Journal: Journal of Biological Chemistry

Article Title: A New Determinant of Endoplasmic Reticulum Localization Is Contained in the Juxtamembrane Region of the Ectodomain of Hepatitis C Virus Glycoprotein E1

doi: 10.1074/jbc.m910400199

Figure Lengend Snippet: FIG. 2. The C-terminal region 99– 192 of HCV E1 protein determines lo- calization in the ER of the reporter CD8. FRT cells stably expressing CD8 (a–b) or CD8-E199–192 (c–f) were analyzed by double indirect immunofluorescence microscopy as detailed under “Experi- mental Procedures” and “Results.” a, c, e, and f, permeabilized cells; b and d, not permeabilized cells. a, c, and e, anti-CD8 mAb OKT8; b and d, polyclonal antibody anti-CD8; f, polyclonal antibody anti- SSRa; a, c, and e, Texas Red-conjugated anti-mouse secondary antibody; b, d, and f, fluorescein-conjugated anti-rabbit sec- ondary antibody.

Article Snippet: The following antibodies were used: mouse mAb OKT8 (anti-CD8 protein), mouse mAb N1 (anti-CD8 protein), rabbit polyclonal anti-CD8 and rabbit polyclonal anti-SSra (16); mouse mAb G10-1 (anti-CD8 protein) (24)2; rabbit polyclonal anti-calnexin (17); rabbit polyclonal anti-calreticulin (Stress-Gene, Canada); rabbit polyclonal anti-alkaline phosphatase (Rockland, Gilbertsville, PA); mouse mAb anti-placental alkaline phosphatase (Chemicon International, Inc., Temecula, CA); peroxidaseconjugated anti-mouse and anti-rabbit IgG (Sigma-Aldrich); Texas Redconjugated anti-mouse IgG and fluorescein-conjugated anti-rabbit IgG (Jackson Immunoresearch Lab, West Grove, PA).

Techniques: Stable Transfection, Expressing, Immunofluorescence, Microscopy

Journal: Journal of Biological Chemistry

Article Title: A New Determinant of Endoplasmic Reticulum Localization Is Contained in the Juxtamembrane Region of the Ectodomain of Hepatitis C Virus Glycoprotein E1

doi: 10.1074/jbc.m910400199

Figure Lengend Snippet: FIG. 6. The juxtamembrane region 99–142 of the ectodomain of HCV E1 protein contains another determi- nant for ER localization. FRT cells sta- bly expressing CD8-E199–142 (a–d) and CD8-SV (e–f) were analyzed by double in- direct immunofluorescence microscopy as detailed under “Experimental Proce- dures” and “Results.” a, c–e, permeabi- lized cells; b and f, non-permeabilized cells. a, c, and e, anti-CD8 OKT8 mAb; b and f, polyclonal antibody anti-CD8; d, polyclonal antibody anti-calnexin; a, c, and e, Texas Red-conjugated anti-mouse secondary antibody; b, d, and f, fluores- cein-conjugated anti-rabbit secondary antibody.

Article Snippet: The following antibodies were used: mouse mAb OKT8 (anti-CD8 protein), mouse mAb N1 (anti-CD8 protein), rabbit polyclonal anti-CD8 and rabbit polyclonal anti-SSra (16); mouse mAb G10-1 (anti-CD8 protein) (24)2; rabbit polyclonal anti-calnexin (17); rabbit polyclonal anti-calreticulin (Stress-Gene, Canada); rabbit polyclonal anti-alkaline phosphatase (Rockland, Gilbertsville, PA); mouse mAb anti-placental alkaline phosphatase (Chemicon International, Inc., Temecula, CA); peroxidaseconjugated anti-mouse and anti-rabbit IgG (Sigma-Aldrich); Texas Redconjugated anti-mouse IgG and fluorescein-conjugated anti-rabbit IgG (Jackson Immunoresearch Lab, West Grove, PA).

Techniques: Expressing, Immunofluorescence, Microscopy

Journal: Journal of Biological Chemistry

Article Title: A New Determinant of Endoplasmic Reticulum Localization Is Contained in the Juxtamembrane Region of the Ectodomain of Hepatitis C Virus Glycoprotein E1

doi: 10.1074/jbc.m910400199

Figure Lengend Snippet: FIG. 5. The TMD region of HCV E1 protein determines localization in the ER of the reporter CD8. FRT cells stably expressing CD8-E1155–192 were analyzed by double indirect immunofluorescence microscopy as detailed under “Experimental Procedures” and in the Results. a, c, and d, permeabilized cells; b, non-permeabilized cells. a and c, anti-CD8 mAb OKT8; b, polyclonal antibody anti-CD8; d, polyclonal antibody anti-calnexin; a and c, Texas red-conjugated anti-mouse secondary antibody; b and d, fluorescein-conjugated anti-rabbit secondary antibody.

