Journal: Communications Biology

Article Title: Activated protein C reverses epigenetically sustained p66 Shc expression in plaque-associated macrophages in diabetes

doi: 10.1038/s42003-018-0108-5

Figure Lengend Snippet: p66 Shc and CD36 expressions are increased in plaque-associated macrophages in diabetic mice. a , b Representative reverse-transcriptase-PCR gel images showing expression of CD68, p66 Shc , CD36, β-actin (loading control), and SMCα-actin (reflecting purity of laser-dissected macrophages, a , left panel) and dot plot summarizing data for CD36 ( a , middle panel) and p66 Shc ( a , right panel). Representative images showing co-immunofluorescence staining for p66 Shc (red), MOMA-2 (green), and DAPI nuclear counterstain (blue) within lesions of the brachiocephalic artery ( b , left panel) and dot plot summarizing data using automated digital co-localization analyses, yielding a correlation index ( I corr ) ( b , right panel). c Representative immunoblot images of CD36 (out of 4 independent repeat experiments with two technical replicates each, top) and dot plot summarizing data (bottom) in ApoE −/− and ApoE −/− p66 Shc−/− bone marrow-derived macrophages (BMDMs); NG normal glucose (5 mM), HG high glucose (25 mM), GAPDH: loading control. d Representative co-immunofluorescence images for CD36 (red), MOMA-2 (green), DAPI nuclear counterstain (blue) within lesions of the brachiocephalic artery in ApoE −/− HFD and ApoE −/− DM mice (left panel) and dot plot summarising data ( I corr , correlation index, right panel). DM: hyperglycemic ApoE −/− mice; HFD: ApoE −/− mice with high-fat diet (HFD). Data shown as dot plots represent mean ± SEM of 10 mice per group ( a , b , d ) or 4 biological distinct replicates ( c ); size bars: b , d : 20 µm; ** P < 0.01; a , b , d : t -test; c : two-way ANOVA with Bonferroni-adjusted post hoc comparison of ApoE −/− (NG, HG) versus p66 Shc−/− ApoE −/− (NG, HG, respectively) BMDMs. Uncropped reverse-transcriptase PCR gel images for Fig. 2a and immunoblots for Fig. 2c are provided in Supplementary Figure

Article Snippet: The following antibodies were used in the current study: rabbit anti-p66 Shc (Merck, Millipore, United States); rabbit anti-CD36, mouse anti-nitrotyrosine, rabbit anti-PAR4 (Santa Cruz, Germany); rabbit anti-DNMT1, rabbit anti-NF-κB p65, rabbit anti-DNMT3a rabbit anti-DNMT3b, rabbit anti-β-actin, goat anti-rabbit IgG HRP (Cell Signaling Technology, Germany); mouse anti-8-hydroxy-2’-deoxyguanosine (Trevigen, United States); rat anti-MOMA-2, rabbit anti-mouse IgG HRP (abcam, Germany); rat anti-endothelial cell protein C receptor (EPCR), rabbit anti-GAPDH (Sigma-Aldrich, Germany).

Techniques: Reverse Transcription, Expressing, Control, Immunofluorescence, Staining, Western Blot, Derivative Assay, Comparison

Journal: Communications Biology

Article Title: Activated protein C reverses epigenetically sustained p66 Shc expression in plaque-associated macrophages in diabetes

doi: 10.1038/s42003-018-0108-5

Figure Lengend Snippet: p66 Shc and CD36 expressions are increased in plaque-associated macrophages in diabetic patients. Representative co-immunofluorescence images for p66 Shc (red, a , left panel), CD36 (red, b , left panel), MOMA-2 (green, a , b , left panels), and DAPI nuclear counterstain (blue) and dot plot summarising data ( I corr ; correlation index, right panels). Co-localization of p66 Shc and CD36 with the macrophage marker MOMA-2 is increased in diabetic patient’s samples ( N = 10) as compared to non-diabetic patient’s samples ( N = 10). DM: diabetic patient’s samples; non-DM: non-diabetic patient’s samples. Data shown as dot plots represent mean ± SEM of 10 patients per group, size bars: 20 µm; ** P < 0.01; a Mann–Whitney U test, b t -test

Article Snippet: The following antibodies were used in the current study: rabbit anti-p66 Shc (Merck, Millipore, United States); rabbit anti-CD36, mouse anti-nitrotyrosine, rabbit anti-PAR4 (Santa Cruz, Germany); rabbit anti-DNMT1, rabbit anti-NF-κB p65, rabbit anti-DNMT3a rabbit anti-DNMT3b, rabbit anti-β-actin, goat anti-rabbit IgG HRP (Cell Signaling Technology, Germany); mouse anti-8-hydroxy-2’-deoxyguanosine (Trevigen, United States); rat anti-MOMA-2, rabbit anti-mouse IgG HRP (abcam, Germany); rat anti-endothelial cell protein C receptor (EPCR), rabbit anti-GAPDH (Sigma-Aldrich, Germany).

