Journal: British Journal of Cancer

Article Title: Hypoxia-associated spontaneous pulmonary metastasis in human melanoma xenografts: involvement of microvascular hot spots induced in hypoxic foci by interleukin 8

doi: 10.1038/sj.bjc.6600052

Figure Lengend Snippet: Immunohistochemical preparations of adjacent sections of a D-12 tumour. An avidin-biotin peroxidase-based method was used for staining. Haematoxylin was used for counterstaining. IL-8 positive foci visualized by anti-IL-8 antibody staining ( A ) and hypoxic foci visualized by anti-pimonidazole antibody staining ( B ).

Article Snippet: Membranes were incubated with anti-human VEGF rabbit polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or anti-human IL-8 mouse monoclonal antibody (R&D Systems, Abingdon, UK).

Techniques: Immunohistochemical staining, Avidin-Biotin Assay, Staining

Journal: British Journal of Cancer

Article Title: Hypoxia-associated spontaneous pulmonary metastasis in human melanoma xenografts: involvement of microvascular hot spots induced in hypoxic foci by interleukin 8

doi: 10.1038/sj.bjc.6600052

Figure Lengend Snippet: Hypoxia, IL-8 expression and neovascularization in metastatic and non-metastatic D-12 primary tumours. Points represent single tumours. Density of hypoxic foci ( A ), density of IL-8 positive foci ( B ) and density of vascular hot spots ( C ).

Article Snippet: Membranes were incubated with anti-human VEGF rabbit polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or anti-human IL-8 mouse monoclonal antibody (R&D Systems, Abingdon, UK).

Techniques: Expressing

Journal: British Journal of Cancer

Article Title: Hypoxia-associated spontaneous pulmonary metastasis in human melanoma xenografts: involvement of microvascular hot spots induced in hypoxic foci by interleukin 8

doi: 10.1038/sj.bjc.6600052

Figure Lengend Snippet: Effects of anti-VEGF treatment, anti-IL-8 treatment, and combined anti-VEGF and anti-IL-8 treatment on development of hypoxia, neovascularization and spontaneous pulmonary metastasis in D-12 tumours. ( A ) Percentage of mice with metastasis. Points represent single experiments involving 10 mice each. ( B ) Densities of hypoxic foci and vascular hot spots. Columns represent mean values of 20 mice. Bars represent s.e.m.

Article Snippet: Membranes were incubated with anti-human VEGF rabbit polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or anti-human IL-8 mouse monoclonal antibody (R&D Systems, Abingdon, UK).

Techniques:

Journal:

Article Title: Campylobacter -Induced Interleukin-8 Secretion in Polarized Human Intestinal Epithelial Cells Requires Campylobacter -Secreted Cytolethal Distending Toxin- and Toll-Like Receptor-Mediated Activation of NF-?B ▿

doi: 10.1128/IAI.01317-07

Figure Lengend Snippet: Campylobacter inoculation increased the secretion of IL-8 and TNF-α in polarized human intestinal epithelial T84 cells. T84 cells were cultured on transwells until the transepithelial resistance reached 1,400 Ω/cm2. C. jejuni 81-176 was inoculated from the apical chamber at an MOI of ∼10 and incubated at 37°C for 0, 4, 6, or 24 h. For the 24-h time point, the cells were washed at 6 h to remove unattached bacteria and cultured in fresh medium for another 18 h. No significant difference was detected between the transepithelial resistance before incubation and that after incubation at any time point. The supernatants from the apical and basolateral chambers were collected separately; the bacteria were removed by centrifugation; and protease inhibitors were added to prevent protein degradation. The concentrations of IL-8 (A) and TNF-α (B) were determined by ELISA. Averages for three independent experiments are shown; error bars, standard deviations. Asterisks represent significant (P < 0.05) differences from control cells that were not inoculated with the bacteria.

Article Snippet: Mouse anti-human IL-8 and anti-human TNF-α monoclonal antibodies (BD Bioscience, San Jose, CA) were used as capturing antibodies; biotinylated mouse anti-human IL-8 and anti-human TNF-α monoclonal antibodies (BD Bioscience) were used as detecting antibodies; and streptavidin-alkaline phosphatase (SouthernBiotech Inc., Birmingham, AL) and the substrate p -nitrophenylphosphate disodium (Sigma-Aldrich) were used to visualize bound antibodies.

