1 antibody (55131‐1‐AP, proteintech), Histone H3K9/14ac (Acetyl H3K9/14) Antibody (A‐4021‐025, epigentek), Histone H3K27me3 (H3K27 Trimethyl) Antibody (A‐4039‐025, epigentek), Histone H3K9me3 (H3K9 Trimethyl) Antibody (A‐4036‐025, epigentek), IL‐6 (21865‐1‐AP, proteintech), TGF‐beta1 (21898‐1‐AP, proteintech) and Secondary antibody anti‐Mouse Alexa‐Fluro 555 ( A31570 , Thermo‐Fischer) and anti‐Rabbit Alexa‐Fluro 488 (A11008, Thermo‐Fischer), TUNEL Assay Kit (25879, cell signaling) and Senescence β‐Galactosidase (SA‐β‐gal) Staining Kit (9860, cell signaling) were purchased.

Techniques: Activation Assay, Immunofluorescence, Analysis, Expressing, Quantitative RT-PCR, Two Tailed Test

Journal: Journal of Biochemical and Molecular Toxicology

Article Title: Renal Tubular Epithelial Cells Senescence Induced by 1‐Aminopyrene Activates Fibroblast via BRD4 Driven Senescence‐Associated Secretory Phenotype Signaling

doi: 10.1002/jbt.71124

Figure Lengend Snippet: BRD4 recruitment in RTECs sustains SASP transcription and paracrine fibroblast activation. (A) Representative images and quantification of immunofluorescence analysis of BRD4 expression in RTECs following 1‐AP treatment ± JQ1 (0.5 μM) after 48 h. Scale bars, 20 µm. (B) Heatmap for RT‐qPCR analysis of senescence regulators ( p16, p21, p53 ) and SASP genes ( IL‐1β, IL‐6, IL‐8, MCP‐1, TGF‐β1, CXCL1 ) in RTECs following 1‐AP treatment ± JQ1 after 48 h. Conditioned media (CM) transfer from NRK‐52E/RTECs treated with 1‐AP ± JQ1 to NRK‐52F fibroblasts after 48 h showing (C) Heatmap for RT‐qPCR analysis of fibroblast activation and extracellular matrix genes ( α‐SMA, POSTN, Col1a2, TIMP‐1, CTGF ), (D–F) Quantification of immunofluorescence analysis of α‐SMA, POSTN and COL1A1 in NRK52F fibroblasts. Data are presented as mean ± SD, n = 3. Statistical analysis was performed using two‐tailed unpaired Student's t‐test. Exact p ‐values are indicated in the graphs; ns, not significant. DAPI used as nuclear counterstain.

Article Snippet: Primary antibodies for AhR antibody (182705‐10 G, Meck), Phospho‐Histone H2A.X (Ser139) Antibody (20E3, cell signaling), BRD4 antibody (ER1901‐02, Thermo‐Fischer), Ki‐67 antibody (27309‐1‐AP, proteintech), p21 (2947, cell signaling), α‐SMA antibody (14395‐1‐AP, proteintech), periostin (19899‐1‐AP, proteintech), COL1A1 antibody (55131‐1‐AP, proteintech), Histone H3K9/14ac (Acetyl H3K9/14) Antibody (A‐4021‐025, epigentek), Histone H3K27me3 (H3K27 Trimethyl) Antibody (A‐4039‐025, epigentek), Histone H3K9me3 (H3K9 Trimethyl) Antibody (A‐4036‐025, epigentek), IL‐6 (21865‐1‐AP, proteintech), TGF‐beta1 (21898‐1‐AP, proteintech) and Secondary antibody anti‐Mouse Alexa‐Fluro 555 ( A31570 , Thermo‐Fischer) and anti‐Rabbit Alexa‐Fluro 488 (A11008, Thermo‐Fischer), TUNEL Assay Kit (25879, cell signaling) and Senescence β‐Galactosidase (SA‐β‐gal) Staining Kit (9860, cell signaling) were purchased.

Techniques: Activation Assay, Immunofluorescence, Analysis, Expressing, Quantitative RT-PCR, Two Tailed Test