Fig 2: Extrinsic or intrinsic apoptotic inducers differentially trigger mitochondrial depolarization.(A to D) Analysis of mitochondria membrane depolarization using TMRE measurements by flow cytometry in WT, caspase-9, and caspase-8 knockout NALM6 cells, upon intrinsic [(A) 5 µM ABT263, quantitation in (B)] or extrinsic trigger [(C) Birinapant (50 nM), TNFa (10 ng/ml), quantitation in (D)]. Graphs show the mean ± SEM for three repeated experiments performed in duplicate. Student’s t test was used for comparison between single time point values as indicated.

Fig 3: hUsp27xL or mUsp27xS overexpression sensitizes 1205Lu and WM1158 human melanoma cells to TNF- and pIC induced apoptosis. A, Overexpression of enzymatic active mUsp27xS or hUsp27xL specifically sensitizes 1205Lu to TNF and pIC induced apoptosis. Wilde Type (WT), mUsp22, Usp27xS/L-inducible (mUsp27xS-C87A is catalytically inactive [20]) 1205Lu cells were treated 72 h with doxycycline (Dox) and/or 100 ng/ml TNF / 50 µg/ml poly-I:C (pIC). Active caspase-3 as a marker for apoptosis was determined by flow cytometry. Shown is the rate of active-caspase-3 positive cells (n = 5). B, Induction of mUsp27xS or hUsp27xL enhances apoptosis in TNF treated WM1158 cells in a dose dependent manner. WT or inducible WM1158 cells were pre-stimulated 24 h with doxycycline to induce Usp27x or Usp22 and afterwards indicated concentrations of TNF were added for additional 24 h. Active caspase-3 was determined by flow cytometry (WT, n = 7; TetR-GFP-mUsp27xS, n = 8; TetR-GFP-hUsp27xL, n = 5; TetR-GFP-mUsp22, n = 3). C, Induction of hUsp27xL enhances apoptosis in pIC treated WM1158 cells in a dose dependent manner independently of the mitochondrial pathway. GFP-hUsp27xL-inducible WM1158, Bax-/-/Bak-/- GFP-hUsp27xL- inducible WM1158 or constitutional overexpressing Bcl-XL GFP-hUsp27xL-inducible WM1158 cells were pre-stimulated 48 h with Dox to induce Usp27x and afterwards indicated concentrations of pIC were added for 4 h. Again, active caspase-3 was determined by flow cytometry (TetR-GFP-hUsp27xL treated with 50/ 100 ng/ml pIC, n = 3; other samples: n = 4). D, The mitochondrial pathway does not restrict caspase-8 processing or cFLIPL decrease in hUsp27xL overexpressing and pIC treated WM1158 cells. DISC-lysates of indicated doxycycline (Dox) inducible WM1158 cells. Cells were or were not pre-treated 48 h with Dox and then pIC was added to indicated samples for 4 h. Proteins were detected by Western blot. Arrow-heads indicate specific signal (n = 1). E, pIC-induced apoptotic cell death in Usp27x over-expressing WM1158 melanoma cells is not dependent on RIPK1 kinase activity. GFP-hUsp27xL inducible WM1158 cells were pre-stimulated 48 h with Dox. Cells were then pre-treated 30 min with Nec1 [10 µM] to inhibit RIPK1 kinase activity. Afterwards 50 ng/ml pIC was added for 4 h. QVD [10 µM] was added as indicated. Active caspase-3 was determined by flow cytometry (n = 4). A-C, E, Data (means, ns: not significant (adjusted p value > 0.05)). Error bars represent SEM. *, **, ***: significant, adjusted p values, see 2 section

Fig 4: miR-96-5p reduces TNF-α production by inhibiting IKKβ expression. (A) Reverse transcription-quantitative PCR was used for confirmation of knockdown and overexpression of IKKβ. (B) miR-96-5p mimic reduced TNF-α release from OGD-treated BV-2 cells, while IKKβ overexpression rescued this effect. (C) miR-96-5p inhibitor enhanced TNF-α release from OGD-treated BV-2 cells while IKKβ inhibition counteracted this effect. (D) miR-96-5p was negatively correlated with IKKβ in middle cerebral artery occlusion-treated mouse cerebral tissues. Spearman's rank correlation coefficient was used. *P<0.05 and **P<0.01. miR-96-5p, microRNA-96-5p; TNF, tumor necrosis factor; IKKβ, inhibitor of nuclear factor κB kinase subunit β; OGD, oxygen/glucose deprivation; Ctrl, control; sh-, short hairpin.

Fig 5: APG-1387 induces cell death partly through TNF-alpha signaling pathway. a ELISA analysis of the secretion of TNF-alpha after different time points of APG-1387 on SKOV3 cells streatment. b Cells were treated with different time points of APG-1387, and the secretion of TNF-alpha were analyzed by ELISA. c Cells were transfected with TNFR1 siRNAs, cell viability was determined by the CCK-8 assay. d Cells were transfected with TNF-alpha siRNAs, Cell viability was determined by the CCK-8 assay. e APG-1387 was coadministered with or without TNF-alpha neutralizing antibody, cell viability was determined by the CCK-8 assay. f Cells were treated with different concentrations of APG-1387 on cells after 24 h treatment and the expression levels of NF-?B1/p50 and NF-?B2/p52 were determined by western blot. g The protein levels of NF-?B1/p50 and NF-?B2/p52 in cells by western blot after APG-1387 was coadministered with or without Necrostatin-1