Techniques: Cell Culture, Incubation, Centrifugation, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: Campylobacter -Induced Interleukin-8 Secretion in Polarized Human Intestinal Epithelial Cells Requires Campylobacter -Secreted Cytolethal Distending Toxin- and Toll-Like Receptor-Mediated Activation of NF-?B ▿

doi: 10.1128/IAI.01317-07

Figure Lengend Snippet: Campylobacter-stimulated IL-8 secretion is not dependent on the secretion of TNF-α. (A) Polarized T84 cells were incubated with different concentrations of TNF-α (0, 5, 10, 50, and 100 ng/ml) basolaterally at 37°C for 24 h. The basolateral media were collected, and the concentrations of IL-8 were measured using ELISA. (B) IL-8 concentrations in the basolateral medium were measured using ELISA at 24 h after T84 cells were incubated under the following conditions: TNF-α (50 ng/ml) applied apically (Apical) (bar I), TNF-α applied basolaterally (Basol) (bar II), (III) TNF-α applied basolaterally plus mouse anti-human TNF-α antibody (5 μg/ml) applied both apically and basolaterally (Both) (bar III), or live C. jejuni 81-176 applied apically plus mouse anti-human TNF-α antibody applied both apically and basolaterally (bar IV). Averages from three independent experiments are shown; error bars, standard deviations.

Article Snippet: Mouse anti-human IL-8 and anti-human TNF-α monoclonal antibodies (BD Bioscience, San Jose, CA) were used as capturing antibodies; biotinylated mouse anti-human IL-8 and anti-human TNF-α monoclonal antibodies (BD Bioscience) were used as detecting antibodies; and streptavidin-alkaline phosphatase (SouthernBiotech Inc., Birmingham, AL) and the substrate p -nitrophenylphosphate disodium (Sigma-Aldrich) were used to visualize bound antibodies.

Techniques: Incubation, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: Campylobacter -Induced Interleukin-8 Secretion in Polarized Human Intestinal Epithelial Cells Requires Campylobacter -Secreted Cytolethal Distending Toxin- and Toll-Like Receptor-Mediated Activation of NF-?B ▿

doi: 10.1128/IAI.01317-07

Figure Lengend Snippet: Comparison of IL-8 secretion induced by different Campylobacter strains inoculated apically or basolaterally. (A and B) Polarized T84 cells were inoculated from either the apical (A) or the basolateral (B) side with chicken meat isolates (C. coli [C] or C. jejuni [J] strains) or with C. jejuni 81-176 or its flaA mutant at an MOI of ∼10 and were incubated for 24 h. At 6 h, T84 cells were washed and placed in fresh medium. No significant difference was detected between the transepithelial resistance before incubation and that after incubation. Uninfected T84 cells were used as controls. The apical and basolateral media were collected separately at 24 h, and the concentrations of IL-8 were measured by ELISA. Averages for three independent experiments are shown; error bars, standard deviations. (C) Pearson's correlation coefficients between IL-8 secretion and Campylobacter adherence, invasion, and transcytosis efficiencies (expressed as percentages of the inocula) were calculated and tested for significance (α = 0.05 by a two-tailed test).

Article Snippet: Mouse anti-human IL-8 and anti-human TNF-α monoclonal antibodies (BD Bioscience, San Jose, CA) were used as capturing antibodies; biotinylated mouse anti-human IL-8 and anti-human TNF-α monoclonal antibodies (BD Bioscience) were used as detecting antibodies; and streptavidin-alkaline phosphatase (SouthernBiotech Inc., Birmingham, AL) and the substrate p -nitrophenylphosphate disodium (Sigma-Aldrich) were used to visualize bound antibodies.

