Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Wogonin protects against cisplatin-induced acute kidney injury by targeting RIPK1-mediated necroptosis.

doi: 10.1038/labinvest.2017.115

Figure Lengend Snippet: Figure 5 Wogonin fails to further reduce cisplatin-induced cell injury and inflammatory response in HK2 cells where RIPK1 is blocked. (a) Western blot analysis and quantitative data of RIPK1/RIPK3/MLKL signaling in Nec-1-treated HK2 cells. (b) Western blot analysis and quantitative data of KIM-1. Results indicated that when RIPK1 was blocked, wogonin failed to further suppress the protein levels of KIM-1. (c) Real-time PCR showed that when RIPK1 was blocked, wogonin failed to further decrease IL-6 in mRNA level. Independent experiments were performed throughout the in vitro studies in triplicate or quadruplicate. *Po0.05, **Po0.01, ***Po0.001 compared with the control. ##Po0.01, ###Po0.001 compared with cisplatin-treated group. $$Po0.01, $$

Article Snippet: Then, western blot analysis was performed as described previously.16,17 After blocking nonspecific binding with 5% BSA (room temperature, 1 h), membranes were consequently incubated with the primary antibody against KIM-1, RIPK1, RIPK3, p-MLKL, cleaved-caspase-3, cleaved-caspase-8 and β-actin overnight at 4 °C, followed by treated with IRDye 800-conjugated secondary antibody (Rockland immunochemicals, Gilbertsville, PA, USA).

Techniques: Western Blot, Real-time Polymerase Chain Reaction, In Vitro, Control

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Wogonin protects against cisplatin-induced acute kidney injury by targeting RIPK1-mediated necroptosis.

doi: 10.1038/labinvest.2017.115

Figure Lengend Snippet: Figure 6 Wogonin fails to further reduce cisplatin-induced cell injury and inflammatory response in HK2 cells where RIPK3 is disrupted. (a) Western blot analysis and quantitative data of RIPK3 in HK2 cells. Results showed that RIPK3 was downregulated by transfection of RIPK3 shRNA plasmid. (b) Western blot analysis and quantitative data of RIPK1/RIPK3/MLKL signaling in RIPK3 knockdown HK2 cells. (c) Western blot analysis and quantitative data of KIM-1. Results indicated that when RIPK3 was knockdown, wogonin failed to further decrease the protein levels of KIM-1. (d) Real-time PCR identified that when RIPK3 was disrupted, wogonin failed to further decrease mRNA levels of TNF-α and IL-6. Independent experiments were performed throughout the in vitro studies in triplicate or quadruplicate. **Po0.01,***Po0.001 compared with the control. #Po0.05, ##Po0.01, ###Po0.001 compared with cisplatin-treated group. $$Po0.01,$$$Po0.001 compared with RIPK3 EV group. Cis, cisplatin; Wog, wogonin; EV, empty vector; KD, knockdown.

Article Snippet: Then, western blot analysis was performed as described previously.16,17 After blocking nonspecific binding with 5% BSA (room temperature, 1 h), membranes were consequently incubated with the primary antibody against KIM-1, RIPK1, RIPK3, p-MLKL, cleaved-caspase-3, cleaved-caspase-8 and β-actin overnight at 4 °C, followed by treated with IRDye 800-conjugated secondary antibody (Rockland immunochemicals, Gilbertsville, PA, USA).

Techniques: Western Blot, Transfection, shRNA, Plasmid Preparation, Knockdown, Real-time Polymerase Chain Reaction, In Vitro, Control

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Wogonin protects against cisplatin-induced acute kidney injury by targeting RIPK1-mediated necroptosis.

doi: 10.1038/labinvest.2017.115

Figure Lengend Snippet: Figure 9 Wogonin prevents RIPK1-mediated necroptosis in cisplatin nephropathy. (a) Electron microscope. Results clearly demonstrated that wogonin largely reduced nuclear structure damage and abnormal cell organelle content in injured kidney. (b) Western blot analysis of RIPK1/RIPK3/MLKL signaling. Results showed that treatment of wogonin significantly suppressed the activation of RIPK1/RIPK3/MLKL axis in cisplatin-injured kidney. Data represent the mean ± s.e.m. for 6–8 mice. ***Po0.001 compared with control. ##Po0.01, ###Po0.001 compared with model. Cis, cisplatin; Wog, wogonin.

Article Snippet: Then, western blot analysis was performed as described previously.16,17 After blocking nonspecific binding with 5% BSA (room temperature, 1 h), membranes were consequently incubated with the primary antibody against KIM-1, RIPK1, RIPK3, p-MLKL, cleaved-caspase-3, cleaved-caspase-8 and β-actin overnight at 4 °C, followed by treated with IRDye 800-conjugated secondary antibody (Rockland immunochemicals, Gilbertsville, PA, USA).

