Fig. 3. TUFM suppresses CASP8 activation induced by vIRF-1 depletion.
A Immunoblots of total-cell extracts derived from the indicated iBCBL-1 cell lines that were left untreated or treated with 1 µg/ml Dox for 3 days. CASP8 cleavage products p41/43 (marked by the red dotted rectangle) in the different extracts were quantified relative to each other, after normalization to LDH; the values are provided below the CASP8 blot. In addition, the relative levels of the cleavage fragment (cCASP3) of CASP3, detected by antibody specific to cleaved CASP3, were determined as above and noted under the blot. B Immunoblots of total-cell extracts of the iBCBL-1 cell lines transduced with empty lentiviruses (EV) or lentivirus expressing TUFM-Flag and 1 day later treated with Dox for 3 days. The relative levels of the p41/43 fragments of CASP8 are noted under the blot. C Detection of CASP8 activity using SR-FLICA® CASP8 (C8-FLICA) reagent in vIRF-1 KD or vIRF-1/TUFM dKD lytic iBCBL-1 cells treated with Dox for 3 days. After C8-FLICA staining, the cells were subjected to TOM20-IFA and DAPI nuclear staining. Scale bar, 15 µm. D Determination of the percentages of cells with altered mitochondria. Images were taken of four random fields for each culture: vIRF-1 KD (n = 570) and vIRF-1/TUFM dKD (n = 472). In addition, the percentage of C8-FLICA-negative or positive cells among cells showing altered mitochondria was determined. Statistical significance of vIRF-1 KD vs vIRF-1/TUFM dKD in C8 FLICA-negative or -positive cells was determined using student t-test. **p < 0.01; ns not significant. E The fluorescence intensities of TOM20 and C8-FLICA were measured in the cells with altered mitochondria above, vIRF-1 KD (n = 27) and vIRF-1/TUFM dKD (n = 47), using ImageJ software, and background-corrected integrated densities (IntDen) were plotted along with an arbitrary threshold (dotted blue line) of 10,000. F Functional relationship between altered mitochondria and CASP8 activation in the context of apoptosis. A representative image showing vIRF-1/TUFM dKD cells with either C8-FLICA (red dotted circle) or altered mitochondria (green dotted circle), or both (white dotted circle) after lytic reactivation is presented along with a DAPI image. White arrow indicates co-localization of C8-FLICA and mitochondria. Apoptosis was assessed by nuclear condensation (green arrow) or fragmentation (blue arrow). Scale bar, 10 µm. The chart shows the percentage of apoptotic vIRF-1 KD or vIRF-1/TUFM dKD cells with both C8-FLICA and altered mitochondria. It is noteworthy that the few cells (<0.1%) that are TOM20-negative and not nucleus-fragmented still showed CASP8 activation, supporting a requirement for mitochondria in CASP8-mediated apoptosis. G Immunoblots of mitochondrial extracts of the iBCBL-1 cell lines left untreated or treated with 1 µg/ml Dox for 3 days. HSP60 was used as a mitochondrial-protein loading control. The relative levels of the p41/43 fragments normalized to HSP60 are noted under the blot. H C8-FLICA assays in control (shLuc) and TUFM KD iBCBL-1 cell lines lentivirally transduced with EV or Flag-tagged DRP1K38A. The transduced cells were treated with or without Dox for 2 days, stained with C8-FLICA reagent, and subjected to Flag-IFA (Fig. S5). The percent of C8-FLICA-positive cells were determined and depicted in the graph. The data present the mean ± SD of three different images. *p < 0.05; ***p < 0.001. “n” indicates the total-cell numbers counted. I Immunoblots of cell extracts of the indicated shRNA-expressing HeLa.Kyoto cell lines left untreated or treated with 50 ng/ml of TRAIL and/or 20 µM mdivi-1 for 6 h. For induction of shRNA expression, the HeLa.Kyoto cell lines were pretreated with Dox for 3 days.