mouse monoclonal anti il 8  (R&D Systems)

Journal: bioRxiv

Article Title: Epithelial cytokeratin 6a restricts secretory autophagy of proinflammatory cytokines by interacting with Sec16A

doi: 10.1101/2024.01.04.574264

Figure Lengend Snippet: ( A-B ) hTCEpi cells transfected with non-targeting siRNA (siNTC) and K6a-specific siRNA (siK6a) were exposed to 20% sterile P. aeruginosa PAO1 culture supernatant or 20% tryptic soy broth (TSB) in KGM-2 media for 18-20 h. Cell culture supernatants were collected for cytokine and chemokine quantification (n = 3 independent experiments). ( A ) Multiplex assay was performed; data normalized to baseline control (siNTC+TSB) is represented by a heat map with gradient color bars showing fold changes. ( B ) ELISA of IL-1α, IL-8, CXCL1 and CCL20 (mean ± SD). Statistical significance was determined by a two-tailed unpaired t-test. *P < 0.05, **P < 0.01, ***P < 0.001; ns, P > 0.05. ( C ) Anti-Flag immunoprecipitation of whole-cell lysate from hTCEpi cells expressing HA-K6a-Flag followed by LC-MS/MS and pathway analysis of identified proteins. Bar graph representation shows each enriched pathway with the number of identified proteins and p-value. ( D-E ) LC-MS/MS identification of Sec16A was verified by anti-HA immunoprecipitation of whole-cell lysate from hTCEpi cells expressing HA-K6a-Flag. ( D ) Eluted proteins were analyzed by SDS-PAGE and Colloidal Blue staining. The arrow indicates the band of HA-K6a-Flag. ( E ) Immunoblotting of eluted proteins confirmed Sec16A as an interacting protein of K6a.

Article Snippet: For immunostaining, rabbit polyclonal anti-LC3 [GTX127375] (GeneTex) (1:250), mouse monoclonal anti-IL-8 [O-IL8-15] (Abcam) (1:100), rabbit polyclonal anti-Sec16A (cat. no. A300-648A, Bethyl Laboratories) (1:250), and mouse monoclonal anti-GORASP2/GRASP55 [1C9A3] (Proteintech) (1:500) were used to incubate cells for 2-4 hours at room temperature.

Techniques: Transfection, Sterility, Cell Culture, Multiplex Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Immunoprecipitation, Expressing, Liquid Chromatography with Mass Spectroscopy, SDS Page, Staining, Western Blot

Journal: bioRxiv

Article Title: Epithelial cytokeratin 6a restricts secretory autophagy of proinflammatory cytokines by interacting with Sec16A

doi: 10.1101/2024.01.04.574264

Figure Lengend Snippet: (A-D) hTCEpi cells transfected with non-targeting siRNA (siNTC) and K6a-specific siRNA (siK6a) were exposed to 20% sterile P. aeruginosa PAO1 culture supernatant or 20% tryptic soy broth (TSB) for 18-20 h. (A) Representative images of confocal immunofluorescence microscopy using anti-LC3 antibody. scale bar = 25µm. LC3-II puncta/cell was quantified by ImageJ. Data (median with interquartile range) was pooled from 3 independent experiments with 2-3 fields per experiment and 10-20 cells per field (totaling 88-95 cells per group). The Kruskal-Wallis with Dunn’s multiple comparisons test was used for statistical analysis. (B-D) hTCEpi cells were stimulated in the presence of Bafilomycin A (BafA1, 100nM) or DMSO (vehicle control). (B) The levels of LC3 and p62 were detected by immunoblotting. Integrated densities of (C) LC3-II and (D) p62 protein bands were quantified by ImageJ and normalized to β-actin. Data is shown as mean ± SD from three independent experiments (n=3). Two-tailed unpaired t-test was used for statistical analysis. (E-I) hTCEpi cells were subjected to transfection with NTC and K6a siRNA, followed by transduction with baculovirus carrying RFP-GFP-LC3-II tandem probe at a multiplicity of infection (MOI) of 1:80 for 6 hours. Subsequently, cells were treated with 20% TSB or 20% sterile PAO1 culture supernatant for 24 h before imaging by confocal microscopy. RFP (red) and GFP (green) puncta of each cell were quantified by ImageJ. Data was pooled from two independent experiments and represented as median and interquartile range. Statistical significance was determined by Wilcoxon matched-pairs test. ( J) Co-localization of LC3-II and IL-8. K6a was knocked down by siRNA in hTCEpi cells and stimulated with 20% sterile PAO1 culture supernatant for 20 h. Cell were fixed and stained with anti-LC3 and IL-8 antibodies. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns: P > 0.05.