Techniques: Microscopy, Western Blot, Activation Assay, Control

Journal: International journal of molecular sciences

Article Title: Activation of the TNF-α-Necroptosis Pathway in Parvalbumin-Expressing Interneurons of the Anterior Cingulate Cortex Contributes to Neuropathic Pain.

doi: 10.3390/ijms242015454

Figure Lengend Snippet: Figure 2. TNF-α-necroptosis pathway is activated in the ACC of neuropathic pain models. Volcano map (A) and heat map (B) showing the expression of TNF-α, RIP1, RIP3, and MLKL in the ACC of CCI model of neuropathic pain (n = 3/group) and controls (n = 3/group) (data from GEO dataset GSE212311). The Hub gene expression data of CCI model (red dots) and control (green dots) for TNF-α, RIP1, RIP3, and MLKL are shown in (C). * p < 0.05 versus the control (unpaired t test). Volcano map (D) and heat map (E) showing the expression of TNF-α, IL-6, RIP1, and MLKL in the ACC of SNI model (n = 3/group) and controls (n = 3/group) (data from GEO dataset GSE228065). (F) Representative triple staining shows the overlap of TNF-α (green) with p-RIP3 (red) and PV (magenta), or p-MLKL (red) and PV (magenta) on PO day 7. Enlarged and color-split images of the area enclosed in white boxes are shown below. White arrows indicate co-localization. Blue fluorescence corresponds to DAPI.

Article Snippet: To seal it at room temperature, 5% skimmed milk was used to for almost 1 h, then end up placing the membranes in p-RIP1 antibody (1:1000, Affinity, Cincinnati, OH, USA), anti-p-RIP3 antibody (1:1000, CST, Danvers, MA, USA), anti-p-MLKL antibody (1:1000, Affinity, Cincinnati, OH, USA), anti-RIP1 antibody (1:1000, Novus, Littleton, CO, USA), anti-RIP3 antibody (1:1000, Novus, Littleton, CO, USA), anti-MLKL antibody (1:1000, Millipore, Billerica, MA, USA), and anti-TNF-α antibody (1:1000, Abcam, Cambridge, UK) overnight in the refrigerator at 4 ◦C.

Techniques: Expressing, Gene Expression, Control, Staining

Journal: International journal of molecular sciences

Article Title: Activation of the TNF-α-Necroptosis Pathway in Parvalbumin-Expressing Interneurons of the Anterior Cingulate Cortex Contributes to Neuropathic Pain.

doi: 10.3390/ijms242015454

Figure Lengend Snippet: Figure 3. SNI increases the expression of necroptosis-related protein p-RIP1 in the contralateral ACC (A) Representative Western blot of RIP1 and p-RIP1 expression in the bilateral ACC is shown in the top panel. The quantitative results of Western blotting protein are shown below. SNI induces the expression of p-RIP1in the contralateral ACC at postoperative (PO) day 7. ** p < 0.01 versus the sham group (two-way ANOVA). (B) Representative double staining shows the overlap (yellow) of p-RIP1 (red) with NeuN (neuronal marker, green) and PV (PV-IN marker, green), but not with GFAP (astrocyte marker, green) or Iba1 (microglia marker, green), on PO day 7. Enlarged and color- split images of the area enclosed in white boxes are shown in the middle. White arrows indicate co-localization (yellow). Blue fluorescence corresponds to DAPI. The fluorescence intensity curves for red and green from boxed areas are shown on the right side of each group.

Article Snippet: To seal it at room temperature, 5% skimmed milk was used to for almost 1 h, then end up placing the membranes in p-RIP1 antibody (1:1000, Affinity, Cincinnati, OH, USA), anti-p-RIP3 antibody (1:1000, CST, Danvers, MA, USA), anti-p-MLKL antibody (1:1000, Affinity, Cincinnati, OH, USA), anti-RIP1 antibody (1:1000, Novus, Littleton, CO, USA), anti-RIP3 antibody (1:1000, Novus, Littleton, CO, USA), anti-MLKL antibody (1:1000, Millipore, Billerica, MA, USA), and anti-TNF-α antibody (1:1000, Abcam, Cambridge, UK) overnight in the refrigerator at 4 ◦C.

Techniques: Expressing, Western Blot, Double Staining, Marker

Journal: Experimental Hematology & Oncology

Article Title: Tetrahydromagnolol targets TRIM38 to mediate PANoptosis in cancer cells and has the potential for synergistic cancer therapy

doi: 10.1186/s40164-025-00734-4

Figure Lengend Snippet: THM induced PANoptosis by assembling PANoptosome. A Immunoblot analysis was used to detect the activation markers of pyroptosis (GSDME, AIM2), apoptosis (PARP, Cleaved-CASP3, Cleaved-CASP8) and necroptosis (p-RIPK1, p-RIPK3, p-MLKL) in the cell lysates of HT29, HCT116, A549 and H1299 cells after treatment with THM. β-actin was used as a loading control. B Representative images of IHC staining for Ki67, Cleaved-PARP, Cleaved-CASP3, AIM2, p-MLKL and p-RIPK1 in tumor tissues, scale bar = 50 μm. C-D HCT116 (75 µM) and A549 (90 µM) cells were treated with THM for 24 h, then immunofluorescence confocal laser microscopy analysis was performed after staining with the specified PANoptosome complex antibodies. Representative images illustrated the pairwise co-localization of ASC with CASP8, RIPK3, and AIM2 respectively. Scale bar = 20 μm