Article Snippet: The following antibodies were used: mouse mAb OKT8 (anti-CD8 protein), mouse mAb N1 (anti-CD8 protein), rabbit polyclonal anti-CD8 and rabbit polyclonal anti-SSra (16); mouse mAb G10-1 (anti-CD8 protein) (24)2; rabbit polyclonal anti-calnexin (17); rabbit polyclonal anti-calreticulin (Stress-Gene, Canada); rabbit polyclonal anti-alkaline phosphatase (Rockland, Gilbertsville, PA); mouse mAb anti-placental alkaline phosphatase (Chemicon International, Inc., Temecula, CA); peroxidaseconjugated anti-mouse and anti-rabbit IgG (Sigma-Aldrich); Texas Redconjugated anti-mouse IgG and fluorescein-conjugated anti-rabbit IgG (Jackson Immunoresearch Lab, West Grove, PA).

Techniques: Stable Transfection, Expressing, Immunofluorescence, Microscopy

Journal: Journal of Biological Chemistry

Article Title: A New Determinant of Endoplasmic Reticulum Localization Is Contained in the Juxtamembrane Region of the Ectodomain of Hepatitis C Virus Glycoprotein E1

doi: 10.1074/jbc.m910400199

Figure Lengend Snippet: FIG. 9. CD8-E199–142M protein is ex- pressed on the plasma membrane. HuH-7 cells were transfected with plas- mids expressing CD8-SV (a and b), CD8- E199–142 (c and d), and CD8-E199–142M (e and f) proteins. 36 h post-transfection, the cells were analyzed by single (a and b) and double (c and f) indirect immunoflu- orescence microscopy as detailed under “Experimental Procedures.” a, c, and e, permeabilized cells; b, d, and f, not per- meabilized cells; a–c and e, anti-CD8 mAb OKT8, Texas Red-conjugated anti-mouse secondary antibody; d and f, anti-CD8 polyclonal antibody, fluorescein-conju- gated anti-rabbit secondary antibody.

Article Snippet: The following antibodies were used: mouse mAb OKT8 (anti-CD8 protein), mouse mAb N1 (anti-CD8 protein), rabbit polyclonal anti-CD8 and rabbit polyclonal anti-SSra (16); mouse mAb G10-1 (anti-CD8 protein) (24)2; rabbit polyclonal anti-calnexin (17); rabbit polyclonal anti-calreticulin (Stress-Gene, Canada); rabbit polyclonal anti-alkaline phosphatase (Rockland, Gilbertsville, PA); mouse mAb anti-placental alkaline phosphatase (Chemicon International, Inc., Temecula, CA); peroxidaseconjugated anti-mouse and anti-rabbit IgG (Sigma-Aldrich); Texas Redconjugated anti-mouse IgG and fluorescein-conjugated anti-rabbit IgG (Jackson Immunoresearch Lab, West Grove, PA).

Techniques: Clinical Proteomics, Membrane, Transfection, Expressing, Microscopy

Journal: Journal of Biological Chemistry

Article Title: A New Determinant of Endoplasmic Reticulum Localization Is Contained in the Juxtamembrane Region of the Ectodomain of Hepatitis C Virus Glycoprotein E1

doi: 10.1074/jbc.m910400199

Figure Lengend Snippet: FIG. 12. The juxtamembrane region 99–142 of the ectodomain of HCV E1 determines intracellular localization of the AP protein. HuH-7 cells were transfected with plasmids expressing AP8 (a and b), AP8-SV (c and d), AP8- E199–142 (e and f), and AP8-E199–142M (g and h) proteins. 36 h post-transfection, the cells were analyzed by double indirect immunofluorescence microscopy as de- tailed under “Experimental Procedures.” a, c, e, and g, permeabilized cells, anti-AP mAb, Texas red-conjugated anti-mouse secondary antibody; b, d, f, and h, not permeabilized cells, anti-AP polyclonal antibody, fluorescein-conjugated anti- rabbit secondary antibody.

Article Snippet: The following antibodies were used: mouse mAb OKT8 (anti-CD8 protein), mouse mAb N1 (anti-CD8 protein), rabbit polyclonal anti-CD8 and rabbit polyclonal anti-SSra (16); mouse mAb G10-1 (anti-CD8 protein) (24)2; rabbit polyclonal anti-calnexin (17); rabbit polyclonal anti-calreticulin (Stress-Gene, Canada); rabbit polyclonal anti-alkaline phosphatase (Rockland, Gilbertsville, PA); mouse mAb anti-placental alkaline phosphatase (Chemicon International, Inc., Temecula, CA); peroxidaseconjugated anti-mouse and anti-rabbit IgG (Sigma-Aldrich); Texas Redconjugated anti-mouse IgG and fluorescein-conjugated anti-rabbit IgG (Jackson Immunoresearch Lab, West Grove, PA).

