Fig 2: High throughput fluorescence microscopy workflow of C28/I2 human chondrocyte cell line transfected with CaMPARI2-F391W. (A) The IncuCyte S3 high-throughput microscope of Sartorius took five images per well of 96-well plates. The shown cells were treated with 100 µM histamine directly after the parabolic flight and were illuminated for 8 s by photoconversion light 15 s after the histamine stimulus. The centered image (red frame in (A)) was used as an example to show the mask created with the IncuCyte 2021A software in (B,D). (B,D) Green and Red fluorescence of CaMPARI2-F391W is depicted as an orange merge. The red frame is the image section displayed in (C,E). (C,E) Cells only identified as green fluorescent are circled cyan. Cells only identified as red fluorescent are circled purple. Cells identified by the IncuCyte mask as green and red fluorescent are colored white. The scales in (B,C) represent 500 µm; the scales in (D,E) represent 100 µm.

Fig 3: NIT-1 and INS-1 ß cells are susceptible to RIPK1- and cIAP-mediated TNFa-induced cell death. A Sartorius IncuCyte S3 live cell imaging and analysis instrument was used to monitor cell death via quantification of Sytox green positive cells. Cell culture treatment conditions are indicated by color (black: vehicle, blue: 40 ng/mL TNFa, red: 5 µM BV6, purple: TNFa+BV6) and ß-cell lines are indicated by shapes (NIT-1 CTL: circles, NIT-1 RIPK1?: triangles, INS-1: squares). A) Cell death was monitored for 24 h in NIT-1 CTL and NIT-1 RIPK1? cells following TNFa treatment, and B) cell death was quantified 24 h post treatment (n = 6). C) Percent NIT-1 CTL cell death was quantified 24 h after treatment with vehicle or TNFa using the Sartorius IncuCyte S3 advanced label-free classification analysis software module, as described (n = 6). D) NIT-1 CTL and NIT-1 RIPK1? cell caspase 3/7 activity was quantified 24 h after treatment and expressed relative to vehicle treated NIT-1 CTL cells (n = 6). E) NIT-1 CTL and NIT-1 RIPK1? cells were treated with BV6, TNFa or a combination thereof, then F) cell death was quantified 4 h post treatment (n = 5–6). For comparisons between cell lines, cell death was reported as Sytox green positive cell objects normalized to phase positive cell area for each time point. G) Percent NIT-1 CTL cell death was quantified 4 h after treatment with BV6, TNFa or a combination thereof using the Sartorius IncuCyte S3 advanced label-free classification analysis software module (n = 4–5). H) NIT-1 CTL and NIT-1 RIPK1? cell caspase 3/7 activity was quantified 4 h after treatment and expressed relative to vehicle treated NIT-1 CTL cells (n = 4–6). I) Genomic DNA was isolated from NIT-1 CTL cells following treatment with vehicle or TNFa+BV6 for 4 h, then visualized on an agarose gel. J) For INS-1 cells, cell death was monitored for 24 h following treatment with BV6, TNFa or a combination thereof, then K) cell death was quantified 24 h post treatment (n = 5). For comparisons within a cell line, cell death was reported as Sytox green positive cell objects relative to time t = 0. L) Percent INS-1 cell death was quantified 24 h after treatment using the Sartorius IncuCyte S3 advanced label-free classification analysis software module, as described (n = 4). M) Representative IncuCyte images illustrate Sytox green positive INS-1 cells 24 h post treatment. Data are presented as mean ± SEM and were analyzed by one-way ANOVA followed by Sidák post-test and multiple comparisons correction. *p < 0.05; **p < 0.01; ***p < 0.001 as indicated. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig 4: RIPK3-deficient macrophages are competent to perform efferocytosis and antibody-dependent cellular phagocytosis.A Primary peritoneal macrophages isolated from Ripk3+/+ and Ripk3-/- mice were co-incubated in 1:5 ratio with apoptotic (dexamethasone-killed, DEX) or non-apoptotic (no DEX) thymocytes stained with pH-sensitive dye, CypHER 5E. Live cell imaging was performed with IncuCyte® S3 (Sartorius). Representative images of macrophages which engulfed DEX-treated or no DEX-treated thymocytes become positive for CypHER 5E (red) can be seen. The difference between Ripk3+/+ and Ripk3-/- at 8 h of co-incubation is shown as fold change relative to the no DEX control (three independent experiments). B M2e-coated fluorescent polystyrene beads were incubated in 2.5:1 ratio with primary alveolar macrophages isolated form Ripk3+/+ or Ripk3-/- mice in the absence or presence of 0.1 µg/mL of isotype control antibody, Anti-SHE or an anti-M2e mouse IgG2a monoclonal antibody (Mab65) specific antibody, Anti-M2e. Live cell imaging was performed with Operetta high content imaging system and analysis was done with Harmony software. The difference between Ripk3+/+ and Ripk3-/- at 8 h of co-incubation is shown as fold increase in antibody-mediated phagocytosis relative to no antibody control (three independent experiments). Significance was determined using one-way ANOVA with Tukey correction (GraphPad Prism 8). Error bars represent SEM.

Fig 5: RIPK1 and cIAPs regulate TNFa-induced NIT-1 and INS-1 ß-cell death when caspases are inhibited. A) NIT-1 CTL and NIT-1 RIPK1? cell death was monitored over 48 h, and B) quantified at 48 h post treatment (n = 5–6). Cell culture treatment conditions are indicated by color (black: vehicle, green: 40 ng/ml TNFa + 50 µM zVAD, white: TNFa+zVAD + 5 µM BV6) and ß-cell lines are indicated by shapes (NIT-1 CTL: circles, NIT-1 RIPK1?: triangles, INS-1: squares). C) Percent NIT-1 CTL cell death was quantified 48 h after treatment with vehicle, TNFa +zVAD or TNFa+BV6+zVAD using the Sartorius IncuCyte S3 advanced label-free classification analysis software module, as described (n = 6). D) NIT-1 CTL and RIPK1? cell caspase 3/7 activity was quantified 24 h post treatment and expressed relative to vehicle treated NIT-1 CTL cells (n = 4–5). E) Genomic DNA was isolated from NIT-1 CTL cells following treatment with vehicle or TNFa+BV6+zVAD for 8 h, then visualized on an agarose gel. F) INS-1 cell death was monitored over 24 h, and G) quantified at 24 h post treatment (n = 5–6). H) Percent INS-1 cell death was quantified 24 h after treatment using the Sartorius IncuCyte S3 advanced label-free classification analysis software module (n = 4). I) Representative IncuCyte images illustrate Sytox green positive INS-1 cells 24 h post treatment. Data are presented as mean ± SEM and were analyzed by one-way ANOVA followed by Sidák post-test and multiple comparisons correction. *p < 0.05; **p < 0.01; ***p < 0.001 as indicated. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)