Article Snippet: Primary antibodies were incubated with slides overnight at 4 °C, including Ki67 (CST, 9449, 1:1500), Cleaved-PARP (CST, 5625, 1:50), Cleaved-CASP3 (CST, 9664, 1:2000), AIM2 (Proteintech, 20590-1-AP, 1:100), p-MLKL (UpingBio, YP-Ab-10353, 1:200), p-RIPK1 (Proteintech, 28252-1-AP, 1:100) and TRIM38 (Proteintech, 13405-1-AP, 1:100).

Techniques: Western Blot, Activation Assay, Control, Immunohistochemistry, Immunofluorescence, Microscopy, Staining

Journal: EMBO Molecular Medicine

Article Title: An immunohistochemical atlas of necroptotic pathway expression

doi: 10.1038/s44321-024-00074-6

Figure Lengend Snippet: Reagents and tools table

Article Snippet: A range of working dilutions were trialed for the following antibodies, although no conditions could be optimized for specificity and intensity: rabbit anti-phospho-RIPK1 (clone D813A; RRID:AB_2799268; Cell Signaling Technology Cat#44590 S); mouse anti-RIPK1 (clone 38/RIP; RRID:AB_397831; 0.25 g/L BD Biosciences Cat#610459); mouse anti-RIPK1 (clone 334640; RRID:AB_2253447; 0.5 g/L; R&D Systems Cat#MAB3585); rabbit anti-RIPK3 (clone 18H1L23; RRID: AB_2866471; 0.5 g/L; Thermo Fisher Scientific Cat#703750); rabbit anti-phospho-RIPK3 (clone D6W2T; RRID:AB_2800206; Cell Signaling Technology Cat#93654); rabbit anti-phospho-MLKL (clone D6E3G; RRID:AB_2799112; Cell Signaling Technology Cat# 37333); rabbit anti-phospho-MLKL (clone EPR9514; RRID:AB_2619685; Abcam Cat#ab187091; (Wang et al, )); mouse anti-MLKL (clone 3D4C6; RRID:AB_2882029; 1.957 g/L; ProteinTech Cat#66675-1-IG); rat anti-MLKL (clone 3H1; RRID:AB_2820284; 2 g/L produced in-house (Murphy et al, ) and available from Millipore Cat# MABC604); rabbit anti-mouse MLKL (clone D6W1K; RRID:AB_2799118; Cell Signaling Technology Cat#37705); rabbit anti-MLKL (clone 2B9; RRID:AB_2717284; 1 g/L; Thermo Fisher Scientific Cat#MA5-24846); mouse anti-Caspase-10 (clone 4C1; RRID:AB_590721; 1 g/L; MBL International Cat# M059-3); mouse anti-Caspase-8 (clone 5D3; RRID:AB_590761; 1 g/L; MBL International Cat#M058-3); rat anti-human RIPK3 (clone 1H2; RRID:AB_2940816; 2 g/L produced in-house (Petrie et al, ) and available from Millipore Cat# MABC1640); rabbit anti-human RIPK3 (clone E1Z1D; RRID:AB_2687467; Cell Signaling Technology Cat# 13526); rat anti-human MLKL (clone 7G2; RRID:AB_2940818; 2 g/L produced in-house (Samson et al, ) and available from Millipore Cat# MABC1636).

Techniques: Derivative Assay, Polymer, Plasmid Preparation, Blocking Assay, Membrane, Staining, RNA HS Assay, dsDNA Assay, Protease Inhibitor, Software, Imaging, Enzyme-linked Immunosorbent Assay

Journal: Nature Communications

Article Title: Single-cell transcriptomics identifies the differentiation trajectory from inflammatory monocytes to pro-resolving macrophages in a mouse skin allergy model