Techniques: Transfection, Expressing, Immunofluorescence, Microscopy

Journal: International Journal of Biological Sciences

Article Title: NAT10 Promotes Tubular Epithelial Cell Senescence in Cisplatin-Induced Acute Kidney Injury by Regulating DDX17

doi: 10.7150/ijbs.127909

Figure Lengend Snippet: NAT10 expression was increased in Cisplatin-induced AKI. ( A and B) Serum creatinine (Scr) (A) and blood urea nitrogen (BUN) (B) in mice treated with Cisplatin for 0, 1, 2 and 3 days (n=6). (C-E) Representative western blot images and quantification of NAT10 and NGAL in the kidney cortex (n=6). (F) qPCR analysis of Nat10 in the kidney cortex (n=6). (G) Representative immunohistochemical staining images of NAT10 in the kidney of control and Cisplatin-treated mice. Scale bar: 50 µm. (H) Representative immunofluorescence (IF) co-staining images of NAT10 and lotus tetragonolobus lectin (LTL) in the kidney. Scale bar: 25 µm. (I) qPCR analysis of NAT10 in HK-2 cells treated with 0, 5, 10 and 20 μM Cisplatin (n=3). (J and K) Representative western blot images and quantification of NAT10 and KIM-1 in HK-2 cells (n=3). (L) qPCR analysis of NAT10 in HK-2 cells exposed to 20 μM Cisplatin for 0, 6, 12 and 24 hours (n=3). (M and N) Representative western blot images and quantification of NAT10 and KIM-1 in HK-2 cells (n=3). (O) Representative IF staining images of NAT10 in control and Cisplatin-treated HK-2 cells. Scale bar: 20 µm. Data were mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001. ns, not significant. One-way ANOVA followed by Tukey's post-test (A, B, D-F, I, K, L and N).

Article Snippet: The primary antibodies for NAT10 (1:200, Proteintech), NGAL (1:200, Abcam), γ-H2A.X (1:200, Cell Signaling Technology) and IL-1β (1:200, 12242, Cell Signaling Technology) were used.

Techniques: Expressing, Western Blot, Immunohistochemical staining, Staining, Control, Immunofluorescence

Journal: International Journal of Biological Sciences

Article Title: NAT10 Promotes Tubular Epithelial Cell Senescence in Cisplatin-Induced Acute Kidney Injury by Regulating DDX17

doi: 10.7150/ijbs.127909

Figure Lengend Snippet: Lentivirus mediated NAT10 knockdown attenuated Cisplatin-induced AKI. ( A) qPCR analysis of LCN2 in Cisplatin treated HK-2 cells transfected with negative control lentivirus (shNC) or NAT10 knockdown lentivirus (shNAT10) (n=3). (B and C) Representative western blot images and quantification of NAT10 and KIM-1 in HK-2 cells (n=3). (D and E) Serum creatinine (Scr) (D) and blood urea nitrogen (BUN) (E) in Cisplatin-induced mice with kidney cortex injection of shNC or shNAT10 lentivirus (n=6). (F) qPCR analysis of Lcn2 in the kidney cortex (n=6). (G and H) Representative western blot images and quantification of NAT10, GFP and NGAL in the kidney cortex (n=6). (I) Representative hematoxylin and eosin (H&E) staining images (upper panel), immunohistochemical staining images of NGAL (middle panel) and immunofluorescence staining images of F4/80 (lower panel) in the kidney. Scale bar: 50 µm. (J) Tubular injury scores were calculated according to H&E staining images (n=6). (K) The percentages of NGAL positive area were calculated (n=6). (L) The percentages of F4/80 positive area were calculated (n=6). Data were mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001. One-way ANOVA followed by Tukey's post-test (A, C-F, H and J-L).

Article Snippet: The primary antibodies for NAT10 (1:200, Proteintech), NGAL (1:200, Abcam), γ-H2A.X (1:200, Cell Signaling Technology) and IL-1β (1:200, 12242, Cell Signaling Technology) were used.

Techniques: Knockdown, Transfection, Negative Control, Western Blot, Injection, Staining, Immunohistochemical staining, Immunofluorescence

Journal: International Journal of Biological Sciences

Article Title: NAT10 Promotes Tubular Epithelial Cell Senescence in Cisplatin-Induced Acute Kidney Injury by Regulating DDX17

doi: 10.7150/ijbs.127909

Figure Lengend Snippet: Treatment with NAT10 inhibitor Remodelin alleviated Cisplatin-induced AKI. (A) qPCR analysis of LCN2 in Cisplatin-induced HK-2 cells treated with DMSO or Remodelin (n=3). (B and C) Representative western blot images and quantification of KIM-1 in HK-2 cells (n=3). (D) Schematic diagram of Remodelin treatment in Cisplatin-induced AKI. (E and F) Serum creatinine (Scr) (E) and blood urea nitrogen (BUN) (F) in mice (n=6). (G) qPCR analysis of Lcn2 in the kidney cortex (n=6). (H and I) Representative western blot images and quantification of NAT10 and NGAL in the kidney cortex (n=6). (J) Representative hematoxylin and eosin (H&E) staining images (upper panel) and immunohistochemical staining images of NGAL (lower panel) in the kidney. Scale bar: 50 µm. (K) Tubular injury scores were calculated according to H&E staining images (n=6). (L) The percentages of NGAL positive area were calculated (n=6). Data were mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001. One-way ANOVA followed by Tukey's post-test (A, C, E-G, I, K and L).

