Journal: Scientific Reports
Article Title: HIFα independent mechanisms in renal carcinoma cells modulate divergent outcomes in fibronectin assembly mediated by hypoxia and CoCl 2
doi: 10.1038/s41598-020-75756-5
Figure Lengend Snippet: Integrins α4 and α5 are required for FN fibril assembly in response to CoCl 2 . ( a ) Caki-1 cells cultured at 21% O 2 , exposed to 1% O 2 or treated with 50 µM CoCl 2 for the indicated times. Cells were lysed and immunoblotted for pFAK and total FAK. β-Actin was used as the loading control. ( b ) Cell lysates as in ( a ) were immunoblotted for pMLC. β-Actin was used as the loading control. ( c ) Caki-1 cells were pretreated with 10 µM ATN-161 (Integrin α5 inhibitor), 1 µg/ml BIO5192 (Integrin α4 inhibitor) or a combination of both for 30 min followed by treatment with 50 µM CoCl 2 for 2 h and immunostained for FN (red) and counterstained with the nuclear stain DAPI (blue). Scale bar = 10 µm. ( d ) Caki-1 cells transduced with shScr (200 MOI) or shalpha4 (2000 MOI), shalpha5 (2000 MOI) or a combination of shalpha4 (1000 MOI) and shalpha5 (1000 MOI) expressing the GFP reporter were left untreated or treated with 50 µM CoCl 2 for 2 h and immunostained for FN (red) and DAPI (blue). Transduced cells are shown in green (GFP reporter). The bar graph below the figure quantifies the % ‘fibril-containing’ cells in GFP reporter-expressing virus transduced cells. Data is representative of two independent experiments. More than 80 cells were counted in each condition.
Article Snippet: Integrin α5 inhibitor ATN-161 (MedKoo Biosciences, #200350a) and Integrin α4 inhibitor BIO5192 (Tocris, #5051) were reconstituted and stored according to manufacturer’s recommendations.
Techniques: Cell Culture, Control, Staining, Transduction, Expressing, Virus