doi: 10.1038/s41467-024-46148-4

Figure Lengend Snippet: WT and Ccr2 −/− BALB/c mice were treated as in Fig. to induce IgE-CAI. a Ear samples prepared on days 1, 3, and 5 were subjected to HE staining. Bars indicate 500 μm. b Ear specimens collected from Ccr2 −/− BALB/c mice on day 3 post-challenge were subjected to immunostaining with anti-Ly6G antibody or its isotype-matched control antibody. Data are representative of three independent experiments. Bars indicate 200 μm. c – e Neutrophil depletion antibody (anti-Ly6G) or control antibody (rIgG) was intraperitoneally administered to mice on days 0, 1, 2, 3, and 4 post-challenge. In ( c ), time course of ear swelling (Δ ear thickness) is shown (mean ± SEM, n = 2, n = 4 and n = 4 biologically independent animals for WT rIgG group, Ccr2 −/− rIgG group and Ccr2 −/− anti-Ly6G group, respectively). ** p = 0.0037 (for day 1), **** p = 2.93 × 10 -7 (for day 2), **** p = 2.09 × 10 −10 (for day 3), **** p = 1.37 × 10 -8 (for day 4), and **** p = 3.02 × 10 −9 (for day 5) measured by two-way ANOVA with Tukey’s multiple comparison test. In d, the number of hematopoietic cells and neutrophils in the ear skin on day 5 is shown (mean ± SEM; n = 2, n = 4, and n = 4 biologically independent animals for WT rIgG group, Ccr2 −/− rIgG group and Ccr2 −/− anti-Ly6G group, respectively). In the left panel, * p = 0.042 (WT rIgG vs. Ccr2 −/− rIgG), * p = 0.0125 ( Ccr2 −/− rIgG vs. Ccr2 −/− anti-Ly6G) measured by two-way ANOVA with Tukey’s multiple comparison test. In the right panel * p = 0.0325 (WT rIgG vs. Ccr2 −/− rIgG), * p = 0.0071 ( Ccr2 −/− rIgG vs. Ccr2 −/− anti-Ly6G) measured by two-way ANOVA with Tukey’s multiple comparison test. In ( e ), HE-stained ear specimens collected on day 5 are shown. Bars indicate 500 μm. Ear specimens collected from Ccr2 −/− mice on day 3 post-challenge were subjected to TUNEL staining ( f ), immunostaining with anti-RIPK1 antibody ( g ) or its isotype-matched control antibody. Data are representative of three independent experiments. Bars in ( f ) indicate 200 (upper panel) and 50 μm (lower panels), respectively. Bars in ( g ) indicate 100 μm. h , i RIPK1 inhibitor (Necrostatin-1s; Nec-1s) or control PBS was intraperitoneally administered to mice on days 0, 1, 2, 3, and 4 post-challenge. In ( h ), time course of ear swelling (Δ ear thickness) is shown (mean ± SEM, n = 3, n = 4, and n = 3 biologically independent animals for WT PBS group, Ccr2 −/− PBS group and Ccr2 −/− Nec-1s group, respectively). **** p = 6.12 × 10 −5 (for day 1), **** p = 8.96 × 10 −7 (for day 2), **** p = 2.43 × 10 −10 (for day 3), **** p = 1.42 × 10 −12 (for day 4), and **** p = 1.06 × 10 −12 (for day 5) measured by two-way ANOVA with Tukey’s multiple comparison test. In ( i ), the number of hematopoietic cells and neutrophils in the ear skin on day 5 is shown (mean ± SEM, n = 3, n = 4, and n = 3 biologically independent animals for WT PBS group, Ccr2 −/− PBS group and Ccr2 −/− Nec-1s group, respectively) In the left panel, *** p = 0.001 (WT PBS vs. Ccr2 −/− PBS), * p = 0.043 ( Ccr2 −/− PBS vs. Ccr2 −/− Nec-1s) measured by two-way ANOVA with Tukey’s multiple comparison test. In the right panel *** p = 0.0005 (WT PBS vs. Ccr2 −/− PBS), ** p = 0.0075 ( Ccr2 −/− PBS vs. Ccr2 −/− Nec-1s) measured by two-way ANOVA with Tukey’s multiple comparison test. Data shown in ( a – i ) are representative of three independent experiments. Source data are provided as a file.

Article Snippet: Ear sections were then incubated with the following antibodies at 4 °C for 18 h: rat anti-Ly6G antibody (1 μg/mL; clone: 1A8, catalog#: 127602; BioLegend), rat IgG2a isotype control antibody (1 μg/mL; clone: RTK2758, catalog#: 400502; BioLegend), rabbit polyclonal anti-RIPK1 antibody (2.5 μg/mL, catalog#: NBP1-77077, Novus Biologicals), and rabbit polyclonal IgG isotype control antibody (2.5 μg/mL, catalog#: ab37415, Abcam).

Techniques: Staining, Immunostaining, Control, Comparison, TUNEL Assay

Journal: FEBS letters

Article Title: Phospho-ΔNp63α-responsive microRNAs contribute to the regulation of necroptosis in squamous cell carcinoma upon cisplatin exposure.

doi: 10.1016/j.febslet.2015.04.020

Figure Lengend Snippet: Fig. 3. Expression of protein targets in SCC cells upon cisplatin exposure. All SCC cells were exposed to 10 lg/ml cisplatin (CIS) for 16 h. (A, left panels) SCC-11 cells versus SCC-11M cells. (A, right panels). SCC-25 cells versus SCC-25CP cells. (A) Immunoblotting was performed with the indicated antibodies. Each blot was first probed with the indicated antibodies and after stripping with an antibody to a-tubulin used as a loading control. Lines between blots indicate the separate gel runs. Our data showed that the expression of pro-CASP8 was downregulated, while the expression of CYLD, RIPK1, MLKL, and DAP3 was upregulated in SCC-11 cells and SCC-25 cells compared to SCC-11M cells and SCC-25CP cells, respectively. (B) Co-immunoprecipitation of RIPK3, and p-MLKL with the antibody to RIPK1 showed the formation of RIPK1/RIPK3/p-MLKL protein complexes in SCC-11 cells and SCC-25 cells upon cisplatin exposure. Blots were scanned and quantified in triplicate. Values (shown above the blots) normalized by a-tubulin levels were expressed as a fold change to a scrambled control defined as 1.

