Fig 2: Caspase-4 contributes to the arrest in cell proliferation in OIS.A IMR90 ER:STOP and ER:RAS cells were transfected with control (NTP), two individual CASP4-targeting siRNAs (CASP4-1 and CASP4-2) or a pool of 4 different siRNA sequences targeting CASP4 (CASP4-p), and treated with 4OHT or not as indicated. BrdU incorporation was measured by immunofluorescence 5 days after 4OHT addition. Graph bars, error bars and dots represent respectively the mean ± s.e.m. and the individual values of 3 independent experiments. Statistical analysis was performed using two-tailed Student’s t test. B, C IMR90 cells were transduced with an empty retroviral vector (vector) or shRNAs targeting either CASP4 (shCASP4) or TP53 (shP53). B Lysates were subjected to immunoblotting analyses with the indicated antibodies eight days after 4OHT addition. C On day 0, equal number of cells were subjected to 4OHT treatment. Fifteen days after 4OHT addition, plates were fixed and stained with crystal violet. Crystal violet was extracted and used to quantify cell content. Graph bars, error bars and dots represent respectively the mean ± s.e.m. and the individual values of 4 independent experiments. Statistical analysis was performed using two-tailed Student’s t test. D Related to Fig. 5A. DEG analysis was performed between control ER:STOP and CASP4-knockdown ER:RAS compared to control ER:RAS after 5 days of 4OHT treatment. Heatmap of the log2FC values from the indicated genes. E IMR90 ER:STOP and ER:RAS cells were transfected with control (NTP), CASP1 or CASP4- targeting siRNAs and treated with 4OHT during 4 days. Cell lysates were subjected to immunoblotting analyses with the indicated antibodies. F IMR90 ER:RAS cells were transfected with control (NTP), CASP1 or CASP4-targeting siRNA and mRNA relative expression of the indicated genes was quantified by RT-qPCR after 5 days of 4OHT treatment. Graph bars, error bars and dots represent respectively the mean ± s.e.m. and the individual values of 3 independent experiments. Statistical analysis was performed using one-way analysis of variance (ANOVA). ***P < 0.001, **P < 0.01, *P < 0.05 and ns, not significant.

Fig 3: The caspase-4 noncanonical inflammasome is activated in oncogene-induced senescence.A IMR90 cells were infected with RASG12V expression vector to induce OIS. BrdU incorporation and SA-ß-Galactosidase activity were measured 4 days after equal number of cells were seeded (left). Representative images (right) for SA-ß-Galactosidase activity are shown. Graph bars, error bars and dots represent respectively the mean ± s.e.m. and the individual values of 3 independent experiments. Statistical analysis was performed using two-tailed Student’s t test. B RASG12V-OIS was induced as in (A) and CASP4 mRNA relative expression was quantified by RT-qPCR. Graph bars, error bars and dots represent respectively the mean ± s.e.m. and the individual values of 3 independent experiments. Statistical analysis was performed using two-tailed Student’s t test. C RASG12V-OIS was induced as in (A) and BrdU incorporation and caspase-4 protein levels were measured by immunofluorescence in RASG12V-OIS and control cells 4 days after equal number of cells were seeded. Graph bars, error bars and dots represent respectively the mean ± s.e.m. and the individual values of 3 independent experiments. Statistical analysis was performed using two-tailed Student’s t test. D IMR90 cells were infected with a control (ER:STOP) or an ER:RAS vector. Upon addition of 4OHT, ER:RAS cells undergo OIS. OIS can be detected by a reduction in BrdU incorporation (lower left) measured by immunofluorescence 5 days after 4OHT addition and an increase in SA-ß-Galactosidase activity (lower right) one week after 4OHT addition. Graph bars, error bars and dots represent respectively the mean ± s.e.m. and the individual values of 3 independent experiments. Statistical analysis was performed using two-tailed Student’s t test. E Time-course experiment of CASP4 mRNA relative expression in OIS. IMR90 ER:STOP and ER:RAS cells were treated with 4OHT and CASP4 mRNA relative expression was quantified by RT-qPCR 0, 2, 4, 6 and 8 days after 4OHT addition. Graph lines and dots represent respectively the mean and the individual values of 3 independent experiments. Statistical analysis was performed using two-tailed Student’s t test. F Time-course experiment of IL1B mRNA relative expression in OIS. IMR90 ER:STOP and ER:RAS cells were treated with 4OHT and IL1B mRNA relative expression was quantified by RT-qPCR 0, 2, 4, 6, and 8 days after 4OHT addition. Graph lines and dots represent respectively the mean and the individual values of 3 independent experiments. Statistical analysis was performed using two-tailed Student’s t test. G IMR90 ER:STOP and ER:RAS were treated or not with 4OHT during eight days. Caspase-4, IL-1ß and IL-8 protein levels were analyzed by immunoblotting. H To detect caspase-4 oligomers, IMR90 ER:STOP and ER:RAS cells were treated with 4OHT for five days, then cells were collected and subjected to disuccinimidyl suberate (DSS) crosslinking. After SDS-PAGE separation, DSS-cross-linked samples were probed for caspase-4 by immunoblotting. (I) Caspase-4 activity in OIS was measured using a fluorometric assay. IMR90 ER:STOP and ER:RAS cells were treated with 4OHT and LEVD-AFC cleavage was measured in low serum (0.5% FBS) cultured cells 0, 4, and 8 days after 4OHT addition. Graph bars, error bars and dots represent respectively the mean ± s.e.m. and the individual values of 3 independent experiments. Statistical analysis was performed using two-tailed Student’s t test. ***P < 0.001, **P < 0.01, and *P < 0.05 and ns, not significant. Scale bar = 250 µm as indicated.

