ripk1 inhibitors  (Denali Therapeutics)

Journal: Cell Death & Disease

Article Title: RIPK3 promotes skin inflammation by enhancing IL-36α signaling and necroptosis in keratinocytes

doi: 10.1038/s41419-025-08096-9

Figure Lengend Snippet: Western blot ( A ) and RT-PCR ( B ) analysis of the expression level of RIPK3 in primary keratinocytes from RIPK3 f/f and RIPK3 ΔKC mice. C Cell viability of RIPK3-deficient primary keratinocytes following TSZ treatment was detected by CCK-8 kit. D Representative western blot analysis of the necroptosis pathway after TSZ treatment for 12 hours in primary keratinocytes. E Representative immunofluorescence analysis of Occludin in primary keratinocytes after TSZ treatment for 6 hours in primary keratinocytes. Scale bars, 100 μm. F RT-PCR analysis of mRNA levels of chemokines, cytokines and antimicrobial peptides after TSZ treatment for 6 hours in primary keratinocytes. G Cell viability of HaCaT cells treated with GSK’2982772 (100, 10 and 1 nM) and GSK’872 (10, 5 and 2.5 μM) was detected by CellTiter kit. Representative western blot analysis of the necroptosis pathway ( H ) in HaCaT cells treated with TSZ for 24 hours, and tight junction proteins ( I ) in HaCaT cells treated with TSZ for 6 hours. Representative immunofluorescence ( J ) and mRNA expression levels of IL-1α , IL-1β and CXCL8 measured by RT-PCR ( K ) in HaCaT cells treated with TSZ for 6 hours. Scale bars, 100 μm. These results are representative of three independent experiments. All dates are shown as means ± SEM. *** P < 0.001, compared as indicated, ### P < 0.001, compared with BLANK, were measured by Student’s t -test, one-way or two-way ANOVA. There was a significant difference between the RIPK3 f/f and RIPK3 f/f TSZ groups.

Article Snippet: Subsequently, application of 100 ng/ml murine TNF-α (Peprotech, London, UK) or human TNF-α (Peprotech), 50 nM SM-164 (MedChemExpress, NJ, USA), 20 μM Z-VAD-FMK (MedChemExpress) and different concentrations of RIPK1 inhibitors GSK’2982772 (MedChemExpress) and RIPK3 inhibitor GSK’872 (MedChemExpress) were treated for different times.

Techniques: Western Blot, Reverse Transcription Polymerase Chain Reaction, Expressing, CCK-8 Assay, Immunofluorescence

Journal: Infection and Immunity

Article Title: Rickettsia heilongjiangensis suppresses RIPK1 kinase-mediated host cell death during the infection

doi: 10.1128/iai.00158-25

Figure Lengend Snippet: Inhibition of RIPK1-mediated apoptosis and necroptosis in HMEC-1. (a) Immunoblot assay shows increased levels of cleaved caspase-3 (P17) protein associated with apoptosis in Rh -B8-infected cells. HMEC-1 cells were infected with Rh -B8 and harvested at 3, 6, 9, 12, 24, 48, and 72 hpi. The levels of proteins associated with necroptosis and pyroptosis, including phosphorylated MLKL (S358) and cleaved GSDMD (P30), were not activated throughout infection. (b) Western blot assay indicates that auto-phosphorylated RIPK1 at Serine 166 was activated during early infection. Throughout the infection, RIPK1 did not activate cleaved caspase-8 (P18) or induce phosphorylation of RIPK3 (as indicated by phosphorylation at Serine 227). Instead, the upregulation of phosphorylation at Ser 320 and Ser 416 indicates the inhibition of RIPK1’s kinase activity. Representative results from three independent experiments are presented for the blotting assays described above.

Article Snippet: Necrostain-1, a RIPK1 inhibitor (MedChemExpress HY-15760), was used at 25 μM.

