A toolbox for imaging RIPK1, RIPK3, and MLKL in mouse and human cells

Abstract

Necroptosis is a lytic, inflammatory cell death pathway that is dysregulated in many human pathologies. The pathway is executed by a core machinery comprising the RIPK1 and RIPK3 kinases, which assemble into necrosomes in the cytoplasm, and the terminal effector pseudokinase, MLKL. RIPK3-mediated phosphorylation of MLKL induces oligomerization and translocation to the plasma membrane where MLKL accumulates as hotspots and perturbs the lipid bilayer to cause death. The precise choreography of events in the pathway, where they occur within cells, and pathway differences between species, are of immense interest. However, they have been poorly characterized due to a dearth of validated antibodies for microscopy studies. Here, we describe a toolbox of antibodies for immunofluorescent detection of the core necroptosis effectors, RIPK1, RIPK3, and MLKL, and their phosphorylated forms, in human and mouse cells. By comparing reactivity with endogenous proteins in wild-type cells and knockout controls in basal and necroptosis-inducing conditions, we characterise the specificity of frequently-used commercial and recently-developed antibodies for detection of necroptosis signaling events. Importantly, our findings demonstrate that not all frequently-used antibodies are suitable for monitoring necroptosis by immunofluorescence microscopy, and methanol- is preferable to paraformaldehyde-fixation for robust detection of specific RIPK1, RIPK3, and MLKL signals.

Fig. 1. Methanol fixation is optimal for the immunofluorescent detection of human MLKL.

a Human MLKL domain architecture showing the immunogens used to raise the tested anti-MLKL antibodies. b Demonstration of how signal-to-noise ratios were used to quantify the abundance and brightness of specific immunofluorescent signals generated by different antibodies. The 5th and 95th percentile of each signal-to-noise curve defines the gate where specific immunosignals were observed. As indicated by the pseudocolour look-up-table, only immunosignals within this gate were visualised. c Chart exemplifying how the amount of signal within the gate, relative to the total amount of detectable signal, provides another gauge of antibody specificity for immunofluorescence. d Quantitation of the percentage of gated signals for the tested MLKL antibodies. e Quantitation of specific signal abundance produced by the tested MLKL antibodies on methanol-fixed (MeOH) or paraformaldehyde-fixed (PFA) HT29 cells. The number of cells imaged (N) to generate each signal-to-noise curve is shown. f Micrographs of immunofluorescent signals for the tested MLKL antibodies on HT29 cells. As indicated by each pseudocolour look-up-table, only immunosignals within the respective gate in panel e were visualised. Data are representative of n = 3 (clones 10C2, 7G2) and n = 2 (Abcam clone EPR9514, Novus Biological MAB9187/clone 954702, WEHI clone 3H1, MyBiosource clone 6B4 and Sigma-Aldrich M6697) independent experiments. Nuclei were detected by Hoechst 33342 staining and are demarked by white outlines in micrographs. T, TNF; S, Smac mimetic Compound A; I, pan-Caspase inhibitor IDN-6556. g Immunoblot using the tested MLKL antibodies against wild-type and MLKL^−/− HT29 cell lysates. Closed arrowheads indicate the main specific band. An asterisks indicate non-specific bands that could otherwise confound data interpretation. Immunoblots were re-probed for GAPDH as loading control.

Fig. 2. Better antibodies are needed for imaging human RIPK3.