Article Snippet: We also used antibodies against a-tubulin (ab7291, 1:5000), RIPK3 (ab56164, 1:500), MLKL (ab172868, 1:1000), and p-S358MLKL (ab187091, 1:1000), all purchased from Abcam, and finally antibody against RIPK1 (NB100-56160, 1:1000, Novus Biologicals).

Techniques: Expressing, Western Blot, Stripping Membranes, Control, Immunoprecipitation

Journal: FEBS letters

Article Title: Phospho-ΔNp63α-responsive microRNAs contribute to the regulation of necroptosis in squamous cell carcinoma upon cisplatin exposure.

doi: 10.1016/j.febslet.2015.04.020

Figure Lengend Snippet: Fig. 4. Silencing of RIPK1 modulated the survival of SCC-11/11M cells and SCC-25/ 25CP cells upon cisplatin exposure. (A) SCC-11 cells cells were transfected with the scrambled (Scr) RNA, miR-101-3p mimic and siRNA against RIPK2. SCC-11M cells were transfected with the Scr RNA and miR-101-3p inhibitor. After 36 h post- transfection, cells were exposed to control media (Con) and 10 lg/ml cisplatin (CIS) for 16 h. SCC-11 cells were also treated with the RIPK1 inhibitor, necrostatine (NEC- 1), and MLKL inhibitor, necrosulfonamide (NSA). (B) SCC-25 cells were transfected with the Scr RNA, miR-101-3p mimic and siRNA against RIPK2. SCC-25CP cells were transfected with the Scr RNA and miR-101-3p inhibitor. After 36 h post-transfec- tion, cells were exposed to control media (Con) and 10 lg/ml cisplatin (CIS) for 16 h. SCC-25 cells were also treated with the RIPK1 inhibitor, necrostatine (NEC-1), and MLKL inhibitor, necrosulfonamide (NSA). All cells were also exposed to control media (Con) and 10 lg/ml cisplatin (CIS) for 16 h, as indicated. Cell viability assay. Assay was performed using five independent experiments in triplicate and compared to control conditions (*, P < 0.05).

Article Snippet: We also used antibodies against a-tubulin (ab7291, 1:5000), RIPK3 (ab56164, 1:500), MLKL (ab172868, 1:1000), and p-S358MLKL (ab187091, 1:1000), all purchased from Abcam, and finally antibody against RIPK1 (NB100-56160, 1:1000, Novus Biologicals).

Techniques: Transfection, Control, Viability Assay

Journal: Cell Death & Disease

Article Title: Tubular epithelial cells in renal clear cell carcinoma express high RIPK1/3 and show increased susceptibility to TNF receptor 1-induced necroptosis

doi: 10.1038/cddis.2016.184

Figure Lengend Snippet: ( A ) Histology of untreated cultures (UT) show (a) low-grade RCC with small round tumor cells and a clear cytoplasm surrounded by a distinct cell membrane and round uniform nuclei with inconspicuous or absent nucleoli. (b) R1TNF- and (c) wtTNF-treated cultures show elevated level of death in mTECs compared with (d) R2TNF-treated cultures. ( B ) Morphological features of necrosis are evident in R1TNF-treated cultures such as large/small cytoplasmic vacuoles (x), condensation of the chromatin into small, irregular, circumscribed patches (white arrow), increasing translucent cytoplasm, swollen organelles (orange arrow) and disruption of the plasma membrane (black arrows). ( C ) Expression of RIPK1 and RIPK3 in tissue biopsies comprising RCC and NK; NK show a rare signal for RIPK1 and RIPK3 in MNCs within glomeruli (arrows) and with interstitium (small arrows). In contrast, RCC grade 3 shows marked signal in mTECs (arrows), MNCs (small arrows) and in some VECs (arrowheads). ( D and E ) Representative immunoblot of samples from nine patients with similar results and bar graph of relative RIPK1 and RIPK3 protein levels in grades 3/4 RCC (G3/G4) and non-tumor kidney (NK). Bars=mean+S.E.M.; * P <0.05 versus NK; paired Student's t -test. Glom-glomeruli; MNCs-mononuclear cells; TECs-tubular epithelial cells

Article Snippet: Blocking peptides for RIPK1 (cat~NBP1-77077PEP) are from Novus Biologicals and for RIPK3 (~ab178834) and pMLKL Ser358 (ab206929) are from Abcam. zVAD.fmk (pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone; cat~G7231) is from Promega, Madison, WI, USA; PDTC, 3,3′-diaminobenzidine substrate (DAB), Necrostatin-1 (Nec-1, cat~N9037) and mdivi-1 (3-(2, 4-dichloro-5-methoxyphenyl)-2-sulfanyl-4(3H)-quinazolinone) are from Sigma-Aldrich (Gillingham, UK).

