Journal: bioRxiv

Article Title: Tuberculosis-associated IFN-I induces Siglec-1 on tunneling nanotubes and favors HIV-1 spread in macrophages

doi: 10.1101/836155

Figure Lengend Snippet: (A) Vertical scatter plot showing the relative abundance of IFN-I in cmCTR (white) and cmMTB (black) media, as measured indirectly after 24h exposure to the HEK-Blue IFN-α/β reporter cell line yielding reporter activity in units (U) per mL. (B-D) For 3 days, monocytes were differentiated into macrophages either with cmMTB (black) or cmCTR (white), the indicated recombinant cytokines (B), the presence of an IL-10 depletion (α-IL-10) or a control (α-IgG) antibodies (C), or the presence of an IFNAR-2 blocking (α-IFNAR) or control (α-IgG) antibodies (D). (E) Representative serial immunohistochemical images of lung biopsies of a co-infected (ATB-SIV) NHP stained for Siglec-1 (brown, top) and pSTAT1 (brown, bottom). Scale bar, 250 μm. Insets are 10× zooms. (F) Correlation of the percentage of cells positive for Siglec-1 and pSTAT1, as measured per mm 2 of lung tissue from the indicated NHP groups. Mean value is represented as a black line. (G) Representative immunohistochemical images of lung biopsies from the indicated NHP group stained for pSTAT1 (brown). Arrowheads show pSTAT1-positive nuclei. Scale bar, 500 μm. (H) Vertical Box and Whisker plot illustrating the percentage of pSTAT1 + alveolar macrophages in lung biopsies from the indicated NHP groups. N=450 alveolar macrophages analyzed per condition from one representative animal per NHP group. (A-D) Each circle within vertical scatter plots represents a single donor and histograms median value. (B-D) Vertical scatter plots displaying the median fluorescent intensity (MFI) of Siglec-1 cell-surface expression. Statistical analyses: Two-tailed, Wilcoxon signed-rank test (A-D), Spearman correlation (F), and Mann-Whitney unpaired test (H). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ns: not significant.

Article Snippet: Different antigen unmasking methods where used on tissue slides for immunohistochemical staining, which was performed using anti-CD163 (Leica/Novocastra), anti-Siglec-1 (Novus Biologicals) and anti-pSTAT1 (Cell Signaling Technology).

Techniques: Activity Assay, Recombinant, Blocking Assay, Immunohistochemical staining, Infection, Staining, Whisker Assay, Expressing, Two Tailed Test, MANN-WHITNEY

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