Programmed necrosis, not apoptosis, is a key mediator of cell loss and DAMP-mediated inflammation in dsrna-induced retinal degeneration

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Programmed necrosis, not apoptosis, is a key mediator of cell loss and DAMP-mediated inflammation in dsrna-induced retinal degeneration The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citation Murakami, Y, H Matsumoto, M Roh, A Giani, K Kataoka, Y Morizane, M Kayama, et al. 2013. Programmed Necrosis, Not Apoptosis, Is a Key Mediator of Cell Loss and DAMP-Mediated Inflammation in dsrna-induced Retinal Degeneration. Cell Death & Differentiation 21 (2) (August 16): 270 277. doi:10.1038/cdd.2013.109. Published Version 10.1038/cdd.2013.109 Citable link http://nrs.harvard.edu/urn-3:hul.instrepos:34961435 Terms of Use This article was downloaded from Harvard University s DASH repository, and is made available under the terms and conditions applicable to Open Access Policy Articles, as set forth at http:// nrs.harvard.edu/urn-3:hul.instrepos:dash.current.terms-ofuse#oap

Legends to supplementary figures. Figure S1. Effect of subretinal injection on the baseline expression of RIP3. (A) Quantitative real-time PCR analysis for RIP3 in the untreated retinas and the retinas 2 days after subretinal injection of PBS (n = 4 each). NS: not significant. (B) Western blot analysis for RIP3 in the untreated retinas and the retinas 2 days after subretinal injection of PBS (n = 4 each). Levels normalized to!-actin. The bar graphs indicate the relative level of RIP3 to!-actin by densitometric analysis. NS: not significant. Figure S2. Effect of Z-VAD on caspase-3 cleavage after subretinal dsrna injection in WT mice. (A and B) Immunofluorescence for cleaved caspase-3 (A) and quantification of cleaved caspase-3-positive cells (B) in the WT retinas 2 days after subretinal poly(i:c) injection (n = 6 in WT and n = 8 in Rip3 -/- ). Treatment with Z-VAD suppressed caspase-3 cleavage after poly(i:c) injection. Scale bar, 50 µm. *, P < 0.05. (B) The graphs show mean ± SEM. Figure S3. Nec-1 protects photoreceptors and the RPE against dsrna-induced retinal injury in mice. (A and B) TUNEL (green) and DAPI (blue) staining (A) and quantification of TUNELpositive cells (B) in the WT retinas 2 days after subretinal injection of poly(i:c) plus vehicle (n = 5), poly(i:c) plus Z-VAD (n = 6), poly(i:c) plus Nec-1 (n = 6), or poly(i:c), Nec-1 plus Z-VAD (n = 5). Scale bar, 50 µm. *, P < 0.05, **, P < 0.01, vs. poly(i:c) plus vehicle treatment. (C and D) Retinal histology (C) and quantification of ONL thickness (D) in the WT retinas 2 days after subretinal injection of poly(i:c) plus vehicle (n = 6), poly(i:c) plus Z- VAD (n = 6), poly(i:c) plus Nec-1 (n = 7), or poly(i:c), Nec-1 plus Z-VAD (n = 7). Scale bar, 50 µm. *, P < 0.05, **, P < 0.01, vs. poly(i:c) plus vehicle treatment. (E and F) ZO-1 staining (E) and quantification of ZO-1-positive RPE cells (F) in the

WT retinas 2 days after subretinal injection of poly(i:c) plus vehicle (n = 6), poly(i:c) plus Z-VAD (n = 6), poly(i:c) plus Nec-1 (n = 6), or poly(i:c), Nec-1 plus Z-VAD (n = 5). Rip3 deficiency attenuated the loss of RPE cells after poly(i:c) injection. Scale bar, 50 µm. *, P < 0.05, **, P < 0.01, vs. poly(i:c) plus vehicle treatment. (B, D and F) The graphs show mean ± SD. Figure S4. Nec-1 suppresses inflammatory cell infiltration in dsrna-induced retinal degeneration in mice. (A and B) Immunofluorescence for CD11b (A) and quantification of CD11b-positive cells (B) in the WT retinas 2 days after subretinal injection of poly(i:c) plus vehicle (n = 6), poly(i:c) plus Z-VAD (n = 6), poly(i:c) plus Nec-1 (n = 8), or poly(i:c), Nec-1 plus Z-VAD (n = 8). Scale bar, 50 µm. *, P < 0.05, **, P < 0.01, vs. poly(i:c) plus vehicle treatment. (B) The graphs show mean ± SD. Figure S5. RIP3 promotes microglial infiltration in the inner retina after dsrna injection in mice. (A and B) Retinal wholemount staining for Iba-1 (A) and quantification of Iba-1-positive microglial cells (B) in the area injected with PBS or poly(i:c) in WT and Rip3 -/- mice (n = 6 each). Microglial infiltration in the inner retina after poly(i:c) injection was relatively decreased in Rip3 -/- mice compared with WT mice. Scale bar, 50 µm. *, P < 0.01, NS: not significant. (B) The graphs show mean ± SD. Figure S6. RIP3 is not essential for NF-κB activation and cytokine production induced by dsrna in RPE cells and macrophages. (A and D) Western blotting for phosphorylated NF-κB after 5 µg/ml poly(i:c) treatment in WT- and Rip3 -/- mouse-derived RPE cells (A) or peritoneal macrophages (D).

(B, C, E and F) ELISA to measure TNF-α (B and E) and IL-6 (C and F) in the supernatants of RPE cells (B and C) or macrophages (E and F) 24 hours after poly(i:c) stimulation (n = 4 each). There was no difference in TNF-α or IL-6 production between WT- and Rip3 -/- mouse-derived cells. NS: not significant. UD: undetectable. (B, C, E and F) The graphs show mean ± SEM. Figure S7. RIP3 is not essential for NF-κB activation and cytokine production induced by TNF-α in RPE cells and macrophages. (A and C) Western blotting for phosphorylated NF-κB after 30 ng/ml TNF-α treatment in WT- and Rip3 -/- -derived RPE cells (A) or peritoneal macrophages (C). (B and D) ELISA to measure IL-6 in the supernatants of RPE cells (B) or macrophages (D) 24 hours after TNF-α stimulation (n = 4 each). NS: not significant. (B and D) The graphs show mean ± SEM. Supplementary methods. Iba-1 staining. Eyes were enucleated and fixed in 4% PFA for 1 h. After washing with PBS, the neuroretinas were dissected, blocked with 10% nonfat dried milk and 0.3% triton X in PBS for 1 h, and then incubated with anti-iba-1 antibody (1:300; Wako, Osaka, Japan) at 4 C overnight. Anti-rabbit Alexa Fluor 488 (1:300; Life Technologies, Grand Island, NY) was used as a secondary antibody. Five points in PBS- or poly(i:c)-injected areas were randomly selected and the inner retina was photographed at x40 magnification with a Leica SP2 confocal microscopy. The number of Iba-1 positive cells in the five regions was counted using ImageJ software and averaged by a masked observer.

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