Techniques: Inhibition, Western Blot, Infection, Phospho-proteomics, Activity Assay

Journal: Infection and Immunity

Article Title: Rickettsia heilongjiangensis suppresses RIPK1 kinase-mediated host cell death during the infection

doi: 10.1128/iai.00158-25

Figure Lengend Snippet: Inhibition of caspase-8 does not sensitize Rh -B8-infected HMEC-1 cells to necroptosis. (a and b) Rh -B8-infected cells treated with the caspase-3 inhibitor (Z-DEVD-FMK) showed a significant reduction in cell death during the late stages of infection. An immunofluorescence assay showcased images of TUNEL (green) and DAPI (blue) in HMEC-1 cells infected with Rickettsia (red) at 72 hpi. The growth curve illustrates the replication of Rh -B8 and cell viability of HMEC-1 treated or untreated with Z-DEVD-FMK. (c and d) Rh -B8-infected HMEC-1 cells treated with the caspase-8 inhibitor (Z-IETD-FMK) did not show protection against apoptosis at 72 hpi. The replication of Rh -B8 and cell viability of HMEC-1 between the Z-IETD-FMK-treated and untreated groups exhibited no significant difference. (e and f) The activity of RIPK1 kinase, blocked by necrostatin-1, also did not influence rickettsial growth or host cell viability. (g) The Western blot assay demonstrated that HMEC-1 cells infected with Rh -B8 and treated with the RIPK1 inhibitor necrostatin-1 (Nec-1) at various stages continued to undergo apoptosis. Representative results from three independent experiments are presented for the blotting assays. Uninfected cells treated with each inhibitor for 72 hours were included as controls in panels (a), (c), and (e). Data in (b), (d), and (f) were shown as mean ± SD (each time point has three biological replicates). *** P < 0.001 relative to normal infection, P -values were calculated using an unpaired t -test (two-tailed).

Article Snippet: Necrostain-1, a RIPK1 inhibitor (MedChemExpress HY-15760), was used at 25 μM.

Techniques: Inhibition, Infection, Immunofluorescence, TUNEL Assay, Activity Assay, Western Blot, Two Tailed Test

Journal: Infection and Immunity

Article Title: Rickettsia heilongjiangensis suppresses RIPK1 kinase-mediated host cell death during the infection

doi: 10.1128/iai.00158-25

Figure Lengend Snippet: Nuclear factor κB protects against RIPK1-mediated host cell apoptosis and necroptosis during Rh -B8 infection. (a) The GSEA enrichment analysis, conducted based on the significantly upregulated genes between the infection group and the control group, indicates that the length of the bars represents the number of genes enriched in each pathway. (b) GSEA analysis indicates the NF-κB pathway was significantly associated with Rh-B8 infection at 24 hpi, 48 hpi, and 72 hpi. (c) The bar plot presents the top 14 significantly enriched transcription factors, with NFKB1 ranked as the primary contributor. The horizontal axis shows the −log 10 adjusted P -value scale. (d) Immunoblot assay showcases the levels of total NF-κB in cell lysates and the amount that translocated into the nucleus. Representative results from three independent experiments are presented for the blotting assays. (e) Growth curves generated using quantitative RT-PCR at the indicated time points indicate that SN50 treatment, an inhibitor that restrains NF-κB translocation, significantly reduces cell viability and restricts Rh -B8 replication. * P < 0.1, *** P < 0.001 relative to normal infection, P -values were calculated using an unpaired t -test (two-tailed). (f) Western blot analysis was performed to evaluate the expression levels of key proteins, including cleaved caspase-3, phosphorylated RIPK3 (p-RIPK3, S227), phosphorylated MLKL (p-MLKL, S358), cleaved N-terminal GSDMD (N-GSDMD), caspase-8, and its cleaved isoforms, in HMEC-1 cells treated with SN50 upon infection by Rh -B8. Representative results from three independent experiments are presented for the blotting assays. (g) Densitometric analysis of cleaved caspase-8 (P18) during Rh -B8 infection, both with and without SN50 supplementation, was conducted using ImageJ. Statistical significance was assessed using Bonferroni’s multiple comparisons test. The data presented are representative of three biological replicates from three independent experiments. ** P < 0.01, *** P < 0.001.

Article Snippet: Necrostain-1, a RIPK1 inhibitor (MedChemExpress HY-15760), was used at 25 μM.

Techniques: Infection, Control, Western Blot, Generated, Quantitative RT-PCR, Translocation Assay, Two Tailed Test, Expressing