unc3866  (MedChemExpress)

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: CBX4 regulates lfTSLP protein translation. (A) Schematic sketches of wild-type (WT) CBX4 and its mutants. (B) Immunoblots for the indicated proteins in HBE ectopically expressing the WT or CBX4 mutants for 48h. (C) HBE were treated with indicated concentrations of UNC3866 for 24hr. Expression of lfTSLP was determined by immunoblot analysis. (D) HBE were transfected with WT or CBX4 mutant plasmids for 48hr and lfTSLP mRNA expression was determined by RT-PCR. (E) HDM-treated HBE lysates were subjected to IP with anti-CBX4 antibody or anti-IgG antibody followed by immunoblot analysis with anti-lfTSLP antibody. (F) siNC or siCBX4 was transfected into HBE followed by treatment with 100 μg/mL cycloheximide (CHX) for the indicated amount of time. Expression of lfTSLP was determined by immunoblot analysis. (G) siNC or siCBX4 was transfected into HBE followed by treatment with 5 μg/mL actinomycin D for the indicated amount of time. The lfTSLP mRNA expression was measured by RT-PCR. (H) HBE transfected with shNC or shCBX4 were fractionated into cytoplasmic extracts through sucrose gradients. The distribution of lfTSLP and GAPDH mRNAs was quantified by RT-PCR analysis of RNA isolated from 12 gradient fractions. Data are presented as mean ± SEM. Images show representative results for one of 3 or more experimental replicates. NS, Not significant. (B) One-way ANOVA with Bonferroni’s post hoc test was used. (C) Unpaired two tailed Student’s t-test was used. (F and G) Two-way ANOVA was used. * p

Article Snippet: HBE was treated with 2-D08 (5, 10, 20μM) or UNC3866 (MCE, HY-100832) (50, 100, 200 nM) for 24 hours, then 400U/mL HDM (ALK-Abello A/S, A4963) was added for an additional 24 hours.

Techniques: Western Blot, Expressing, Transfection, Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Isolation, 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: TFII-I is a transcriptional activator of MEX-3B. (A) siNC or siCBX4 was transfected into HBE followed by treatment with 5 μg/mL actinomycin D for the indicated amount of time. MEX-3B mRNA expression was measured by RT-PCR. (B) HBE were treated with the indicated concentrations of UNC3866 200 nM for 24hr. The MEX-3B protein level was determined by immunoblot analysis. (C) Potential transcription factors were scanned by the ALGGEN database. Prediction of an interaction between CBX4 and these factors were conducted by GeneMANIA. (D) HBE lysates were subjected to IP with anti-CBX4 antibody or anti-IgG antibody followed by immunoblot analysis with anti-CBX4 and TFII-I antibody. (E) HBE lysates were subjected to IP with anti-TFII-I antibody or anti-IgG antibody followed by immunoblot analysis with anti-TFII-I and CBX4 antibody. (F) Co-localization of CBX4 and TFII-I was analyzed by immunostaining of HBE with anti-CBX4 and TFII-I via confocal microscopy. (G) Immunoblots for the indicated proteins in HBE transfected with siNC or siCBX4. (H) RT-PCR for the indicated mRNA expression levels in HBE transfected with siNC or siCBX4. (I) Localization of TFII-I-binding sites in MEX-3B promoter. (J) HBE were treated with HDM 400 U 24 hr followed by ChIP with anti-TFII-I antibody or nonrelated IgG. Precipitated DNAs were quantified by RT-PCR using five MEX-3B promoter-specific primers covering five TFII-I-binding sites. (K) Human 293T cells transfected with pcDNA3.1(+)-TFII-I together with firefly luciferase reporter and pRL-tk-renilla plasmids for 24 hr. Data are presented as mean ± SEM. Images show representative results for one of 3 or more experimental replicates. NS, Not significant. (A) Two-way ANOVA was used. (B, G, H and K) Unpaired two tailed Student’s t-test was used. (J) One-way ANOVA with Bonferroni’s post hoc test was used. * p

Article Snippet: HBE was treated with 2-D08 (5, 10, 20μM) or UNC3866 (MCE, HY-100832) (50, 100, 200 nM) for 24 hours, then 400U/mL HDM (ALK-Abello A/S, A4963) was added for an additional 24 hours.

Techniques: Transfection, Expressing, Reverse Transcription Polymerase Chain Reaction, Immunostaining, Confocal Microscopy, Western Blot, Binding Assay, Chromatin Immunoprecipitation, Luciferase, Two Tailed Test