Techniques: Membrane, Disruption, Clinical Proteomics, Expressing, Western Blot

Journal: Cell Death & Disease

Article Title: Tubular epithelial cells in renal clear cell carcinoma express high RIPK1/3 and show increased susceptibility to TNF receptor 1-induced necroptosis

doi: 10.1038/cddis.2016.184

Figure Lengend Snippet: Representative confocal images and light micrographs of the effect of R1TNF on protein and mRNA expression for RIPK1 and RIPK3 in organ cultures of RCC grade 1 and adjacent non-tumor kidney (NK). ( a and b ) Untreated (UT) cultures from RCC show a rare signal for RIPK1 and RIPK3 protein and mRNA. In contrast, R1TNF-treated cultures show a marked signal of both proteins mainly confined to mTECs (arrows), with RIPK3 also present in nuclei (white shaded arrow). Similarly, UT cultures of NK show a rare signal for both proteins; increased expression of both protein and mRNA is detected in R1TNF-treated cultures mainly confined to normal TECs (white arrows), peritubular capillaries (white arrowheads) and in infiltrating mononuclear cells (black arrowhead) but not in glomeruli (Glom). ( c ) Representative mean fluorescence intensity (MFI) for RIPK1 and RIPK3 expression in RCC and NK organ cultures. ** P <0.01 versus UT, * P <0.05 versus UT, ± P <0.05 versus R1TNF; Bars=mean±S.E.M.; n =3 independent experiments from six separate organ culture experiments with similar results. Nuclei stained with Hoechst 33342. Confocal images: × 40 and × 63 original magnifications; photomicrographs: × 400 magnification

Article Snippet: Blocking peptides for RIPK1 (cat~NBP1-77077PEP) are from Novus Biologicals and for RIPK3 (~ab178834) and pMLKL Ser358 (ab206929) are from Abcam. zVAD.fmk (pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone; cat~G7231) is from Promega, Madison, WI, USA; PDTC, 3,3′-diaminobenzidine substrate (DAB), Necrostatin-1 (Nec-1, cat~N9037) and mdivi-1 (3-(2, 4-dichloro-5-methoxyphenyl)-2-sulfanyl-4(3H)-quinazolinone) are from Sigma-Aldrich (Gillingham, UK).

Techniques: Expressing, Fluorescence, Organ Culture, Staining

Journal: Cell Death & Disease

Article Title: Tubular epithelial cells in renal clear cell carcinoma express high RIPK1/3 and show increased susceptibility to TNF receptor 1-induced necroptosis

doi: 10.1038/cddis.2016.184

Figure Lengend Snippet: PLA of organ culture of RCC grade 1. ( a ) In comparison with untreated controls (UT), R1TNF induced a strong interaction of RIPK1-RIPK3 and RIPK3-pMLKL Ser358 appearing as strong red fluorescence spots mainly within the cytoplasm of mTECs. Each individual interacting protein pair observed as a red spot by confocal microscopy is expressed as the number of signals/cell (PLA spots/cell). ( b and c ) Quantification of PLA spots in TECs in the two study groups; RCC (RCCoC) and normal kidney (NKoC) show a statistically significant difference, more pronounced in RCCoC. ** P <0.01 versus UT, * P <0.05 versus UT, ± P <0.05 versus R1TNF (NKoC). ( d and e ) Representative confocal images of pMLKL Ser358 or pDrp1 Ser637 and TUNEL in organ cultures of RCC grade 1. Compared with UT cultures, R1TNF induced an increase in the level of TUNEL+ mTECs ( green ) associated with pMLKL Ser358 and pDrp1 Ser616 ( red ) expression (arrows). ( f and g ) Quantification of TUNEL+ mTECs/pMLKL Ser358+ and TUNEL+ mTECs/pDrp1 Ser616+ shows statistical significant differences between cultures. *** P <0.0001 versus UT, ** P <0.001 versus UT, * P <0.01 versus UT, ± P <0.05 versus R1TNF (NKoC). ( h ) Combined immunofluorescence of R1TNF-treated cultures shows co-localization of pMLKL Ser358 and pDrp1 Ser616 in mTECs (arrow). ( i ) Immunogold electron microscopy demonstrate close proximity of gold particles for pMLKL Ser358 (5 nm) and pDrp1 Ser616 (15 nm) in mitochondria (m) ( inset zoomed × 2.5 ). Bars=mean±S.E.M.; images are representative of n =3 independent experiments from six separate organ culture experiments with similar results

Article Snippet: Blocking peptides for RIPK1 (cat~NBP1-77077PEP) are from Novus Biologicals and for RIPK3 (~ab178834) and pMLKL Ser358 (ab206929) are from Abcam. zVAD.fmk (pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone; cat~G7231) is from Promega, Madison, WI, USA; PDTC, 3,3′-diaminobenzidine substrate (DAB), Necrostatin-1 (Nec-1, cat~N9037) and mdivi-1 (3-(2, 4-dichloro-5-methoxyphenyl)-2-sulfanyl-4(3H)-quinazolinone) are from Sigma-Aldrich (Gillingham, UK).