Techniques: Mutagenesis, Incubation, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal:

Article Title: Campylobacter -Induced Interleukin-8 Secretion in Polarized Human Intestinal Epithelial Cells Requires Campylobacter -Secreted Cytolethal Distending Toxin- and Toll-Like Receptor-Mediated Activation of NF-?B ▿

doi: 10.1128/IAI.01317-07

Figure Lengend Snippet: IL-8 secretion induced by conditioned supernatants generated from Campylobacter-T84 cell coculture. (A) Polarized T84 cells were incubated for 24 h either with bacterium-free conditioned supernatants generated from the apical or basolateral supernatant of polarized T84 cells that had been inoculated with C. jejuni 81-176 for 4 h or with C. jejuni 81-176 alone cultured in the invasion medium for 4 h (bacterial). T84 cells were incubated with the bacterial culture medium in the apical chamber, with the apical conditioned supernatant in the apical chamber, or with the basolateral conditioned supernatant in the basolateral chamber. Polarized T84 cells inoculated apically with live C. jejuni 81-176 for 4 h and 24 h were used as controls. (B) The basolateral conditioned supernatant generated from C. jejuni 81-176-T84 cell coculture either was not pretreated (−) or was pretreated with either DNase I (10 U/ml) at 37°C for 2 h, polymyxin B (20 μg/ml) at 37°C for 30 min (PLXB), protease K (100 μg/ml) overnight followed by a 20-min incubation at 100°C (ProtK), or a 20-min incubation at 100°C without protease K (Boiling). Polarized T84 cells were incubated with the pretreated or untreated conditioned supernatants from wt C. jejuni 81-176-T84 cell coculture in the basolateral chamber at 37°C for 24 h. (C) Polarized T84 cells were treated basolaterally with a C. jejuni 81-176 DNA extract (25 μg/ml) (DNA) and DNase I-treated C. jejuni 81-176 DNA (DNA + DNase) at 37°C for 24 h. (D) Polarized T84 cells were incubated basolaterally with different concentrations of E. coli LPS in the presence or absence of 20 μg/ml PLXB. (E) Polarized T84 cells were incubated with conditioned supernatants from a coculture of T84 cells with wt C. jejuni 81-176 or its flaA, pflA, cdtB, or flaA cdtB mutant in the basolateral chamber at 37°C for 24 h. After different treatments, the apical (filled bars) and basolateral (open bars) media were collected. IL-8 concentrations were determined by ELISA. The data are means for three independent experiments; error bars, standard deviations. The data in panels B and E are expressed as the ratio of the IL-8 level induced by a treated basolateral conditioned supernatant to that induced by an untreated supernatant and as the ratio of the IL-8 level induced by a mutant basolateral conditioned supernatant to that induced by a wt supernatant. *, P < 0.05. (F) The cytotoxicity of CDT was measured by incubating Vero cells with serially diluted C. jejuni culture supernatants that were filtrated and treated with polymyxin B for 48 h. The viability of the Vero cells was monitored using an MTT assay. The CDT titers were expressed as the reciprocal of the highest dilution that caused 50% Vero cell death compared with the level in untreated cells. Data are averages for three independent cytotoxicity assays; error bars, standard deviations.

Article Snippet: Mouse anti-human IL-8 and anti-human TNF-α monoclonal antibodies (BD Bioscience, San Jose, CA) were used as capturing antibodies; biotinylated mouse anti-human IL-8 and anti-human TNF-α monoclonal antibodies (BD Bioscience) were used as detecting antibodies; and streptavidin-alkaline phosphatase (SouthernBiotech Inc., Birmingham, AL) and the substrate p -nitrophenylphosphate disodium (Sigma-Aldrich) were used to visualize bound antibodies.

Techniques: Generated, Incubation, Cell Culture, Mutagenesis, Enzyme-linked Immunosorbent Assay, MTT Assay

Journal:

Article Title: Campylobacter -Induced Interleukin-8 Secretion in Polarized Human Intestinal Epithelial Cells Requires Campylobacter -Secreted Cytolethal Distending Toxin- and Toll-Like Receptor-Mediated Activation of NF-?B ▿