a Human RIPK3 domain architecture showing the immunogens or epitopes for the tested anti-RIPK3 antibodies. b Quantitation of the percentage of gated signals for the tested RIPK3 antibodies. c Quantitation of specific signal abundance produced by the tested RIPK3 antibodies on methanol-fixed (MeOH) or paraformaldehyde-fixed (PFA) HT29 cells. The number of cells imaged (N) to generate each signal-to-noise curve is shown. d Micrographs of immunofluorescent signals for the tested RIPK3 antibodies on HT29 cells. As indicated by each pseudocolour look-up-table, only immunosignals within the respective gate in panel c were visualised. Data are representative of n = 3 (Cell Signaling Technology clone D6W2T and in-house clone 1H2) and n = 2 (ProSci 2283, Novus Biological NBP2-24588, CST clone E1Z1D, Abcam clone EPR9627 and MAB7604/clone 780115) independent experiments. Nuclei were detected by Hoechst 33342 staining and are demarked by white outlines in micrographs. e Immunoblot using the tested RIPK3 antibodies against wild-type and RIPK3^−/− HT29 cell lysates. Closed arrowheads indicate the main specific band. Open arrowheads indicate other specific bands of interest. An asterisk indicates a non-specific band that could otherwise confound data interpretation. Immunoblots were re-probed for GAPDH as loading control.

Fig. 3. Three specific antibodies for imaging endogenous human RIPK1.

a Human RIPK1 domain architecture showing the immunogens or epitopes for the tested anti-RIPK1 antibodies. b Quantitation of the percentage of gated signals for the tested RIPK1 antibodies. c Quantitation of specific signal abundance produced by the tested RIPK1 antibodies on methanol-fixed (MeOH) or paraformaldehyde-fixed (PFA) HT29 cells. The number of cells imaged (N) to generate each signal-to-noise curve is shown. d Micrographs of immunofluorescent signals for the tested RIPK1 antibodies on HT29 cells. As indicated by each pseudocolour look-up-table, only immunosignals within the respective gate in (c) were visualised. Data are representative of n = 2 (Cell Signaling Technology clones D94C12 and D8I3A, BD Transduction laboratories clone 38/RIP) independent experiments. Nuclei were detected by Hoechst 33342 staining and are demarked by white outlines in micrographs. e Immunoblot using the tested RIPK1 antibodies against wild-type and RIPK1^−/− HT29 cell lysates. Closed arrowheads indicate the main specific band. Open arrowheads indicate other specific bands of interest. Asterisks indicate non-specific bands that could otherwise confound data interpretation. Immunoblots were re-probed for GAPDH as loading control.

Fig. 4. A new monoclonal antibody to image endogenous mouse MLKL.

a Mouse MLKL domain architecture showing the immunogens or epitopes for the tested anti-MLKL antibodies. b Quantitation of the percentage of gated signals for the tested MLKL antibodies. c Quantitation of specific signal abundance produced by the tested MLKL antibodies on methanol-fixed (MeOH) or paraformaldehyde-fixed (PFA) MDFs. The number of cells imaged (N) to generate each signal-to-noise curve is shown. d Micrographs of immunofluorescent signals for the tested MLKL antibodies on MDFs. As indicated by each pseudocolour look-up-table, only immunosignals within the respective gate in panel c were visualised. Data are representative of n = 3 (in-house clone 5A6) and n = 2 (Abcam clone EPR9515(2), Millipore MABC1158/clone 7C6.1 and Cell Signaling Technology clone D6E3G) independent experiments. Nuclei were detected by Hoechst 33342 staining and are demarked by white outlines in micrographs. Immunoblot using the tested MLKL antibodies against lysates from wild-type versus Mlkl^−/− MDFs (e) and from lysates of wild-type versus Mlkl^−/− mouse spleen, small intestine and colon (f). Closed arrowheads indicate the main specific band. Asterisks indicate non-specific bands that could otherwise confound data interpretation. Open arrowheads indicate other specific bands of interest. Immunoblots were re-probed for GAPDH as loading control. g Micrographs of clone 5A6 immunosignals on methanol- or paraformaldehyde-fixed sections of the wild-type versus Mlkl^−/− mouse spleen, small intestine and liver. Data are representative of n = 2 mice per genotype. In the spleen, MLKL expression was noted in the red pulp, but not the white pulp. In the small intestine, MLKL expression was observed both in epithelial cells and subepithelial cell types in villi. In comparison, MLKL expression was more restricted in the liver; being primarily observed in perivascular regions such as the portal area and in parenchymal cells that resemble Kupffer cells (exemplified by arrows).