Article Snippet: The primary antibodies for NAT10 (1:200, Proteintech), NGAL (1:200, Abcam), γ-H2A.X (1:200, Cell Signaling Technology) and IL-1β (1:200, 12242, Cell Signaling Technology) were used.

Techniques: Western Blot, Staining, Immunohistochemical staining

Journal: International Journal of Biological Sciences

Article Title: NAT10 Promotes Tubular Epithelial Cell Senescence in Cisplatin-Induced Acute Kidney Injury by Regulating DDX17

doi: 10.7150/ijbs.127909

Figure Lengend Snippet: NAT10 accelerated Cisplatin-induced cellular senescence in HK-2 cells. (A) A volcano plot of differentially expressed genes (DEGs) in Cisplatin-induced HK-2 cells transfected with negative control siRNA or NAT10 siRNA. (B) Heatmap of DEGs associated with senescence. (C) GO enrichment analysis of DEGs. (D and E) Representative western blot images and quantification of p53, p16 and γ-H2A.X in Cisplatin treated HK-2 cells transfected with negative control lentivirus (shNC) or NAT10 knockdown lentivirus (shNAT10) (n=3). (F) qPCR analysis of CDKN1A , CDKN2A , TP53 , IL1B , IL6 , IL8 and TNF in HK-2 cells (n=3). (G) Representative SA-β-gal staining images (upper panel) and immunofluorescence staining images of γ-H2A.X (lower panel) in HK-2 cells. Scale bar: 50 µm (upper panel) and 20 µm (lower panel). (H) The percentages of SA-β-gal positive cells were calculated (n=3). (I) The percentages of γ-H2A.X positive cells were calculated (n=3). Data were mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001. One-way ANOVA followed by Tukey's post-test (E, F, H and I).

Article Snippet: The primary antibodies for NAT10 (1:200, Proteintech), NGAL (1:200, Abcam), γ-H2A.X (1:200, Cell Signaling Technology) and IL-1β (1:200, 12242, Cell Signaling Technology) were used.

Techniques: Transfection, Negative Control, Western Blot, Knockdown, Staining, Immunofluorescence

Journal: International Journal of Biological Sciences

Article Title: NAT10 Promotes Tubular Epithelial Cell Senescence in Cisplatin-Induced Acute Kidney Injury by Regulating DDX17

doi: 10.7150/ijbs.127909

Figure Lengend Snippet: NAT10 knockdown improved cellular senescence in the kidney of Cisplatin-induced AKI. (A-E) qPCR analysis of Cdkn1a (A), Tp53 (B), Il1b (C), Il6 (D) and Tnf (E) in the kidney cortex of Cisplatin-induced mice with kidney cortex injection of negative control lentivirus (shNC) or NAT10 knockdown lentivirus (shNAT10) (n=6). (F and G) Representative western blot images and quantification of p53, p21 and γ-H2A.X in the kidney cortex (n=6). (H) Representative immunohistochemical staining images of γ-H2A.X (upper panel) and IL-1β (middle panel), and SA-β-gal staining images (lower panel) in the kidney. Scale bar: 50 µm. (I) The percentages of γ-H2A.X positive nuclei were calculated (n=6). (J) The percentages of IL-1β positive area were calculated (n=6). (K) The percentages of SA-β-gal positive area were calculated (n=6). (L) Representative DHE staining images of the kidney. Data were mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001. One-way ANOVA followed by Tukey's post-test (A-E, G and I-K).

Article Snippet: The primary antibodies for NAT10 (1:200, Proteintech), NGAL (1:200, Abcam), γ-H2A.X (1:200, Cell Signaling Technology) and IL-1β (1:200, 12242, Cell Signaling Technology) were used.

