Journal: bioRxiv

Article Title: Targeting hypoxia-induced circSTT3A decreases breast cancer stem cell formation via degradation of PGK1 protein and serine synthesis

doi: 10.1101/2023.04.28.538664

Figure Lengend Snippet: a. Silver staining of proteins pulled down by circSTT3a-specific biotin-labeled probe and control probe. HSP70 was ascertained as a candidate protein interacting with circSTT3A by RNA pull-down. b. The interaction between circSTT3A and HSP70 was verified by RIP and qRT-PCR in MDA-MB-231 and BT-549 cell lysate. c. FISH and IF co-staining indicated the co-localization of circSTT3A (red) and HSP70 (green) in MDA-MB-231 and BT-549 cells cultured in hypoxia (Scale bar, 25 μm). d-e . qRT-PCR and Western blotting were used to detect the effects of hypoxia-induced circSTT3A or ectopic circSTT3A overexpression on the expression of HSP70 in BC cells. f. Schematic diagram of HSP70 full-length protein and truncated protein. g. RNA pull-down experiment using biotin-labeled circSTT3A probe in hypoxic MDA-MB-231 cells expressing full-length or truncated HSP70, Western blot revealed the enrichmen of indicated full-length of HSP70 and its deletion mutants. h. RIP assays were performed with anti-Flag in hypoxic MDA-MB-231 cells transfected with indicated full-length or truncated HSP70 plasmids. i. Co-IP assays were carried out in hypoxic MDA-MB-231 cells under indicated conditions using anti-HSP70 and IgG control, followed by silver staining. j. The Co-IP experiment used anti-HSP70 or anti-PGK1 to analyze the direct interaction between HSP70 and PGK1 in MDA-MB-231, respectively. k. Co-IP using anti-Flag was applied to identify the interaction between the truncated HSP70 protein and PGK1 in MDA-MB-231 cells transfected with indicated full-length or truncated HSP70 plasmids. l. IF co-staining showed the co-localization of HSP70 (red) and PGK1 (green) in BC cells (Scale bar, 25 μm). Data were presented as the mean±SD, ***p<0.001.

Article Snippet: After washing with PBS, the cells were incubated with anti-HSP70 antibody at 4 °C overnight, then FITC-labeled goat anti-rabbit IgG was added (Bosterbio, China) at 37 °C for 2 h. DAPI was use to locate nucleus.

Techniques: Silver Staining, Labeling, Control, Quantitative RT-PCR, Staining, Cell Culture, Western Blot, Over Expression, Expressing, Transfection, Co-Immunoprecipitation Assay

Journal: bioRxiv

Article Title: Targeting hypoxia-induced circSTT3A decreases breast cancer stem cell formation via degradation of PGK1 protein and serine synthesis

doi: 10.1101/2023.04.28.538664

Figure Lengend Snippet: a-b. mRNA and protein levels of PGK1 were determined by qRT-PCR (a) and Western blotting (b) in BC cells under indicated conditions. c. co-IP and WB were carried out in hypoxic MDA-MB-231 cells with or without circSTT3 and in normoxic MDA-MB-231 with or without ectopic circSTT3A expression using anti-HSP70 and anti-PGK1, IgG as negative controls. d. The expression of PGK1 was determined after transfection or co-transfection with the indicated vectors or siRNA in MDA-MB-231 cells by Western blotting. e. Western blot analysis of PGK1 expression in hypoxic and normoxic BC cells transfected with corresponding siRNA under MG-132 treatment. f. Ubiquitination of PGK1 was appraised in MG132-treated MDA-MB-231 cells under indicated conditions. g-h. The expression of PGK1 in BC tissues with low or high level of circSTT3A were evaluated by Western blotting (g) and qRT-PCR in 60 BC tissues (h) . i. Correlation analysis of circSTT3A and PGK1 in BC tissues. j-k. The representative images (j) and quantitative analysis (k) of mammosphere formation abilities were showed after transfection or co-transfection with the indicated vectors or siRNA. l. The representative images and volume analysis of xenograft tumors for each group (n=8). Data are presented as the mean±SD; *p<0.05, **p<0.01 and ***p < 0.001.

Article Snippet: After washing with PBS, the cells were incubated with anti-HSP70 antibody at 4 °C overnight, then FITC-labeled goat anti-rabbit IgG was added (Bosterbio, China) at 37 °C for 2 h. DAPI was use to locate nucleus.

Techniques: Quantitative RT-PCR, Western Blot, Co-Immunoprecipitation Assay, Expressing, Transfection, Cotransfection, Ubiquitin Proteomics

Journal: bioRxiv

Article Title: Targeting hypoxia-induced circSTT3A decreases breast cancer stem cell formation via degradation of PGK1 protein and serine synthesis

doi: 10.1101/2023.04.28.538664

Figure Lengend Snippet: a. Cellular 3-phosphoglycerate (3-PG), phosphoserine (pSer) and serine concentrations were measured by HPLC-MS in circSTT3A-knockdown, HSP70-knockdown and PGK1 knockdown MDA-MB-231 derived spheres and control spheres under hypoxia. b-c . Colony formation assays (b) , western blot analyses (c) of stemness-related proteins (CD44, c-Myc, and KLF4) in BC cells under indicated conditions (Scale bar, 200 µm). d-f . The expression of circSTT3A (d) , PGK1 (e) and serine (f) in the chemo-sensitive tissues (n=27) and chemo-resistant tissues (n=27) of BC patients. g-h . Representative pictures of xenograft tumors (n=8) (g) and xenograft tumor volume (h). i-j . Representative images showed PCNA IHC staining (i) , cell apoptotic status checked by TUNEL (i) , KLF4 and OCT4 levels (j) in each group of xenograft tumors (Scale bar, 100 µm). Data are presented as the mean±SD; *p<0.05, **p <0.01 and ***p < 0.001.

Article Snippet: After washing with PBS, the cells were incubated with anti-HSP70 antibody at 4 °C overnight, then FITC-labeled goat anti-rabbit IgG was added (Bosterbio, China) at 37 °C for 2 h. DAPI was use to locate nucleus.

Techniques: Knockdown, Derivative Assay, Control, Western Blot, Expressing, Immunohistochemistry, TUNEL Assay