Article Snippet: For immunostaining, rabbit polyclonal anti-LC3 [GTX127375] (GeneTex) (1:250), mouse monoclonal anti-IL-8 [O-IL8-15] (Abcam) (1:100), rabbit polyclonal anti-Sec16A (cat. no. A300-648A, Bethyl Laboratories) (1:250), and mouse monoclonal anti-GORASP2/GRASP55 [1C9A3] (Proteintech) (1:500) were used to incubate cells for 2-4 hours at room temperature.

Techniques: Transfection, Sterility, Immunofluorescence, Microscopy, Western Blot, Two Tailed Test, Transduction, Infection, Imaging, Confocal Microscopy, Staining

Journal: bioRxiv

Article Title: Epithelial cytokeratin 6a restricts secretory autophagy of proinflammatory cytokines by interacting with Sec16A

doi: 10.1101/2024.01.04.574264

Figure Lengend Snippet: hTCEpi cells transfected with non-targeting siRNA, or K6a– and ATG5-specific siRNA (alone or in combination), were exposed to 20% sterile P. aeruginosa PAO1 culture supernatant or 20% tryptic soy broth (TSB) in KGM-2 media for 18-20 h. Cells were fixed and stained with anti-LC3 antibody, and cell culture supernatants were collected for ELISA. (A-B) Representative images of confocal microscopy and Image J quantification of LC3-II puncta/cell. scale bar=25µm. Data (median and interquartile range) were pooled from 2-3 independent experiments, with a minimum of 2-3 fields per experiment and 10-20 cells per field. Statistical analysis was performed using Kruskal-Wallis with Dunn’s multiple comparisons test. (C) Secreted IL-8 in cell culture supernatants were quantified by ELISA. Data are shown as mean ± SD of three independent experiments. Statistical significance was determined by one-way ANOVA with Sidak’s multiple comparisons test. **P < 0.01, ***P < 0.001, ****P < 0.0001, ns: P > 0.05.

Article Snippet: For immunostaining, rabbit polyclonal anti-LC3 [GTX127375] (GeneTex) (1:250), mouse monoclonal anti-IL-8 [O-IL8-15] (Abcam) (1:100), rabbit polyclonal anti-Sec16A (cat. no. A300-648A, Bethyl Laboratories) (1:250), and mouse monoclonal anti-GORASP2/GRASP55 [1C9A3] (Proteintech) (1:500) were used to incubate cells for 2-4 hours at room temperature.

Techniques: Transfection, Sterility, Staining, Cell Culture, Enzyme-linked Immunosorbent Assay, Confocal Microscopy

Journal: bioRxiv

Article Title: Epithelial cytokeratin 6a restricts secretory autophagy of proinflammatory cytokines by interacting with Sec16A

doi: 10.1101/2024.01.04.574264

Figure Lengend Snippet: (A-C) hTCEpi cells transfected with non-targeting siRNA (siNTC) and K6a-specific siRNA (siK6a) were exposed to KGM-2 containing 20% tryptic soy broth (TSB), or 20% sterile P. aeruginosa PAO1 culture supernatant for 18-20 h. (A-B) Sec16A was immuno-stained and visualized by confocal microscopy. Scale bar=25µm. Fluorescence intensity of Sec16A was measured by ImageJ. Data (mean ± SD) from two independent experiments was analyzed by two-tailed t-test. ( C) Western blots showed the levels of Sec16A and LC3-II in siNTC or siK6a-transfected cells under basal and inflammatory conditions. (D-H) hTCEpi cells were subjected to transfection with non-targeting siRNA, or K6a– and Sec16A-specific siRNA (alone or in combination) prior to stimulation with 20% TSB or 20% PAO1 for 18-20 hours. Cells were fixed and stained with anti-LC3 antibody. (D-E) Representative confocal microscopy images, scale bar=25µm. (F-G) Image J quantification of LC3-II puncta per cell. Data (median with interquartile range) were pooled from three independent experiments (n=3). For each experiment, three fields, each containing 10-20 cells, were analyzed by Kruskal-Wallis with Dunn’s multiple comparisons test. (H) Secreted IL-8 in cell culture supernatants were quantified by ELISA. Data is shown as mean ± SD of three independent experiments. Statistical significance was determined by one-way ANOVA with Sidak’s multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns: P > 0.05.