Techniques: Knockdown, Injection, Negative Control, Western Blot, Immunohistochemical staining, Staining

Journal: International Journal of Biological Sciences

Article Title: NAT10 Promotes Tubular Epithelial Cell Senescence in Cisplatin-Induced Acute Kidney Injury by Regulating DDX17

doi: 10.7150/ijbs.127909

Figure Lengend Snippet: Treatment with NAT10 inhibitor Remodelin attenuated cellular senescence in HK-2 cells and the kidney of Cisplatin-induced AKI. (A) qPCR analysis of CDKN1A , CDKN2A , TP53 , IL6 , IL8 and TNF in Cisplatin-induced HK-2 cells treated with DMSO or Remodelin (n=3). (B and C) Representative western blot images and quantification of p53, p16 and γ-H2A.X in HK-2 cells (n=3). (D) Representative SA-β-gal staining images (upper panel) and immunofluorescence staining images of γ-H2A.X (lower panel) in HK-2 cells. Scale bar: 50 µm. (E) The percentages of SA-β-gal positive cells and γ-H2A.X positive cells were calculated (n=3). (F) qPCR analysis of Cdkn1a , Tp53 , Il1b , Il6 and Tnf in the kidney cortex of Cisplatin-induced mice treated with vehicle or Remodelin (n=6). (G and H) Representative western blot images and quantification of p53, p21 and γ-H2A.X in the kidney cortex (n=6). (I) Representative immunohistochemical staining images of γ-H2A.X (upper panel) and DHE staining images (lower panel) in the kidney. Scale bar: 100 µm. (J) The percentages of γ-H2A.X positive nuclei were calculated (n=6). (K) Relative fluorescence intensity of DHE was calculated (n=6). Data were mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001. One-way ANOVA followed by Tukey's post-test (A, C, E, F, H, J and K).

Article Snippet: The primary antibodies for NAT10 (1:200, Proteintech), NGAL (1:200, Abcam), γ-H2A.X (1:200, Cell Signaling Technology) and IL-1β (1:200, 12242, Cell Signaling Technology) were used.

Techniques: Western Blot, Staining, Immunofluorescence, Immunohistochemical staining, Fluorescence

Journal: International Journal of Biological Sciences

Article Title: NAT10 Promotes Tubular Epithelial Cell Senescence in Cisplatin-Induced Acute Kidney Injury by Regulating DDX17

doi: 10.7150/ijbs.127909

Figure Lengend Snippet: Prevention treatment with NAT10 inhibitor Remodelin attenuated Cisplatin-induced AKI and cellular senescence. (A) Schematic diagram of prevention treatment of Remodelin in Cisplatin-induced AKI. (B) Serum creatinine (Scr) and blood urea nitrogen (BUN) in mice (n=6). (C) qPCR analysis of Lcn2 in the kidney cortex (n=6). (D and E) Representative western blot images and quantification of NGAL in the kidney cortex (n=6). (F) Representative hematoxylin and eosin (H&E) staining images (upper panel) and immunohistochemical (IHC) staining images of NGAL (lower panel) in the kidney. Scale bar: 50 µm. (G) Tubular injury scores were calculated according to H&E staining images (n=6). (H) The percentages of NGAL positive area were calculated (n=6). (I and J) Representative western blot images and quantification of p53, p21 and γ-H2A.X in the kidney cortex (n=6). (K) qPCR analysis of Cdkn1a , Tp53 , Il1b and Il6 in the kidney cortex (n=6). (L) Representative IHC staining images of γ-H2A.X (upper panel) and IL-1β (lower panel). Scale bar: 50 µm. (M) The percentages of γ-H2A.X positive nuclei were calculated (n=6). (N) The percentages of IL-1β positive area were calculated (n=6). Data were mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001. One-way ANOVA followed by Tukey's post-test (B, C, E, G, H, J, K, M and N).

Article Snippet: The primary antibodies for NAT10 (1:200, Proteintech), NGAL (1:200, Abcam), γ-H2A.X (1:200, Cell Signaling Technology) and IL-1β (1:200, 12242, Cell Signaling Technology) were used.

Techniques: Western Blot, Staining, Immunohistochemical staining, Immunohistochemistry

Journal: International Journal of Biological Sciences

Article Title: NAT10 Promotes Tubular Epithelial Cell Senescence in Cisplatin-Induced Acute Kidney Injury by Regulating DDX17

doi: 10.7150/ijbs.127909

Figure Lengend Snippet: NAT10 interacted with DDX17 to regulate its expression. (A) Peptide information of DDX17 identified by mass spectrometry. (B) Co-immunoprecipitation of NAT10 in HK-2 cells. NAT10 and DDX17 were detected by western blot. (C and D) Representative western blot images and quantification of DDX17 in Cisplatin-treated HK-2 cells transfected with negative control lentivirus (shNC) or NAT10 knockdown lentivirus (shNAT10) (n=3). (E and F) Representative western blot images and quantification of DDX17 in Cisplatin-induced HK-2 cells treated with DMSO or Remodelin (n=3). (G and H) Representative western blot images and quantification of DDX17 in the kidney cortex of Cisplatin-induced mice with renal cortex injection of shNC or shNAT10 lentivirus (n=6). (I and J) Representative western blot images and quantification of DDX17 in the kidney cortex of Cisplatin-induced mice treated with vehicle or Remodelin (n=6). (K) Representative western blot images of DDX17 in the kidney cortex of Cisplatin-induced mice with prevention treatment of vehicle or Remodelin. (L) Representative western blot images of DDX17 in Cisplatin-induced HK-2 cells transfected with shNC or shNAT10 lentivirus and then treated with cycloheximide (CHX) for up to 8 hours (n=3). Data were mean ± SEM. *** P < 0.001 and **** P < 0.0001. One-way ANOVA followed by Tukey's post-test (D, F, H and J).