Fig 4: LPS-mediated caspase-4 induced senescence is independent of inflammasome priming.A CASP4, CASP1 or RASG12V were overexpressed in IMR90 cells, IL1A and IL1B mRNA relative expression levels were quantified by RT-qPCR. Graph bars, error bars and dots represent respectively the mean ± s.e.m. and the individual values of 3 independent experiments. Statistical analysis was performed using one-way analysis of variance (ANOVA). B Cells were treated as shown in Fig. 1A. CASP1 or CASP4 expression was targeted by shRNA prior to LPS transfection with 0.1 µg LPS. IL1B mRNA relative expression was quantified by RT-qPCR 48 h after LPS transfection. Graph bars, error bars and dots represent respectively the mean ± s.e.m. and the individual values of 3 independent experiments. Statistical analysis was performed using one-way analysis of variance (ANOVA). C–E IMR90 cells were infected with CASP4 or RASG12V expression vectors or empty vector (vector) control. After 3 h treatment with Pam2CSK4, cells were transfected with 0.1 µg LPS. IL1B mRNA relative expression (C) and BrdU incorporation (D) were measured by IF and RT-qPCR respectively 48 h after LPS transfection. E SA-ß-Galactosidase activity was determined 4 days after LPS transfection. Representative images for SA-ß-Galactosidase activity are shown. Graph bars, error bars and dots represent respectively the mean ± s.e.m. and the individual values of 3 independent experiments. Statistical analysis was performed using one-way analysis of variance (ANOVA). ****P < 0.0001, ***P < 0.001, **P < 0.01, and *P < 0.05. Scale bar = 0.1 mm as indicated.

Fig 5: LPS-mediated caspase-4 activation induces a senescent phenotype in human primary fibroblasts.A Schematic representation of LPS transfection experiments shown in B-D and Supplementary Fig. 1 E–G. IMR90 cells were infected with an empty pGIPZ vector (vector) or shRNA targeting either CASP1 (shC1) or CASP4 (shC4) before transfection with 0.1 µg LPS. The acquisition of senescent features after LPS transfection was assessed by immunofluorescence of senescence markers, mRNA expression analysis by RT-qPCR and SA-ß-Galactosidase activity. B SA-ß-Galactosidase activity was determined in IMR90 cells 4 days after LPS transfection. Representative images for SA-ß-Gal activity are shown. Graph bars, error bars, and dots represent respectively the mean ± standard error of the mean (s.e.m.) and the individual values of 4 independent experiments. Statistical analysis was performed using one-way analysis of variance (ANOVA). C BrdU incorporation and p16INK4a, p21CIP1, and caspase-4 protein expression levels were measured by immunofluorescence in IMR90 cells 48 h after LPS transfection. Graph bars, error bars and dots represent respectively the mean ± s.e.m. and the individual values of 4 independent experiments. Statistical analysis was performed using two-tailed Student’s t test. D CDKN1A (p21CIP1) and CDKN2A (p16INK4a) mRNA relative expression was quantified by RT-qPCR in IMR90 cells 48 h after transfection. Graph bars, error bars and dots represent respectively the mean ± s.e.m. and the individual values of 3 independent experiments. Statistical analysis was performed using one-way analysis of variance (ANOVA). E CASP4 was overexpressed prior to LPS transfection in IMR90 cells. 5 ×105 control (vector) and CASP4 expressing IMR90 cells were transfected with increasing concentrations of LPS (0.1 or 1 µg), and cell proliferation was measured by BrdU incorporation 48 h after transfection. Graph bars, error bars, and dots represent respectively the mean ± s.e.m. and the individual values of 3 independent experiments. Statistical analysis was performed using one-way analysis of variance (ANOVA). F IMR90 cells were treated as in (E) and SA-ß-Galactosidase activity was determined 4 days after LPS transfection. Representative images for SA-ß-Galactosidase assay are shown. Graph bars, error bars, and dots represent respectively the mean ± s.e.m. and the individual values of 3 independent experiments. Statistical analysis was performed using one-way analysis of variance (ANOVA). G IMR90 cells were infected with an empty pGIPZ vector (vector) or shRNA targeting either CASP4 (shC4), GSDMD (shGSDMD) or TP53 (shP53) and protein expression analysis for Caspase-4, Gasdermin-D, p53, and ß-Actin as loading control was performed by western blot. H–J IMR90 cells were transduced with an empty pGIPZ vector (vector) or shRNAs targeting either CASP4 (shC4), GSDMD (shGSDMD) or TP53 (shP53) prior to transfection with 0.1 µg LPS. BrdU incorporation (H) and the levels of p21CIP1 (I) and p16INK4a (J) were measured by immunofluorescence in IMR90 cells 48 h after LPS transfection. Graph bars, error bars and dots represent respectively the mean ± s.e.m. and the individual values of 3 independent experiments. Statistical analysis was performed using one-way analysis of variance (ANOVA). ****P < 0.0001, ***P < 0.001, **P < 0.01, and *P < 0.05. ns, not significant. Scale bar = 0.1 mm as indicated.