Journal: bioRxiv

Article Title: Sex differences in IL-17 determine chronicity in male versus female urinary tract infection

doi: 10.1101/449124

Figure Lengend Snippet: ( A-C ) Female and male mice were infected with 10 7 CFU of UPEC UTI89-RFP-kan R and bladders analyzed at 24 hours PI. Graphs show ( A ) IL-33 protein levels in homogenized bladder, ( B ) IL-4Rα expression on bladder resident macrophages, and ( C ) the number of ILCs (CD90 + CD25 + CD3 − CD4 − NK1.1 − MHC II − CD11b − ) in bladders. ( D-F ) Female mice were implanted with empty tubing (Mock) or slow release tubing containing testosterone (T tube) and allowed to recover 1 week before infection with 10 7 CFU UPEC strain UTI89-RFP-kan R . Graphs show ( D ) IL-33 protein levels in homogenized bladder tissue, ( E ) IL-4Rα expression on bladder resident macrophages, and ( F ) the number of ILCs in bladders. Data are pooled from 2-3 experiments, n=4-5 mice/group in each experiment. Each dot is one mouse, red dots depict female mice and blue dots are male mice, lines are medians. ns = not significant, ** p<0.01, *** p<0.001, **** p<0.0001 Mann-Whitney test. Analyses in this figure were corrected for multiple testing by Holm–Bonferroni method, all p<0.05 had q< 0.05.

Article Snippet: For IL-33 neutralization, 3.6 µg/mouse α-IL-33 (catalog number AF3626, BioTechne/R&D Systems) or 3.6 µg/mouse polyclonal goat IgG isotype control (catalog number AB-108-C, BioTechne/R&D Systems) were delivered intraperitoneally in 100 µL PBS ( ).

Techniques: Infection, Expressing, MANN-WHITNEY

Journal: bioRxiv

Article Title: Sex differences in IL-17 determine chronicity in male versus female urinary tract infection

doi: 10.1101/449124

Figure Lengend Snippet: ( A ) Experimental design in which male mice (blue) were supplemented with recombinant IL-33 (rIL-33) or female mice (red) were treated with α-IL-33 neutralizing antibody. All mice were infected with 10 7 CFU UPEC and bacteriuria was assessed over time. ( B-G ) Graphs show ( B ) the number of infected male mice over time and ( C ) CFU at 24 hours post primary (1°) or challenge (2°) infection in the bladder, in PBS or rIL-33 treated male mice; ( D ) the number of infected mice over time and ( E ) CFU at 24 hours post primary (1°) or challenge (2°) infection in the bladder, in isotype or α-IL-33 treated female mice; and the absolute number of the ( F ) indicated immune cell populations and ( G ) UPEC + immune cell populations in bladders 24 hours PI in isotype or α-IL-33 treated female mice. Data in B and D are representative experiments of 2 independent experiments, n=6 mice/group/experiment. In C, E-G , data are pooled from 2 experiments, n=4-5 mice/group in each experiment, each dot is one mouse, lines are medians. Mann-Whitney test, no statistically significant differences found.

Article Snippet: For IL-33 neutralization, 3.6 µg/mouse α-IL-33 (catalog number AF3626, BioTechne/R&D Systems) or 3.6 µg/mouse polyclonal goat IgG isotype control (catalog number AB-108-C, BioTechne/R&D Systems) were delivered intraperitoneally in 100 µL PBS ( ).

Techniques: Recombinant, Infection, MANN-WHITNEY

Journal: bioRxiv

Article Title: CBX4 regulates long-form thymic stromal lymphopoietin-mediated airway inflammation through SUMOylation in HDM-induced asthma

doi: 10.1101/2021.05.24.445396

Figure Lengend Snippet: (A) Immunoblot analysis of IL-25, IL-33 and TSLP in mice lung protein extract. (B-D) Lung sections were stained with IL-25, IL-33 and TSLP antibody by immunocytochemistry. (E) lfTSLP protein levels were detected by Western blotting in HBE treated with different concentrations of 2-D08. (F) then analyzed lfTSLP and sfTSLP mRNA expression in HBE treated with 20 μM 2-D08 was measured by RT-PCR. Data are presented as mean ± SEM. Images show representative results for one of 3 or more experimental replicates. NS, Not significant. (A) One-way ANOVA with Bonferroni’s post hoc test was used. (E and F) Unpaired two tailed Student’s t-test was used. * p < 0.05, ** p < 0.01, and *** p < 0.001. Scale bar, 100 µm.