Article Snippet: For immunostaining, rabbit polyclonal anti-LC3 [GTX127375] (GeneTex) (1:250), mouse monoclonal anti-IL-8 [O-IL8-15] (Abcam) (1:100), rabbit polyclonal anti-Sec16A (cat. no. A300-648A, Bethyl Laboratories) (1:250), and mouse monoclonal anti-GORASP2/GRASP55 [1C9A3] (Proteintech) (1:500) were used to incubate cells for 2-4 hours at room temperature.

Techniques: Transfection, Sterility, Staining, Confocal Microscopy, Fluorescence, Two Tailed Test, Western Blot, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Epithelial cytokeratin 6a restricts secretory autophagy of proinflammatory cytokines by interacting with Sec16A

doi: 10.1101/2024.01.04.574264

Figure Lengend Snippet: (A-E) hTCEpi cells transfected with non-targeting siRNA (siNTC) and K6a-specific siRNA (siK6a) were exposed to KGM-2 containing 20% tryptic soy broth (TSB), or 20% sterile P. aeruginosa PAO1 culture supernatant for 18-20 hours. (A-B) GRASP55 was stained and visualized by confocal microscopy. Scale bar=25µm. Fluorescence intensity of GRASP55 was measured by ImageJ. Data (mean ± SD) from two independent experiments was analyzed by two-tailed t-test. (C) GRASP55 and LC3-II protein levels were detected by western blotting. GRASP55 band intensities were measured by ImageJ and normalized to β-actin. Data from two independent experiments was analyzed by one-way ANOVA. (D-E) Co-localization of LC3-II and GRASP55 was examined by confocal immunofluorescence microscopy. (D) Representative images and (E) ImageJ Coloc2 analysis of LC3-II and GRASP55 staining. Pearson correlation coefficients (mean ± SD) were computed from three independent experiments, and compared between groups using Welch ANOVA with Dunnett’s T3 multiple comparisons test. (F-J) hTCEpi cells underwent transfection with non-targeting siRNA, or K6a– and GRASP55-specific siRNA (alone or in combination) prior to stimulation with 20% TSB or 20% sterile PAO1 culture supernatant for 18-20 h. Representative confocal microscopy images of LC3-II puncta and ImageJ quantification under (F-G) basal condition or (H-I) inflammatory condition. scale bar = 25µm. LC3-II puncta/cell (median with interquartile range) was quantified by pooling data from three independent experiments with 2-3 fields per experiment and 10-20 cells per field. Statistical significance was determined by Kruskal-Wallis with Dunn’s multiple comparisons test. (J) Concentration of secreted IL-8 was assessed using ELISA. Data was pooled from four independent experiments and shown as mean ± SD. Statistical significance was determined by one-way ANOVA with Sidak’s multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns: P > 0.05.

Article Snippet: For immunostaining, rabbit polyclonal anti-LC3 [GTX127375] (GeneTex) (1:250), mouse monoclonal anti-IL-8 [O-IL8-15] (Abcam) (1:100), rabbit polyclonal anti-Sec16A (cat. no. A300-648A, Bethyl Laboratories) (1:250), and mouse monoclonal anti-GORASP2/GRASP55 [1C9A3] (Proteintech) (1:500) were used to incubate cells for 2-4 hours at room temperature.

Techniques: Transfection, Sterility, Staining, Confocal Microscopy, Fluorescence, Two Tailed Test, Western Blot, Immunofluorescence, Microscopy, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Epithelial cytokeratin 6a restricts secretory autophagy of proinflammatory cytokines by interacting with Sec16A

doi: 10.1101/2024.01.04.574264

Figure Lengend Snippet: hTCEpi cells were transfected with non-targeting siRNA, or K6a-specific siRNA alone or in combination with (A) Rab8a, or (B) Rab8b, prior to stimulation with 20% TSB or 20% sterile PAO1 culture supernatant for 18-20 h. Concentration of secreted IL-8 in culture supernatants was assessed using ELISA. Data was pooled from three independent experiments and shown as mean ± SD. Statistical significance was assessed using one-way ANOVA with Sidak’s multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001, ns: P > 0.05.

Article Snippet: For immunostaining, rabbit polyclonal anti-LC3 [GTX127375] (GeneTex) (1:250), mouse monoclonal anti-IL-8 [O-IL8-15] (Abcam) (1:100), rabbit polyclonal anti-Sec16A (cat. no. A300-648A, Bethyl Laboratories) (1:250), and mouse monoclonal anti-GORASP2/GRASP55 [1C9A3] (Proteintech) (1:500) were used to incubate cells for 2-4 hours at room temperature.

Techniques: Transfection, Sterility, Concentration Assay, Enzyme-linked Immunosorbent Assay