Techniques: Organ Culture, Comparison, Fluorescence, Confocal Microscopy, TUNEL Assay, Expressing, Immunofluorescence, Electron Microscopy

Journal: Cell Death & Disease

Article Title: Tubular epithelial cells in renal clear cell carcinoma express high RIPK1/3 and show increased susceptibility to TNF receptor 1-induced necroptosis

doi: 10.1038/cddis.2016.184

Figure Lengend Snippet: Expression of necrosomal signaling components regulated by wtTNF, R1TNF and R2TNF in malignant tubular epithelial cells in human RCC grade 1 organ cultures

Article Snippet: Blocking peptides for RIPK1 (cat~NBP1-77077PEP) are from Novus Biologicals and for RIPK3 (~ab178834) and pMLKL Ser358 (ab206929) are from Abcam. zVAD.fmk (pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone; cat~G7231) is from Promega, Madison, WI, USA; PDTC, 3,3′-diaminobenzidine substrate (DAB), Necrostatin-1 (Nec-1, cat~N9037) and mdivi-1 (3-(2, 4-dichloro-5-methoxyphenyl)-2-sulfanyl-4(3H)-quinazolinone) are from Sigma-Aldrich (Gillingham, UK).

Techniques: Expressing, Immunostaining

Journal: Cell Death & Disease

Article Title: Tubular epithelial cells in renal clear cell carcinoma express high RIPK1/3 and show increased susceptibility to TNF receptor 1-induced necroptosis

doi: 10.1038/cddis.2016.184

Figure Lengend Snippet: ( a ) Effect of necrosulfonamide (NSA) and mdivi-1 (m) in grade 1 RCC organ cultures treated with R1TNF. R1TNF alone without NSA (no NSA) induced increased levels of TUNEL+ mTECs, which were significantly reduced with 50 μ M NSA, and to a lesser extent, with 10 μ M or 20 μ M NSA (arrows). Cultures pretreated with 5 μ M NSA showed comparable levels of TUNEL+ mTECs as untreated cultures (UT). ( b ) The percentage of TUNEL+ mTECs and ( c ) pMLKL Ser358 expression presented as mean fluorescent intensity (MFI) in similar cultures. *** P <0.0001 versus UT, ** P <0.001 versus R1TNF; * P <0.05 versus R1TNF; ¥ P <0.05 versus R1TNF (+20 or 10 μ M); ± P <0.001 versus R1TNF (+20 or 10 μ M); ns, not significant. ( d ) In comparison with UT, which show a rare TUNEL+ mTECs, R1TNF alone (without m) induced increased levels of TUNEL+ mTECs, significantly reduced by m (10 μ M) with no effect by zVAD.fmk (m+z) but a marked reduction by nec-1 (m+n) comparable with cultures pretreated with a combination of zVAD.fmk and nec-1 (m+n+z). ( e ) The percentage of TUNEL+ mTECs and ( f and g ) the mean fluorescence intensity (MFI) for pDrp1 Ser616 and pDrp1 Ser637 in similar cultures. *** P <0.0001 versus UT ( e ), ** P <0.01 versus UT ( f ), * P <0.05 versus R1TNF, ± P <0.05 versus R1TNF+m. ( g ) * P <0.05 versus UT, ± P <0.5 versus R1TNF, ¥ P <0.05 versus R1TNF+m; ns, not significant. Bars=mean±S.E.M.; n =3 independent experiments from six separate organ culture experiments with similar results. ( h ) Schematic diagram of the consequences of R1TNF-mediated necroptosis in mTEC in RCC; ligation of TNFR1 results in the recruitment of RIPK1, facilitating its interaction with RIPK3, which in turn recruits and phosphorylates MLKL at Ser358 and Drp1 at Ser616 thus causing their co-localization with the mitochondria. A separate process causes a reduction in pDrp1 at ser637. Nec-1 inhibits RIPK1, and NSA inhibits MLKL and mdivi-1 inhibits Drp1 inhibiting cell death

Article Snippet: Blocking peptides for RIPK1 (cat~NBP1-77077PEP) are from Novus Biologicals and for RIPK3 (~ab178834) and pMLKL Ser358 (ab206929) are from Abcam. zVAD.fmk (pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone; cat~G7231) is from Promega, Madison, WI, USA; PDTC, 3,3′-diaminobenzidine substrate (DAB), Necrostatin-1 (Nec-1, cat~N9037) and mdivi-1 (3-(2, 4-dichloro-5-methoxyphenyl)-2-sulfanyl-4(3H)-quinazolinone) are from Sigma-Aldrich (Gillingham, UK).

