atf6 inhibitor  (MedChemExpress)

Journal: bioRxiv

Article Title: UPR-induced intracellular C5aR1 promotes adaptation to the hypoxic tumour microenvironment by regulating tumour cell fate

doi: 10.1101/2024.09.27.615431

Figure Lengend Snippet: For the whole figure: Individual biological replicates (large points) represent the average of the technical replicates (small points). p values were calculated using biological replicates by two-tailed paired Student’s t test (A), two-tailed unpaired Student’s t test (B-D), two-way ANOVA with uncorrected Fisher’s LSD test (E-G and I), or one-way ANOVA with Dunnett’s test (H). (A) HIF-1α-KO and WT HCT116 cells were cultured under normoxia or hypoxia (<0.1% O 2 ) for 24 hr and subjected to qRT-PCR. n=3 and 4. (B-D) HCT116 cells were treated with either 200 µM cobalt chloride (CoCl 2 ) for 24 hr (B), 10 µg/mL tunicamycin (Tuni) for 24 hr (C), 2 µM thapsigargin (Thap) for 16 hr (D), or its vehicle (Ctrl), and subjected to qPCR. n=3. (E-G) HCT116 cells were cultured under normoxia or hypoxia (<0.1% O 2 ) in the presence of either IRE1-inhibitor (E), PERK-inhibitor (F), or ATF6-inhibitor (G), and subjected to qPCR. n=3. (H) HCT116 cells were transfected with the indicated siRNA or scramble siRNA (siScr), cultured under normoxia or hypoxia (<0.1% O 2 ) for 24 hr, and subjected to qPCR. n=4. (I) After simultaneously silencing XBP1, ATF4, and ATF6 using siRNA mixtures, HCT116 cells were cultured under normoxia or hypoxia (<0.1% O 2 ) for 24 hr, and subjected to qPCR. n=3. (J) Enrichment Analysis with public Chip-seq data on Chip-Atlas website is shown ( https://chip-atlas.org/peak_browser ). Colours represent copy numbers of the indicated transcription factors binding to C5aR1 gene locus in all cell lines. (K) Pearson’s correlation of C5AR1 mRNA expression and Xhu UPR signature in TCGA colorectal cancer and glioblastoma samples. R score and p value are shown. (L and M) Serial sections of HCT116 spheroids treated with EF5 (L) or HCT116 tumour xenografts (M) were stained with the indicated antibodies; (L) Section 1, C5aR1(red), BiP (green), or DAPI (blue); Section 2, BiP (red), EF5 (green), or DAPI (blue). (M) C5aR1 (red), BiP (green), or DAPI (blue). Scale bar, 50 µm.

Article Snippet: Before hypoxic culture, cells were pretreated with IRE1α inhibitor (4μ8c, Sigma-Aldrich, SML0949), PERK inhibitor (AMG PERK 44, Tocris, 5517), ATF6 Inhibitor (Ceapin-A7, Sigma-Aldrich, SML2330), and Dynasore (Sigma-Aldrich, D7693) for 1 hr, or with C5aR1 antagonists, PMX205 (Tocris, 5196), JPE-1375 (MedChem Express, HY-148141) and Avacopan (Cayman Chemical, CAY36639) for 8 hr, respectively.

Techniques: Two Tailed Test, Cell Culture, Quantitative RT-PCR, Transfection, ChIP-sequencing, Binding Assay, Expressing, Staining