Article Snippet: The primary antibodies for NAT10 (1:200, Proteintech), NGAL (1:200, Abcam), γ-H2A.X (1:200, Cell Signaling Technology) and IL-1β (1:200, 12242, Cell Signaling Technology) were used.

Techniques: Expressing, Mass Spectrometry, Immunoprecipitation, Western Blot, Transfection, Negative Control, Knockdown, Injection

Journal: International Journal of Biological Sciences

Article Title: NAT10 Promotes Tubular Epithelial Cell Senescence in Cisplatin-Induced Acute Kidney Injury by Regulating DDX17

doi: 10.7150/ijbs.127909

Figure Lengend Snippet: NAT10-induced cellular injury and senescence was dependent on DDX17. (A-D) Representative western blot images and quantification of NAT10, DDX17, KIM-1, p53, p16 and γ-H2A.X in Cisplatin-induced HK-2 cells transfected with NAT10 knockdown lentivirus (shNAT10) and DDX17 overexpression plasmids (oeDDX17) (n=3). (E) qPCR analysis of LCN2 , CDKN1A , CDKN2A , TP53 , IL1B , IL8 and TNF in HK-2 cells (n=3). (F) Representative immunofluorescence staining images of γ-H2A.X in HK-2 cells. Scale bar: 25 µm. (G) The percentages of γ-H2A.X positive cells were calculated (n=3). Data were mean ± SEM. * P < 0.05, ** P < 0.01 and *** P < 0.001. ns, not significant. One-way ANOVA followed by Tukey's post-test (B-E and G).

Article Snippet: The primary antibodies for NAT10 (1:200, Proteintech), NGAL (1:200, Abcam), γ-H2A.X (1:200, Cell Signaling Technology) and IL-1β (1:200, 12242, Cell Signaling Technology) were used.

Techniques: Western Blot, Transfection, Knockdown, Over Expression, Immunofluorescence, Staining

Journal: International Journal of Biological Sciences

Article Title: NAT10 Promotes Tubular Epithelial Cell Senescence in Cisplatin-Induced Acute Kidney Injury by Regulating DDX17

doi: 10.7150/ijbs.127909

Figure Lengend Snippet: Schematic diagram illustrating that NAT10 interacted with DDX17 to regulate Cisplatin-induced tubular epithelial cell senescence. DDR, DNA damage response. SA-β-gal, senescence-associated-β-galactosidase.

Article Snippet: The primary antibodies for NAT10 (1:200, Proteintech), NGAL (1:200, Abcam), γ-H2A.X (1:200, Cell Signaling Technology) and IL-1β (1:200, 12242, Cell Signaling Technology) were used.

Techniques:

Journal: Gut Microbes

Article Title: Ultra-processed foods sourced 7-ketositosterol aggravates colitis through gut dysbiosis induced-PDLIM3 activation

doi: 10.1080/19490976.2025.2587980

Figure Lengend Snippet: KS upregulates the expression of PDLIM3 and activates the p38MAPK/NF-κB pathway. (a) GO enrichment analysis of genes significantly upregulated in colon tissues. ( n = 3). (b) Heatmap representation of RNA sequencing data from colon tissues ( n = 3). (c) Relative mRNA level of PDLIM3 ( n = 6). (d) Immunofluorescence staining for PDLIM3 (red) and nuclei by DAPI (blue) in the colon. Scale bar, 20 μm. (e) Protein levels of PDLIM3, p38, p -p38, p65, p -p65, IκBα, and p -IκBα were measured via Western blotting ( n = 6), and GAPDH was used as a loading control. (f) Immunofluorescence staining for PDLIM3 (red) and nuclei by DAPI (blue) in the colon of UC patients with low or high KS intake and correlation analysis between KS intake and fluorescence intensity ( n = 13). GO gene ontology, PDLIM3 PDZ and LIM domain 3. Data are presented as the mean ± SEM; * P < 0.05, ** P < 0.01, *** P < 0.001 by two-tailed one-way ANOVA.

Article Snippet: The membranes were subsequently incubated overnight at 4 °C with primary antibodies against CLND−3 (rabbit anti-mouse; Affinity Biosciences), ZO-1 (rabbit anti-mouse; Proteintech Group), PDLIM3 (rabbit anti-mouse; Proteintech Group), p38 (anti-mouse monoclonal antibody, Affinity Biosciences), p -p38 (rabbit anti-mouse; Affinity Biosciences), p65 (anti-mouse monoclonal antibody, Affinity Biosciences), p -p65 (rabbit anti-mouse; Affinity Biosciences), IκBα (rabbit anti-mouse; Affinity Biosciences), and p -IκBα (rabbit anti-mouse; Affinity Biosciences) and then incubated with HRP-conjugated secondary antibodies (Beyotime Biotechnology).

