Journal: Scientific Reports

Article Title: Spautin-1 inhibits mitochondrial complex I and leads to suppression of the unfolded protein response and cell survival during glucose starvation

doi: 10.1038/s41598-022-15673-x

Figure Lengend Snippet: Autophagy inhibitors fail to show preferential cytotoxicity and suppress induction of ATF4 and XBP1s in glucose-starved or 2DG-stressed HT1080 cells. ( a ) HT1080 cells were treated with spautin-1 (10 μM) or SAR405 (1 μM) in HBSS containing HCQ (30 μM) for 4 h. Autophagosomes were visualized with the CYTO-ID Autophagy detection kit 2.0. ( b ) Effects of bafilomycin A1 (Baf), HCQ, and SAR405 on cell viability in GS-stressed HT1080 cells were determined by the CellTiter-Glo luminescent cell viability assay. Data are shown as mean ± SD ( n = 3). ( c ) Effects of spautin-1 (10 μM) and other autophagy inhibitors (Baf; 10 nM, HCQ; 30 μM, SAR405; 10 μM) on nuclear ATF4 and XBP1s induction under 2DG-stressed conditions were visualized using the Operetta CLS. Blue, red, and green fluorescent signals indicate nuclei, ATF4, and XBP1s, respectively. ( d ) Mean intensities of nuclear ATF4 and XBP1s intensities were determined using Harmony high-content analysis software. Data are shown as mean ± SD ( n = 3).

Article Snippet: Fluorescent images (nine fields per well) were acquired using a 20 × water objective lens by Operetta CLS (Perkin Elmer).

Techniques: Cell Viability Assay, High Content Screening, Software

Journal: Scientific Reports

Article Title: Spautin-1 inhibits mitochondrial complex I and leads to suppression of the unfolded protein response and cell survival during glucose starvation

doi: 10.1038/s41598-022-15673-x

Figure Lengend Snippet: USP10 and USP13 silencing has little effect on the UPR and cell viability under GS- or 2DG-stressed conditions. ( a ) Effects of USP10 and USP13 silencing on GRP78 in HT1080 cells were determined by western blotting. RPS3 was used as a loading control. The blot membranes were cut prior to hybridization with antibodies, according to Full range rainbow molecular weight markers. Original blots were presented in Supplementary Fig. . ( b ) Effects of USP10 and USP13 silencing on ATF4 and XBP1s induction in vehicle- or spautin-1-treated HT1080 cells under 2DG-stressed conditions were visualized using the Operetta CLS. ( c , d ) Mean intensities of nuclear ( c ) ATF4 and ( d ) XBP1s in ( b ) were determined using Harmony high-content analysis software. Data are shown as mean ± SD ( n = 3). ( e ) Effects of USP10 and USP13 silencing on cell viability under GS were determined by the CellTiter-Glo luminescent cell viability assay. Data are shown as mean ± SD ( n = 3). ( f ) Effects of USP10 and USP13 silencing on preferential cytotoxicity of spautin-1 under GS were determined by the CellTiter-Glo luminescent cell viability assay. Data are shown as mean ± SD ( n = 3).

Article Snippet: Fluorescent images (nine fields per well) were acquired using a 20 × water objective lens by Operetta CLS (Perkin Elmer).

Techniques: Western Blot, Control, Hybridization, Molecular Weight, High Content Screening, Software, Cell Viability Assay