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Additional file 1. Search strategies

Additional file 2. Extracted data items

Additional file 4. GRADE framework applied to prognostic factors findings

Additional file 4. GRADE framework applied to prognostic factors findings

Additional file 3. Prognostic factors estimates of included studies

Additional file 3. Prognostic factors estimates of included studies

Additional file 2: Figure S2. Grx1-roGFP1-iEER is not released from the ER upon treatment of cells with thapsigargin, cyclosporine A, or puromycin for 30 min. HEK293 cells stably expressing HA-tagged Grx1-roGFP1-iEER were treated with 0.1 %DMSO, 10 μM cyclosporine A (CsA), 1 μM thapsigargin (TG) or 100 μM puromycin (Puro) for 30 min. Cells were fixed, stained with Hoechst 33342, incubated with anti-Calnexin and anti-HA antibodies followed by green- and red-fluorescent secondary antibodies, and analysed on an epifluorescence microscope. Overlay images including increased magnification frames of selected insets are shown for each treatment in colour. Size bar, 10 μm. (PPTX 2915 kb)

Additional file 4: Figure S4. BiP does not influence the Ca2+ depletion-dependent reductive shift in the ER. Effect of manipulation of BiP on fluorescence ratio changes of Grx1-roGFP1-iEER in HEK293 cells stably expressing the sensor. BiP levels were diminished by the addition of subtilaseAB (SubAB) toxin or by silencing (BiP kd). As control, an inactive subtilaseAA272B mutant (SubAB mut) and a non-silencing control siRNA (control kd) were also used. The effect of subtilaseAB treatment (A) or BiP silencing (B) was checked by immunoblotting. (C) Identical curves of reductive shift were observed upon CsA addition in all cases; SubAB treatment for 60 min, siRNA transfection for 48 h. (PPTX 1031 kb)

Additional file 1.

Additional file 3.

Additional file 1: Figure S1. Short-term thapsigargin, cyclosporine A and puromycin treatment has only minor effects on mitochondrial superoxide production, the mitochondrial membrane potential, and mitochondrial respiration. (A) Mitochondrial superoxide production was measured in HEK cells seeded at a density of 30000 cells per well to a 96 well plate the day before measurement. Cells were loaded with 5 μM MitoSOX red and Hoechst 33342 (both Life Technologies) for 10 minutes. The cells were treated with either 0.1% DMSO, 30% ethanol (positive control), 10 μM cyclosporine A (CsA), 1 μM thapsigargin, or 100 μM puromycin, and mitochondrial superoxide production was measured for the indicated times on a Cellomics ArrayScan VTI HCS Reader (Thermo Scientific). (B) Mitochondrial membrane potential was detected in HEK cells seeded at a density of 30000 cells per well to a 96 well plate the day before measurement. For positive control, cells were treated with 230 nM valinomycin (Sigma) for six hours. The cells were treated with either 0.1% DMSO, 10 μM cyclosporine A, 1 μM thapsigargin or 100 μM puromycin for 15 to 60 minutes in complete medium. The cells were stained for 30 minutes with Hoechst 33342 and MitoTracker Red CMXRos (both Life Technologies) and analyzed on a Cellomics ArrayScan VTI HCS Reader counting at least 1000 cells per well. Values were normalized to 15 minutes DMSO. (C) Left panel: Real-time measurements of oxygen consumption rate (OCR), reflecting mitochondrial respiration were performed on a Seahorse XF96 Analyzer (Agilent Technologies, USA) based on previous description (Nagy et al., Biochim Biophys Acta Bioenerg 2018, 1859, 201-214). Where indicated by arrows, cells were treated with metabolic inhibitors/modulators (oligomycin 2 μM, FCCP 100 nM, and antimycin A + rotenone 1 μM each). TG, puromycin, and CsA were applied 5 min ahead of the recording at the following concentrations: 1 μM, 100 μM, and 10 μM, respectively. Right panel: Basal respiration rate was detected before oligomycin addition; ATP-linked respiration was calculated as a difference between the values before oligomycin and before carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) addition; maximal respiratory capacity is the difference between values before and after rotenone + antimycin A addition; spare capacity difference between values before oligomycin and before rotenone + antimycin A addition; non-mitochondrial respiration is shown by the last value after rotenone + antimycin A addition. Data were obtained from two independent experiments (mean ± s.d). (PPTX 258 kb)

Additional file 3: Figure S3. BSO treatment prevents Hyper-ER reduction upon addition of thapsigargin. Fluorescence ratio changes of HyPer-ER sensor 24 hours after transfection in untreated or BSO treated HeLa cells. (PPTX 5979 kb)