Techniques: Immunofluorescence, Marker, MANN-WHITNEY

Journal: Communications Biology

Article Title: Activated protein C reverses epigenetically sustained p66 Shc expression in plaque-associated macrophages in diabetes

doi: 10.1038/s42003-018-0108-5

Figure Lengend Snippet: p66 Shc has a pivotal function in hyperglycaemia-, but not hyperlipidaemia-induced atherosclerosis. a . Experimental design of bone marrow transplantation experiments. b . Transplantation of p66 Shc−/− deficient bone marrow markedly reduces hyperglycaemia-, but only slightly hyperlipidaemia-induced atherosclerosis. Representative images showing Oil Red O staining of aortic roots (left panel) and dot plot summarizing plaques size (right panel). c . Dot plots summarizing results of necrotic core area (left panel), fibrous cap thickness (middle panel), and frequency of ruptured shoulders (right panel). d . Representative images showing immunofluorescence staining of macrophages (MOMA-2, green; DAPI nuclear counterstain, blue) within lesions (left panel) and dot plot summarizing data (right panel). Cont: normoglycaemic ApoE −/− mice with normal chow diet; DM: hyperglycaemic ApoE −/− mice; HFD: ApoE −/− mice with high-fat diet (HFD). Data shown as dot plots represent mean ± SEM of 6–10 mice per group ( b – d ); size bars: b , d : 20 µm; ** P < 0.01; b – d : two-way ANOVA with Bonferroni-adjusted post hoc comparison of ApoE −/− (Cont, HFD, DM) versus p66 Shc−/− ApoE −/− (Cont, HFD, DM respectively) recipient mice

Article Snippet: The following antibodies were used in the current study: rabbit anti-p66 Shc (Merck, Millipore, United States); rabbit anti-CD36, mouse anti-nitrotyrosine, rabbit anti-PAR4 (Santa Cruz, Germany); rabbit anti-DNMT1, rabbit anti-NF-κB p65, rabbit anti-DNMT3a rabbit anti-DNMT3b, rabbit anti-β-actin, goat anti-rabbit IgG HRP (Cell Signaling Technology, Germany); mouse anti-8-hydroxy-2’-deoxyguanosine (Trevigen, United States); rat anti-MOMA-2, rabbit anti-mouse IgG HRP (abcam, Germany); rat anti-endothelial cell protein C receptor (EPCR), rabbit anti-GAPDH (Sigma-Aldrich, Germany).

Techniques: Transplantation Assay, Staining, Immunofluorescence, Comparison

Journal: Communications Biology

Article Title: Activated protein C reverses epigenetically sustained p66 Shc expression in plaque-associated macrophages in diabetes

doi: 10.1038/s42003-018-0108-5

Figure Lengend Snippet: In vivo silencing of p66 Shc by vivo morpholino reduces hyperglycaemia-induced atherosclerosis. a Experimental design of vivo morpholino p66 Shc silencing experiments. b Representative images of Oil Red-O staining of the aortic root lesions (left panel) and dot plot summarizing plaque size (right panel). c Dot plots summarizing result of necrotic core area (left panel), fibrous cap thickness (middle panel), and frequency of ruptured shoulders (right panel). d Representative images showing immunofluorescence staining of macrophages (MOMA-2, green; DAPI nuclear counterstain, blue) within lesions (left panel) and dot plot summarizing data (right panel). Cont: normoglycaemic control mice; DM: hyperglycaemic ApoE −/− mice treated with PBS; Cont-MO: hyperglycaemic ApoE −/− mice treated with control vivo morpholino; p66 Shc -MO: hyperglycaemic ApoE −/− mice treated with p66 Shc -specific vivo morpholino. Data shown as dot plots represent mean ± SEM of 6–10 mice per group ( b – d ); size bars: b , d 20 µm; ** P < 0.01; one-way ANOVA with Bonferroni-adjusted post hoc comparison of DM and DM-Cont-MO versus Cont and DM-p66 Shc -MO versus DM-Cont-MO ( b , d ) or DM-p66 Shc -MO versus DM-Cont-MO and DM ( c )