doi: 10.1128/IAI.01317-07

Figure Lengend Snippet: Campylobacter-induced IL-8 secretion depends on Campylobacter-secreted CDT and the TLR signaling adaptor MyD88 in polarized intestinal epithelial cells. (A) Polarized T84 cells were pretreated with MyD88 inhibitory peptide (100 μM) (bars III, V, and VII) or its control peptide (bars II, IV, and VI) for 24 h or with chloroquine (5 μM) (bars VIII, IX, and XIII) for 30 min before being incubated with the conditioned supernatant (ConSup) from the coculture of T84 cells with wt C. jejuni 81-176 (bars II, III, and VIII), its cdtB mutant (bars IV, V, and IX), or a C. jejuni 81-176 DNA extract (25 μg/ml) (bars VI and VII) at 37°C for 24 h. MyD88 inhibitory peptide, its control peptide, or chloroquine was also included during the 24-h incubation. Polarized T84 cells incubated with TNF-α only (bar XII) or TNF-α plus chloroquine (bar XIII) were used as controls for the effect of chloroquine on IL-8 secretion. Polarized T84 cells incubated with the medium alone for 24 h (bars I) or with live C. jejuni 81-176 for 4 h (bars X), after which the conditioned supernatants were collected, or 24 h (bars XI) were used as controls. In the 24-h incubation with live bacteria, unbound bacteria were removed at 6 h. No significant difference was detected between the transepithelial resistance before incubation and that after incubation. The apical and basolateral media were collected individually, and the IL-8 concentrations were determined by ELISA. The data are means for three independent experiments; error bars, standard deviations. (B) Polarized T84 cells were pretreated with chloroquine (5 μM) for 30 min before incubation with C. jejuni 81-176 (MOI, ∼10) at 37°C for 4 h. The abilities of C. jejuni 81-176 to adhere to, invade, and transcytose across chloroquine-treated T84 cells were analyzed as described in the legend to Fig. ​Fig.3.3. Averages for three independent experiments with triplicate samples are shown; error bars, standard deviations.

Article Snippet: Mouse anti-human IL-8 and anti-human TNF-α monoclonal antibodies (BD Bioscience, San Jose, CA) were used as capturing antibodies; biotinylated mouse anti-human IL-8 and anti-human TNF-α monoclonal antibodies (BD Bioscience) were used as detecting antibodies; and streptavidin-alkaline phosphatase (SouthernBiotech Inc., Birmingham, AL) and the substrate p -nitrophenylphosphate disodium (Sigma-Aldrich) were used to visualize bound antibodies.

Techniques: Incubation, Mutagenesis, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: Campylobacter -Induced Interleukin-8 Secretion in Polarized Human Intestinal Epithelial Cells Requires Campylobacter -Secreted Cytolethal Distending Toxin- and Toll-Like Receptor-Mediated Activation of NF-?B ▿

doi: 10.1128/IAI.01317-07

Figure Lengend Snippet: Campylobacter-induced IL-8 secretion is dependent on NF-κB activation. (A) T84 cells were incubated with C. jejuni 81-176 (MOI, ∼10) in the presence of cycloheximide for varying lengths of time. T84 cells treated with TNF-α were used as a positive control. −, no treatment. The cells were washed and lysed, and the cell lysates were analyzed by SDS-PAGE and Western blotting with probing for IκB-α. The blots were stripped and reprobed for tubulin as a loading control. Representative results from three independent experiments are shown. (B) Polarized T84 cells were pretreated with the NF-κB inhibitor SN50 (18 μM), quinazoline (28 μM), or TPCK (50 μM) for 30 min and then incubated with C. jejuni 81-176 in the presence of the inhibitor at 37°C for 24 h. No significant difference was detected between the transepithelial resistance before incubation and that after incubation. The apical and basolateral media were collected individually, and the IL-8 concentrations were determined by ELISA. The data are means for three independent experiments; error bars, standard deviations.

Article Snippet: Mouse anti-human IL-8 and anti-human TNF-α monoclonal antibodies (BD Bioscience, San Jose, CA) were used as capturing antibodies; biotinylated mouse anti-human IL-8 and anti-human TNF-α monoclonal antibodies (BD Bioscience) were used as detecting antibodies; and streptavidin-alkaline phosphatase (SouthernBiotech Inc., Birmingham, AL) and the substrate p -nitrophenylphosphate disodium (Sigma-Aldrich) were used to visualize bound antibodies.

Techniques: Activation Assay, Incubation, Positive Control, SDS Page, Western Blot, 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-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