Techniques: TUNEL Assay, Expressing, Comparison, Fluorescence, Organ Culture, Ligation

Journal: Gastroenterology

Article Title: Inhibition of Aurora Kinase A Induces Necroptosis in Pancreatic Carcinoma

doi: 10.1053/j.gastro.2017.07.036

Figure Lengend Snippet: (A) Necrotic-like morphology was observed in PANC1 cells following treatment with CCT137690 (10 μM) for 24 hours. (B) Western blot analysis of indicated proteins in PANC1 and PANC2.03 cells following treatment with CCT137690 (2.5–10 μM) for 24 hours (n=3, *p < 0.05 versus untreated group). (C, D) PANC1 and PANC2.03 cells were treated with CCT137690 (C) or staurosporine (D) or erastin (D) in the absence or presence of indicated cell death inhibitors for 24 hours. Cell viability was assayed (n=3, *p < 0.05). (E) Indicated MEFs were treated with CCT137690 (10 μM) for 24 hours, and then cell viability was assayed (n=3, *p < 0.05). (F–I) Knockdown of RIPK1, RIPK3, and MLKL by shRNAs inhibited CCT137690 (10 μM, 24 hours)-induced cell death and HMGB1 and ATP release (n=3, *p < 0.05 versus control shRNA group).

Article Snippet: The antibodies to RIPK1 (#NB100-56160), LC3 (#NB100-2220), HMGB1 (#H00003146-M08), and AURKA (#NBP2-36737) were purchased from Novus Biologicals.

Techniques: Western Blot, shRNA

Journal: Gastroenterology

Article Title: Inhibition of Aurora Kinase A Induces Necroptosis in Pancreatic Carcinoma

doi: 10.1053/j.gastro.2017.07.036

Figure Lengend Snippet: (A) Western blot analysis of indicated proteins in PDAC cells following treatment with CCT137690 (10 μM) for three to 24 hours (n=3, *p < 0.05 versus untreated group). (B) Western blot analysis of indicated proteins in control and stable AURKA-knockdown PDAC cells (n=3, *p < 0.05 versus control shRNA group). (C) Immunoprecipitation (IP) analysis of the levels of RIPK3 binding to RIPK1 and MLKL in PANC1 and PANC2.03 cells following treatment with CCT137690 (10 μM) for 24 hours. (D) IP analysis of the levels of RIPK3 binding to RIPK1 and MLKL in control and stable AURKA-knockdown PANC1 and PANC2.03 cells. (E) IP analysis of the levels of AURKA binding to RIPK1, RIPK3, and MLKL in PANC1 and PANC2.03 cells following treatment with CCT137690 (10 μM) for 24 hours. (F) IP analysis of the levels of AURKA binding to RIPK1, RIPK3, and MLKL in HEK293 cells after expressed AURKA wild type and D274A mutant. (G) PANC1 cells were treated with CCT137690 (10 μM) or TNF (50 ng/ml) in the absence or presence of anti-TNFR1 antibody (1 mg/ml) for 24 hours and cell death was analyzed (n=3, *p < 0.05).

Article Snippet: The antibodies to RIPK1 (#NB100-56160), LC3 (#NB100-2220), HMGB1 (#H00003146-M08), and AURKA (#NBP2-36737) were purchased from Novus Biologicals.

Techniques: Western Blot, shRNA, Immunoprecipitation, Binding Assay, Mutagenesis

Journal: Gastroenterology

Article Title: Inhibition of Aurora Kinase A Induces Necroptosis in Pancreatic Carcinoma

doi: 10.1053/j.gastro.2017.07.036

Figure Lengend Snippet: (A) Western blot analysis of indicated proteins in PDAC cells following treatment with CCT137690 (10 μM) for three to 24 hours (n=3, *p < 0.05 versus untreated group). (B) Western blot analysis of indicated proteins in control and stable AURKA-knockdown PANC1 and PANC2.03 cells (n=3, *p < 0.05 versus control shRNA group). (C, D) PANC1 and PANC2.03 cells were treated with XXVI (C) or AR-A014418 (D) in the absence or presence of indicated cell death inhibitors for 24 hours. Cell viability was assayed (n=3, *p < 0.05). (E) Indicated MEFs were treated with XXVI or AR-A014418 for 24 hours, and then cell viability was assayed (n=3, *p < 0.05). (F) Knockdown of RIPK3 and MLKL, but not RIPK1, inhibited XXVI- or AR-A014418-induced cell death (n=3, *p < 0.05 versus control shRNA group). (G–I) HEK293 cells were expressed with GSK3β WT and S9A mutant and then treated with CCT137690 (10 μM) for 24 hours. Protein level (G), cell viability (H), caspase-3 activity (H), and complex formation (I) were assayed.

Article Snippet: The antibodies to RIPK1 (#NB100-56160), LC3 (#NB100-2220), HMGB1 (#H00003146-M08), and AURKA (#NBP2-36737) were purchased from Novus Biologicals.

Techniques: Western Blot, shRNA, Mutagenesis, Activity Assay