Article Snippet: The following antibodies were used in the current study: rabbit anti-p66 Shc (Merck, Millipore, United States); rabbit anti-CD36, mouse anti-nitrotyrosine, rabbit anti-PAR4 (Santa Cruz, Germany); rabbit anti-DNMT1, rabbit anti-NF-κB p65, rabbit anti-DNMT3a rabbit anti-DNMT3b, rabbit anti-β-actin, goat anti-rabbit IgG HRP (Cell Signaling Technology, Germany); mouse anti-8-hydroxy-2’-deoxyguanosine (Trevigen, United States); rat anti-MOMA-2, rabbit anti-mouse IgG HRP (abcam, Germany); rat anti-endothelial cell protein C receptor (EPCR), rabbit anti-GAPDH (Sigma-Aldrich, Germany).

Techniques: In Vivo, Staining, Immunofluorescence, Control, Comparison

Journal: Communications Biology

Article Title: Activated protein C reverses epigenetically sustained p66 Shc expression in plaque-associated macrophages in diabetes

doi: 10.1038/s42003-018-0108-5

Figure Lengend Snippet: aPC reverses glucose-induced sustained p66 Shc expression and the pro-atherogenic phenotype of macrophages. a , b Representative immunoblot images (out of four independent repeat experiments with two technical replicates each, left) and dot plot summarizing relative expression levels (right panels) of p66 Shc , CD36, and GAPDH (loading control) in mouse bone marrow-derived macrophages (BMDMs) exposed to different lipid ( a ) or glucose ( b ) concentrations without or with additional aPC treatment. c Representative immunoblot images (out of 6 independent repeat experiments with two technical replicates each, top) and dot plot summarizing data (bottom) of p66 Shc and GAPDH (loading control) in BMDMs isolated from non-hyperglycaemic (control, Cont), hyperglycaemic (DM, 22 weeks persistent hyperglycaemia), transiently hyperglycaemic (DM-NG, 16 weeks of sustained hyperglycaemia followed by 6 weeks of SGLT2-inhbitor treatment), or DM-NG-aPC (DM-NG mice with aPC treatment parallel to SGLT2-inhibitors treatment). d Representative immunoblot images of p66 Shc (out of four independent repeat experiments with two technical replicates each) and GAPDH (loading control) in wild-type (WT), PAR1 −/− , PAR2 −/− , or PAR3 −/− BMDMs ( d , left panel) and in WT-BMDMs exposed to inhibitory antibodies of PAR4 or EPCR ( d , right panels). NL: normal LDL; HL: high LDL (50 µg/ml, 48 h); HL-NL: 50 µg/ml LDL, 48 h, followed by NL, 24 h); NG: normal glucose (5 mM glucose plus 20 mM mannitol, NG, 48 h); HG: high glucose (25 mM, 48 h); HG-NG: high glucose followed by normal glucose (25 mM glucose, 48 h, followed by 5 mM glucose, 24 h), HG-NG-aPC: HG-NG treated in addition with aPC (20 nM for the last 24 h); Data shown as dot plots represent mean ± SEM of at least 3 independent repeat experiments with at least two technical replicates each; ** P < 0.01; one-way ANOVA with Bonferroni-adjusted post hoc comparison of HL versus NL and HL-NL versus HL ( a ), HG and HG-NG versus NG and HG-NG-aPC versus HG-NG ( b , d ), DM and DM-NG versus Cont and DM-NG-aPC versus DM-NG ( c ), HG-NG-aPC (αPAR4 and αEPCR-4 BMDMs) versus HG-NG-aPC ( d , right panel) BMDMs. One-way ANOVA with Bonferroni-adjusted post hoc comparison of WT (HG and HG-NG versus NG and HG-NG-aPC versus HG-NG) and HG-NG-aPC (PAR3 −/− and PAR2 −/− BMDMs) versus HG-NG-aPC (PAR1 −/− BMDMs) ( d , left panel). Uncropped immunoblots for Fig. 6a–d are provided in Supplementary Figure and Supplementary Figure , respectively

Article Snippet: The following antibodies were used in the current study: rabbit anti-p66 Shc (Merck, Millipore, United States); rabbit anti-CD36, mouse anti-nitrotyrosine, rabbit anti-PAR4 (Santa Cruz, Germany); rabbit anti-DNMT1, rabbit anti-NF-κB p65, rabbit anti-DNMT3a rabbit anti-DNMT3b, rabbit anti-β-actin, goat anti-rabbit IgG HRP (Cell Signaling Technology, Germany); mouse anti-8-hydroxy-2’-deoxyguanosine (Trevigen, United States); rat anti-MOMA-2, rabbit anti-mouse IgG HRP (abcam, Germany); rat anti-endothelial cell protein C receptor (EPCR), rabbit anti-GAPDH (Sigma-Aldrich, Germany).

