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: 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: Biochimica et biophysica acta

Article Title: De-ubiquitinating proteases USP2a and USP2c cause apoptosis by stabilising RIP1.

doi: 10.1016/j.bbamcr.2012.05.022

Figure Lengend Snippet: Fig. 4. USP2c interacts with and de-ubiquitinates RIP1. (A) USP2c co-immunoprecipitates with endogenous RIP1. MCF7 cells were transfected with plasmids coding WT USP2a-HA. Total cell lysates were left untreated (Ctrl, Input) or immunoprecipitated with an anti-HA antibody and blots probed with the indicated antibodies. (B) USP2c de‐ubiquitinates RIP1 in vivo. 293T cells were transfected with the indicated plasmids coding for WT ubiquitin-HA, K48 ubiquitin-HA or K63 ubiquitin-HA together with USP2c or its inactive mutant. 24 h after transfection cells were treated with MG132 (10 μM, left panels) for 4 h or with TNF (20 ng/ml, right panels) for 15 min and then lysed in a denaturating dissociation buffer. The cell lysates were immunoprecipitated with an anti-RIP1 antibody and blots were probed with the indicated antibodies.

Article Snippet: Mammalian expression vectors coding for human USP2a, human TRAF2 and human RIP1 were from Origene (USA).

Techniques: Transfection, Immunoprecipitation, In Vivo, Ubiquitin Proteomics, Mutagenesis

Journal: Biochimica et biophysica acta

Article Title: De-ubiquitinating proteases USP2a and USP2c cause apoptosis by stabilising RIP1.

doi: 10.1016/j.bbamcr.2012.05.022

Figure Lengend Snippet: Fig. 8. The ratio of USP2a and TRAF2 determine the apoptosis sensitivity to TNF. (A) USP2a downregulation reduces and TRAF2 downregulation increases apoptosis by TNF. MCF7 cells were transfected with a siRNA construct against TRAF2 or USP2a, respec- tively or with a scrambled control siRNA (sc) (top panels). 48 h post-transfection, cells were treated overnight with TNF (100 ng/ml). Cells were harvested and stained with PI to quantify cell death (bottom panel). Shown are the means of three independent exper- iments done in duplicate for each construct. Bars indicate means+/−SD. *t-test, pb0.05. (B) Effect of TRAF2 knock-out on RIP1 levels. The endogenous protein levels of RIP1 was assessed by immunoblotting using a specific antibody in MEF WT and MEF TRAF2−/−.

Article Snippet: Mammalian expression vectors coding for human USP2a, human TRAF2 and human RIP1 were from Origene (USA).

Techniques: Transfection, Construct, Control, Staining, Knock-Out, Western Blot

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