Journal: bioRxiv

Article Title: Chlamydia trachomatis modulates the expression of JAK-STAT signaling components to attenuate the Type II interferon response of epithelial cells

doi: 10.1101/2024.01.09.574898

Figure Lengend Snippet: (A) Immunoblot analysis of activated STAT1 (pSTAT1) and IDO1 following exposure to 10 U/mL of IFNγ at different exposure lengths. (B) Immunofluorescence assay showing nuclear localization of activated STAT1 in the presence of IFNγ (C) Immunoblot and densitometric analysis of protein and (D) RT-qPCR of transcript expression levels of IDO1 following treatment with IFNγ at 10 U/mL at 24h hpi for 6 hours. All data are representative of 4 independent experiments, and statistical significance was determined using Welch’s t-test. **p < 0.01, *p < 0.05, ns = not significant.

Article Snippet: Fixed monola were blocked with 4% BSA-PBST and probed with primary antibodies for pSTAT1-Tyr701 (ThermoFisher Scientific MA515071, 1:250 dilution) and Chlamydia heat-shock protein 60 (cHsp60) (ThermoFisher Scientific MA3-023, 1:2000 dilution), followed by secondary antibodies AlexaFluor 488 anti-mouse (ThermoFisher Scientific, A28175, 1:400 dilution) and AlexaFluor 594 anti-rabbit (ThermoFisher Scientific A-11012, 1:400).

Techniques: Western Blot, Immunofluorescence, Quantitative RT-PCR, Expressing

Journal: bioRxiv

Article Title: Chlamydia trachomatis modulates the expression of JAK-STAT signaling components to attenuate the Type II interferon response of epithelial cells

doi: 10.1101/2024.01.09.574898

Figure Lengend Snippet: (A) C. trachomatis -infected cells (MOI=2, yellow outline) and uninfected HEp2 cells exposed to IFNγ (10 U/mL) starting at 24 hpi for 6 hours were fixed and stained for phosphorylated STAT1 (pSTAT1) in red and for DNA with DAPI in blue. (B) Nuclear pSTAT1 fluorescence intensities were quantified in both uninfected and infected cells. Measurements were normalized to respective no IFNγ control to account for background pSTAT1 staining and plotted as mean fluorescence. Violin plot represents 6 independent experiments with total of 260 nuclei measured per group. Statistical significance was determined by Wilcoxon Rank-sum. (C) pSTAT1 and IDO1 protein levels were assessed from total protein lysates collected from C. trachomatis -infected (MOI=4) and uninfected cells exposed to IFNγ (10 U/mL) at 24 hpi for 6 hours. (D) Densitometric quantification of IDO1 plotted as ratios to the loading control α-tubulin. Bar graph represents 4 independent experiments, and statistical significance was determined using Welch’s t-test. *p < 0.05, ***p < 0.001.

Article Snippet: Fixed monola were blocked with 4% BSA-PBST and probed with primary antibodies for pSTAT1-Tyr701 (ThermoFisher Scientific MA515071, 1:250 dilution) and Chlamydia heat-shock protein 60 (cHsp60) (ThermoFisher Scientific MA3-023, 1:2000 dilution), followed by secondary antibodies AlexaFluor 488 anti-mouse (ThermoFisher Scientific, A28175, 1:400 dilution) and AlexaFluor 594 anti-rabbit (ThermoFisher Scientific A-11012, 1:400).

Techniques: Infection, Staining, Fluorescence

Journal: bioRxiv

Article Title: Chlamydia trachomatis modulates the expression of JAK-STAT signaling components to attenuate the Type II interferon response of epithelial cells

doi: 10.1101/2024.01.09.574898

Figure Lengend Snippet: (A) RT-qPCR analysis of JAK-STAT pathway components that are required for IFNγ signaling. ΔΔCTs were calculated by comparing to the mock-infected, untreated sampel and normalized to the housekeeping gene HPRT. All data are representative of 4 independent experiments, and statistical significance was determined using Welch’s t-test. (B) Total protein lysates collected from C. trachomatis -infected (MOI=4) and uninfected cells exposed to IFNγ (10 U/mL) at 24 hpi for 6 hours were probed for total JAK2 and STAT1 as well as their phosphorylated species. α-tubulin serves as loading control and vimentin was probed to show that there is no infection-induced general protein cleavage during sample collection. (C) Densitometric quantification of pJAK2 and pSTAT1 expressed as ratio to their respective total proteins, and (D) relative protein expression levels of total JAK2 and STAT1 normalized to the loading control α-tubulin**p < 0.01, ns = not significant.

Article Snippet: Fixed monola were blocked with 4% BSA-PBST and probed with primary antibodies for pSTAT1-Tyr701 (ThermoFisher Scientific MA515071, 1:250 dilution) and Chlamydia heat-shock protein 60 (cHsp60) (ThermoFisher Scientific MA3-023, 1:2000 dilution), followed by secondary antibodies AlexaFluor 488 anti-mouse (ThermoFisher Scientific, A28175, 1:400 dilution) and AlexaFluor 594 anti-rabbit (ThermoFisher Scientific A-11012, 1:400).

Techniques: Quantitative RT-PCR, Infection, Expressing

Journal: British Journal of Cancer

Article Title: Impaired T-bet-pSTAT1 α and perforin-mediated immune responses in the tumoral region of lung adenocarcinoma

doi: 10.1038/bjc.2015.255

Figure Lengend Snippet: Reduced STAT1 phosphorylation in the tumoral region of NSCLC. ( A – F ) Western blot analysis of pSTAT1 and actin expression in lung tissue samples from the tumoral, peri-tumoral and control region of patients with adenocarcinoma (ADC) ( N Control =5; N Peri-tumoral =5, N Tumoral =5). Bar charts show mean values of the protein expression levels of pSTAT1 α -Isoform ( C and D ) and pSTAT1 β -Isoform ( E and F ) relative to actin levels in ADC and in SCC, respectively. ( G ) Flow cytometry analysis of pSTAT1 + cells in the tumoral, the peri-tumoral and the control lung region of one representative patient with NSCLC. pSTAT1 staining was performed with the lung cell suspensions after 20 min of incubation at 37 °C in the presence or absence of IFN- γ . pSTAT1 + cells were gated on CD4 + , CD8 + or CD11b + cells, respectively. ( H ) Flow cytometry analysis of pSTAT1 + CD4 + and pSTAT1 + CD8 + T cells gated on lymphocytes as well as pSTAT1 + CD11b + cells gated on big cells in the control, the peri-tumoral and the tumoral lung region of one representative patient with NSCLC. Data are shown as mean values±s.e.m. using Student's t -test * P =0.05.

Article Snippet: After another cell centrifugation and the aspiration of methanol solution, the cells were incubated with the PE-conjugated pSTAT1 antibody (BD Biosciences), solved in 50 μ l Permeabilisation buffer (BD Biosciences; 30 min., 4 °C, dark).

Techniques: Western Blot, Expressing, Flow Cytometry, Staining, Incubation