Journal: Cell reports

Article Title: Toll-like Receptor 2 Facilitates Oxidative Damage-Induced Retinal Degeneration

doi: 10.1016/j.celrep.2020.01.064

Figure Lengend Snippet: (A–D) qPCR of CFB (A and C) and C3 (B and D) expression in BMDMs or THP1s treated with 20 nM of Pam3Cys4. (E–I) IHC of C3d (purple) in healthy non-disease donor (E and F) and AMD donor eyes (G–I). (J and K) Black arrow and black asterisk denote C3d in CC and basal laminar deposits in AMD donor eye (representative of n = 4 non-disease donor eyes, n = 5 AMD donor eyes). qPCR of CFB (J) and C3 (K) in ARPE-19 cells treated with 20 nm of Pam3Cys4. Data shown are mean ± SD for a representative of three separate experiments. CC, choriocapillaris; RPE, retinal pigment epithelium; BM, Bruch’s membrane. (L) Generation and chemical structure of CEP-adduct from DHA. (M and N) qPCR of (M) C3 and (N) CFB in hfRPE cells treated with 0.1 µg of IgG or anti-TLR2 antibody prior to 10 µM of CEP-HSA for 24 h. (O) IHC of TLR2 (purple) in a healthy donor. Bottom panel (black box) is photobleached to illustrate apical and basolateral RPE immunoreactivity (n = 4 non-disease donor eyes). (P) Secreted CFB at 24 h and C3 at 48 h in hfRPE cells treated with 17.5 µM or 35 µM of CEP-HSA and 20 nM of Pam3Cys4, mean ± SD representative of three independent experiments: *p < 0.05; **p < 0.01; ***p < 0.001. See also and .

Article Snippet: Antibodies for CFB (Atlas Antibodies Sigma) 1:250, C3 (MP Biomedicals-855444), C3d 1:1000 (Dako), ZO-1 (Invitrogen) 1:1000 and C5b-9 (Santa Cruz) 1:500 were incubated overnight at 4°C.

Techniques: Expressing

Journal: Cell reports

Article Title: Toll-like Receptor 2 Facilitates Oxidative Damage-Induced Retinal Degeneration

doi: 10.1016/j.celrep.2020.01.064

Figure Lengend Snippet: (A–D) Three micrograms of anti-TLR2 or anti-IgG was injected IVT into C57Bl6 mice that were then exposed to 100K lux light for 7 days. (A) Quantification of photoreceptor cell rows in anti-IgG versus anti-TLR2 groups. (B and C) Immunofluorescence (IF) of C3 (green) and nuclear DAPI (blue) in mice injected with (B) anti-IgG versus (C) anti-TLR2 (scale bars represent 20 µm). (D) Quantification of outer retinal C3 positive cells/deposits detected in ONL and subretinal space. Data shown are mean ± SEM. The p value was determined by nonparametric t test, p < 0.05. n = 9–10 per experiment, *p < 0.05. (E and F) BMDMs from WT, TLR2 –/– , Mal –/– , or MyD88 –/– mice were treated for 3, 6, and 24 h with 20 nM of Pam3Cys4; expression of (E) C3 and (F) CFB was assayed by RT-PCR. (G and H) HEK293-TLR2 cells were transfected for 24 h with C3 promoter-luciferase (100 ng), Renilla -luciferase (40 ng), and empty vector (EV) or plasmid expressing (G) Mal or (H) MyD88 at 10, 50, and 80 ng. Results are normalized for Renilla luciferase activity and represented as relative stimulation over the non-stimulated EV control, mean ± SD for triplicate determinations p value determined by one-way ANOVA and Tukey post test: *p < 0.05, **p < 0.01, ***p < 0.001. (I and J) Secreted C3 expression in (I) BMDMs and (J) primary mouse microglia treated with 20 nM of Pam3Cys4 for 6 and 24 or 48 h.

Article Snippet: Antibodies for CFB (Atlas Antibodies Sigma) 1:250, C3 (MP Biomedicals-855444), C3d 1:1000 (Dako), ZO-1 (Invitrogen) 1:1000 and C5b-9 (Santa Cruz) 1:500 were incubated overnight at 4°C.

Techniques: Injection, Immunofluorescence, Expressing, Reverse Transcription Polymerase Chain Reaction, Transfection, Luciferase, Plasmid Preparation, Activity Assay

Journal: Cell reports

Article Title: Toll-like Receptor 2 Facilitates Oxidative Damage-Induced Retinal Degeneration

doi: 10.1016/j.celrep.2020.01.064

Figure Lengend Snippet: (A–E) Polarized hfRPE cells (A and B) or ARPE-19 cells (C–E) were treated with 10% Hi-NHS or NHS in combination with HSA or CEP-HSA for 24 h, phase transmission, presence of MAC (red), Phalloidin (green), and DAPI (blue), with representative images from three separate experiments. (B and E) Quantification of MAC+ specks in three 303 frames, data mean ± SD, one-way ANOVA followed by Tukey post-test to determine significance between groups; ***p < 0.001. (F–I) IF of MAC (red), Phalloidin (green), and DAPI (blue) in ARPE-19 cells treated with (F and G) 0.1 µg of IgG or anti-TLR2 antibody for 1 h or (H and I) with DMSO or 40 µm of Mal peptide inhibitor for 2 h prior to CEP-HSA and 10% NHS for 24 h. (G and I) quantification of MAC+ specks in four 20× frames, mean ± SD; p value determined by nonparametric t test; p < 0.05. (J–O) WT and TLR2 –/– mice injected IV, with NaIO 3 (50 mg/kg). (J) Quantification of MAC fluorescent intensity (scale bars represent 20 µm). (K–O) Representative IF images of MAC at 72 h, (K and O) depicts MAC staining in the whole retina section, (L–N) show higher magnifications, (L) shows MAC in the IS and OS, (M) shows MAC in OS and on RPE, (N) shows MAC on RPE. (P and R) Lysed tissue was assayed by western blot for expression of CFB(Bb). C9 and C9b at (P) 24 h and (R) 72 h. (Q and S) Mean pixel density for C9b was quantified at (Q) 24 h and (S) 72 h using the software ImageJ. See also and . *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Antibodies for CFB (Atlas Antibodies Sigma) 1:250, C3 (MP Biomedicals-855444), C3d 1:1000 (Dako), ZO-1 (Invitrogen) 1:1000 and C5b-9 (Santa Cruz) 1:500 were incubated overnight at 4°C.

Techniques: Transmission Assay, Injection, Staining, Western Blot, Expressing, Software

Journal: Cell reports

Article Title: Toll-like Receptor 2 Facilitates Oxidative Damage-Induced Retinal Degeneration

doi: 10.1016/j.celrep.2020.01.064

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Antibodies for CFB (Atlas Antibodies Sigma) 1:250, C3 (MP Biomedicals-855444), C3d 1:1000 (Dako), ZO-1 (Invitrogen) 1:1000 and C5b-9 (Santa Cruz) 1:500 were incubated overnight at 4°C.

Techniques: Plasmid Preparation, Isolation, Recombinant, In Situ, Enzyme-linked Immunosorbent Assay, RNA Extraction, Viability Assay, Luciferase, Software