Techniques: Expressing, RNA Sequencing, Immunofluorescence, Staining, Western Blot, Control, Fluorescence, Two Tailed Test

Journal: Gut Microbes

Article Title: Ultra-processed foods sourced 7-ketositosterol aggravates colitis through gut dysbiosis induced-PDLIM3 activation

doi: 10.1080/19490976.2025.2587980

Figure Lengend Snippet: KS aggravates colitis in mice via the gut microbiota. (a) Schematic illustration of the experimental procedure. Abx: antibiotic cocktail. (b, c) Changes in body weight and DAI score after DSS induction ( n = 6 for the control, KS; n = 8 for the DSS, DSS + KS, and Abx + DSS + KS). (d) Colon length (cm) on day 8 after DSS induction. (e) Representative images of hematoxylin and eosin (H&E)-stained colon sections (left panel) and colon histopathological scores (right panel). ( n = 8) Scale bar, 100 μm. (f) Protein levels of PDLIM3 were measured by Western blotting, and GAPDH was used as a loading control ( n = 4). (g) Schematic illustration of the FMT procedure. Mouse feces from the KS/Control group were transplanted into recipient mice, and then DSS was administered ( n = 5). (h, i) Body weight and DAI score. (j) Colon length (cm) on day 8 after DSS induction. (k) Representative images of hematoxylin and eosin (H&E)-stained colon sections (left panel) and colon histopathological scores (right panel). Scale bar, 100 μm. (l) Protein levels of PDLIM3 were measured by Western blotting ( n = 5). KS 7-ketositosterol, DSS dextran sodium sulfate, FMT fecal microbiota transplantation, PDLIM3 PDZ and LIM domain 3. Data are presented as the mean ± SEM; * P < 0.05, ** P < 0.01, *** P < 0.001, ns not significant by two-tailed one-way ANOVA.

Article Snippet: The membranes were subsequently incubated overnight at 4 °C with primary antibodies against CLND−3 (rabbit anti-mouse; Affinity Biosciences), ZO-1 (rabbit anti-mouse; Proteintech Group), PDLIM3 (rabbit anti-mouse; Proteintech Group), p38 (anti-mouse monoclonal antibody, Affinity Biosciences), p -p38 (rabbit anti-mouse; Affinity Biosciences), p65 (anti-mouse monoclonal antibody, Affinity Biosciences), p -p65 (rabbit anti-mouse; Affinity Biosciences), IκBα (rabbit anti-mouse; Affinity Biosciences), and p -IκBα (rabbit anti-mouse; Affinity Biosciences) and then incubated with HRP-conjugated secondary antibodies (Beyotime Biotechnology).

Techniques: Control, Staining, Western Blot, Transplantation Assay, Two Tailed Test

Journal: Gut Microbes

Article Title: Ultra-processed foods sourced 7-ketositosterol aggravates colitis through gut dysbiosis induced-PDLIM3 activation

doi: 10.1080/19490976.2025.2587980

Figure Lengend Snippet: Staphylococcus lentus exacerbates DSS-induced colitis in mice through interacting with PDLIM3. (a) Experimental design. The mice were administered Staphylococcus lentus before DSS induction ( n = 5 for control, SL; n = 6 for the DSS and DSS + SL). (b, c) Changes in body weight and DAI score. (d) Colon length (cm) on day 10 after DSS induction. (e) Relative mRNA level of IL-1β, IL-6, and TNF- α in colon. (f) Representative images of hematoxylin and eosin (H&E)-stained colon sections (left panel) and colon histopathological scores (right panel). Scale bar, 100 μm. (g) Purification of PDLIM3 and immunoprecipitation analysis of PDLIM3. (h) Predicted docking mode of LPDP and PDLIM3. PDLIM3 is displayed in blue, and LPDP is displayed in green. (i) Microscale thermophoresis analysis of the interaction between PDLIM3 and LPDP. SL Staphylococcus lentus , PDLIM3 PDZ and LIM domain 3, LPDP lysin motif peptidoglycan-binding domain-containing protein, DSS dextran sodium sulfate. Data are presented as the mean ± SEM; * P < 0.05, ** P < 0.01, *** P < 0.001 by two-tailed one-way ANOVA.

Article Snippet: The membranes were subsequently incubated overnight at 4 °C with primary antibodies against CLND−3 (rabbit anti-mouse; Affinity Biosciences), ZO-1 (rabbit anti-mouse; Proteintech Group), PDLIM3 (rabbit anti-mouse; Proteintech Group), p38 (anti-mouse monoclonal antibody, Affinity Biosciences), p -p38 (rabbit anti-mouse; Affinity Biosciences), p65 (anti-mouse monoclonal antibody, Affinity Biosciences), p -p65 (rabbit anti-mouse; Affinity Biosciences), IκBα (rabbit anti-mouse; Affinity Biosciences), and p -IκBα (rabbit anti-mouse; Affinity Biosciences) and then incubated with HRP-conjugated secondary antibodies (Beyotime Biotechnology).