Article Snippet: Antibodies used in this study were as follows: anti-CBX4 (abclonal, A6221), anti-PIAS1 (proteintech, 14242-1-AP), anti-TSLP (abcam, ab188766), anti-IL-25 (abclonal, A8252), anti-IL-33 (abclonal, A8096), anti-MEX-3B (santacruz, sc-515833), anti-FLAG (proteintech, 20543-1-AP), anti-β-actin (proteintech, 60008), anti-sumo1 (santacruz, sc-5308), anti-sumo 2/3 (santacruz, sc-393144), anti-TFII-I (CST, 4562).

Techniques: Western Blot, Staining, Immunocytochemistry, Expressing, Reverse Transcription Polymerase Chain Reaction, Two Tailed Test

Journal: bioRxiv

Article Title: CBX4 regulates long-form thymic stromal lymphopoietin-mediated airway inflammation through SUMOylation in HDM-induced asthma

doi: 10.1101/2021.05.24.445396

Figure Lengend Snippet: (A) HBE were stimulated with 400 U HDM for 24 hr and expression of SUMOylation E3 ligases (PIAS1-4, CBX4, MMS21, ZNF451 and RanBP2) and deSUMOylation enzymes SENPs were measured by RT-PCR. (B and C) CBX4 and PIAS1 protein levels were measured by Western blotting in HDM-treated HBE (B) and mice lung protein extracts (C). (D) Lung sections were stained with CBX4 and PIAS1 antibody by immunocytochemistry. (E-H) Effects of CBX4 (G) and PIAS1(H) knockdown on IL-25, IL-33 and lfTSLP protein expression in HBE. (I and J) Effects of CBX4 (H) and PIAS1(I) knockdown on lfTSLP and sfTSLP mRNA expression. Data are presented as mean ± SEM. Images show representative results for one of 3 or more experimental replicates. NS, Not significant. (B, E and F) One-way ANOVA with Bonferroni’s post hoc test was used. (C, G, H, I and J) Unpaired two tailed Student’s t-test was used. * p < 0.05, ** p < 0.01, and *** p < 0.001. Scale bar, 100 µm.

Article Snippet: Antibodies used in this study were as follows: anti-CBX4 (abclonal, A6221), anti-PIAS1 (proteintech, 14242-1-AP), anti-TSLP (abcam, ab188766), anti-IL-25 (abclonal, A8252), anti-IL-33 (abclonal, A8096), anti-MEX-3B (santacruz, sc-515833), anti-FLAG (proteintech, 20543-1-AP), anti-β-actin (proteintech, 60008), anti-sumo1 (santacruz, sc-5308), anti-sumo 2/3 (santacruz, sc-393144), anti-TFII-I (CST, 4562).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Staining, Immunocytochemistry, Two Tailed Test

Journal: bioRxiv

Article Title: CBX4 regulates long-form thymic stromal lymphopoietin-mediated airway inflammation through SUMOylation in HDM-induced asthma

doi: 10.1101/2021.05.24.445396

Figure Lengend Snippet: Quantitative immunohistochemical analysis was performed for SUMO 1(A), SUMO 2/3(B), IL-25(C), IL-33(D), TSLP(E), CBX4(F), PIAS1(G), TFII-I(H) in airway epithelium. Data are presented as mean ± SEM. Images show representative results for one of 3 or more experimental replicates. NS, Not significant. (C, D, E and H) One-way ANOVA with Bonferroni’s post hoc test was used. (A, B, F and G) Unpaired two tailed Student’s t-test was used. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: Antibodies used in this study were as follows: anti-CBX4 (abclonal, A6221), anti-PIAS1 (proteintech, 14242-1-AP), anti-TSLP (abcam, ab188766), anti-IL-25 (abclonal, A8252), anti-IL-33 (abclonal, A8096), anti-MEX-3B (santacruz, sc-515833), anti-FLAG (proteintech, 20543-1-AP), anti-β-actin (proteintech, 60008), anti-sumo1 (santacruz, sc-5308), anti-sumo 2/3 (santacruz, sc-393144), anti-TFII-I (CST, 4562).

Techniques: Immunohistochemical staining, Two Tailed Test