Fig. 5. Unlike human RIPK3, mouse RIPK3 is highly amenable to detection by immunofluorescence and immunoblotting.

a Mouse RIPK3 domain architecture showing the immunogens or epitopes for the tested anti-RIPK3 antibodies. b Quantitation of the percentage of gated signals for the tested RIPK3 antibodies. c Quantitation of specific signal abundance produced by the tested RIPK3 antibodies on methanol-fixed (MeOH) or paraformaldehyde-fixed (PFA) MDFs. The number of cells imaged (N) to generate each signal-to-noise curve is shown. d Micrographs of immunofluorescent signals for the tested RIPK3 antibodies on MDFs. As indicated by each pseudocolour look-up-table, only immunosignals within the respective gate in (c) were visualised. Data are representative of n = 3 (in-house clone 8G7 and Genentech clone GEN135-35-9) and n = 2 (ProSci 2283, in-house clone 1H12) independent experiments. Nuclei were detected by Hoechst 33342 staining and are demarked by white outlines in micrographs. e Immunoblot using the tested RIPK3 antibodies against lysates from wild-type and Ripk3^−/− MDFs. Closed arrowheads indicate the main specific band. Open arrowheads indicate other specific bands of interest. Immunoblots were re-probed for GAPDH as loading control.

Fig. 6. Three specific antibodies for imaging endogenous mouse RIPK1.

a Mouse RIPK1 domain architecture showing the immunogens or epitopes for the tested anti-RIPK1 antibodies. b Quantitation of the percentage of gated signals for the tested RIPK1 antibodies. c Quantitation of specific signal abundance produced by the tested RIPK1 antibodies on methanol-fixed (MeOH) or paraformaldehyde-fixed (PFA) MDFs. The number of cells imaged (N) to generate each signal-to-noise curve is shown. d Micrographs of immunofluorescent signals for the tested RIPK1 antibodies on MDFs. As indicated by each pseudocolour look-up-table, only immunosignals within the respective gate in panel c were visualised. Data are representative of n = 2 (Cell Signaling Technology clones D94C12 and 31122, and BD Transduction Laboratories clone 38/RIP) independent experiments. Nuclei were detected by Hoechst 33342 staining and are demarked by white outlines in micrographs. e Immunoblot using the tested RIPK1 antibodies against lysates from wild-type and Ripk1^−/− MDFs. Closed arrowheads indicate the main specific band. Open arrowheads indicate other specific bands of interest. Immunoblots were re-probed for GAPDH as loading control.

Fig. 7. Optimised antibody cocktails for visualising endogenous necroptotic signaling in fixed human and mouse cells.

a Cartoon summary of the currently-understood chronology of TNF-induced necroptosis. Recommendations of validated antibodies for immunostaining various steps in the necroptotic pathway are provided. b Summary of validated antibody cocktails and counterstains that can be multiplexed to examine endogenous necroptotic signaling in fixed human and mouse cells. Successful detection of specific signals relies on fixation in methanol, rather than crosslinking fixatives such as paraformaldehyde. c Two-dimensional Airyscan micrographs of Wheat Germ Agglutinin (WGA)-stained membranes, Hoechst-stained DNA and anti-MLKL immunosignals from clone 5A6 and clone D6E3G on methanol-fixed wild-type MDFs that had been left untreated or TSI-treated for 60 min. Arrowhead exemplifies the small clusters of MLKL that form during necroptosis. The box indicates the junctional accumulation of phospho-MLKL. d Three-dimensional orthogonal projections and maximum intensity projection (MIP) of the boxed region from (c) showing a ring-like structure adopted by phospho-MLKL at the WGA-stained plasma membrane. Accompanied by Supplementary Video 1.