Techniques: Control, Staining, Purification, Immunoprecipitation, Microscale Thermophoresis, Binding Assay, Two Tailed Test

Journal: Gut Microbes

Article Title: Ultra-processed foods sourced 7-ketositosterol aggravates colitis through gut dysbiosis induced-PDLIM3 activation

doi: 10.1080/19490976.2025.2587980

Figure Lengend Snippet: Blockade of the interaction between LysM peptidoglycan-binding domain-containing protein and PDLIM3 by tubuloside B alleviates SL-induced colitis in mice. (a) Molecular docking results of high-throughput screening based on the interaction of LPDP and PDLIM3. (b) Predicted docking model of the three-dimensional structure of tubuloside B and the PDLIM3/LPDP complex. PDLIM3 is displayed in yellow, LysM peptidoglycan-binding domain-containing protein is displayed in blue, and tubuloside B is displayed in green. (c) Microscale thermophoresis results for the binding of LPDP to PDLIM3 in the presence of tubuloside B. (d) The experimental design of DSS colitis model ( n = 5 for the control and Tub B groups; n = 6 for the SL and SL + Tub B). (e, f) Body weight and DAI score. (g) Colon length (cm) on day 8 after DSS induction ( n = 4 for the control, n = 5 for Tub B; n = 4 for SL, n = 5 for SL + Tub B). (h) Representative images of hematoxylin and eosin (H&E)-stained colon sections (left panel) and colon histopathological scores (right panel). Scale bar, 100 μm. LPDP, lysin motif peptidoglycan-binding domain-containing protein; PDLIM3 PDZ and LIM domain 3, DSS dextran sodium sulfate, Tub B tubuloside B. Data are presented as the mean ± SEM; * P < 0.05, ** P < 0.01, *** P < 0.001, ns, not significant by two-tailed one-way ANOVA.

Article Snippet: The membranes were subsequently incubated overnight at 4 °C with primary antibodies against CLND−3 (rabbit anti-mouse; Affinity Biosciences), ZO-1 (rabbit anti-mouse; Proteintech Group), PDLIM3 (rabbit anti-mouse; Proteintech Group), p38 (anti-mouse monoclonal antibody, Affinity Biosciences), p -p38 (rabbit anti-mouse; Affinity Biosciences), p65 (anti-mouse monoclonal antibody, Affinity Biosciences), p -p65 (rabbit anti-mouse; Affinity Biosciences), IκBα (rabbit anti-mouse; Affinity Biosciences), and p -IκBα (rabbit anti-mouse; Affinity Biosciences) and then incubated with HRP-conjugated secondary antibodies (Beyotime Biotechnology).

Techniques: Binding Assay, High Throughput Screening Assay, Microscale Thermophoresis, Control, Staining, Two Tailed Test

Journal: Gut Microbes

Article Title: Ultra-processed foods sourced 7-ketositosterol aggravates colitis through gut dysbiosis induced-PDLIM3 activation

doi: 10.1080/19490976.2025.2587980

Figure Lengend Snippet: Schematic summary of the role of KS-induced colitis. Excessive intake of ultra-processed foods means increased exposure to KS. KS exacerbated DSS-induced colitis in a gut microbiota-dependent manner and resulted in gut dysbiosis, especially increasing the abundance of Staphylococcus lentus . Staphylococcus lentus -derived LPDP could interact with PDLIM3 and activate the p38MAPK/NF-κB signaling pathway, and the binding interfaces could be blocked by tubuloside B, a Chinese herbal extract selected by high-throughput screening, to ameliorate colitis. KS 7-ketositosterol, DSS dextran sodium sulfate, LPDP, lysin motif peptidoglycan-binding domain-containing protein, PDLIM3 PDZ and LIM Domain 3.

Article Snippet: The membranes were subsequently incubated overnight at 4 °C with primary antibodies against CLND−3 (rabbit anti-mouse; Affinity Biosciences), ZO-1 (rabbit anti-mouse; Proteintech Group), PDLIM3 (rabbit anti-mouse; Proteintech Group), p38 (anti-mouse monoclonal antibody, Affinity Biosciences), p -p38 (rabbit anti-mouse; Affinity Biosciences), p65 (anti-mouse monoclonal antibody, Affinity Biosciences), p -p65 (rabbit anti-mouse; Affinity Biosciences), IκBα (rabbit anti-mouse; Affinity Biosciences), and p -IκBα (rabbit anti-mouse; Affinity Biosciences) and then incubated with HRP-conjugated secondary antibodies (Beyotime Biotechnology).

Techniques: Derivative Assay, Binding Assay, High Throughput Screening Assay