Techniques: Expressing, Western Blot, Control, Derivative Assay, Isolation, Comparison

Journal: Communications Biology

Article Title: Activated protein C reverses epigenetically sustained p66 Shc expression in plaque-associated macrophages in diabetes

doi: 10.1038/s42003-018-0108-5

Figure Lengend Snippet: aPC epigenetically inhibits glucose-induced sustained p66 Shc expression. a Representative reverse-transcriptase-PCR gel images (out of three independent repeat experiments, top) showing methylated (M) and unmethylated (U) p66 Shc promoter DNA in BMDMs with treatment as indicated and dot plot summarizing the ratio of methylated to unmethylated p66 Shc promoter DNA (bottom). b Glucose-induced sustained p66 Shc mRNA expression is prevented by aPC (20 nM) in BMDMs cells. Representative RT-PCR gel image (out of five independent repeat experiments, top; β-actin: loading control) and dot plot summarizing data (bottom). c Representative immunoblot images (out of 3 independent repeat experiments with two technical replicates each) for p66 Shc and DNMT1 expression in BMDMs with treatment as indicated (left panel; GAPDH: loading control) and dot plot summarizing data (middle and right panels). d dot plot summarizing DNMT activity in BMDMs with treatment as indicated. NG: normal glucose (5 mM glucose plus 20 mM mannitol, 48 h), HG: high glucose (25 mM, 48 h). HG-NG: HG (24 h) followed by NG (24 h) condition; HG-NG-aPC: HG-NG conditions with additional exposure to aPC (20 nM) during the last 24 h); HG-NG-Aza: HG-NG conditions with additional exposure to the DNMT-inhibitor 5-azacytidine (Aza, 5 µM) during the last 24 h; HG-NG-aPC-Aza: HG-NG conditions with additional and concomitant exposure to aPC (20 nM) and 5-azacytidine (5 µM) during the last 24 h. Data shown represent mean ± SEM of at least three independent experiments each with at least two technical replicates ( a – d ); ** P < 0.01; one-way ANOVA with Bonferroni-adjusted post hoc comparison of HG and HG-NG versus NG, HG-NG-aPC versus HG-NG ( a , b , d ), and HG-NG, HG-NG-Aza, HG-NG-aPC-Aza versus DM-NG-aPC). Uncropped reverse-transcriptase-PCR gel images for Fig. 8a, b and immunoblots for Fig. 8c are provided in Supplementary Figure

Article Snippet: The following antibodies were used in the current study: rabbit anti-p66 Shc (Merck, Millipore, United States); rabbit anti-CD36, mouse anti-nitrotyrosine, rabbit anti-PAR4 (Santa Cruz, Germany); rabbit anti-DNMT1, rabbit anti-NF-κB p65, rabbit anti-DNMT3a rabbit anti-DNMT3b, rabbit anti-β-actin, goat anti-rabbit IgG HRP (Cell Signaling Technology, Germany); mouse anti-8-hydroxy-2’-deoxyguanosine (Trevigen, United States); rat anti-MOMA-2, rabbit anti-mouse IgG HRP (abcam, Germany); rat anti-endothelial cell protein C receptor (EPCR), rabbit anti-GAPDH (Sigma-Aldrich, Germany).

Techniques: Expressing, Reverse Transcription, Methylation, Reverse Transcription Polymerase Chain Reaction, Control, Western Blot, Activity Assay, Comparison

Journal: Communications Biology

Article Title: Activated protein C reverses epigenetically sustained p66 Shc expression in plaque-associated macrophages in diabetes

doi: 10.1038/s42003-018-0108-5

Figure Lengend Snippet: aPC reverses hyperglycaemic induced and epigenetically sustained p66 Shc expression, promoting atherosclerotic plaque regression in mice. a Experimental design (left top) and representative Oil Red O-stained images of aortic roots (left bottom) and dot plot summarizing data (right). b , c Representative co-immunofluorescence images for p66 Shc ( b , red), 8-Oxo-dG ( c , red), macrophages (MOMA-2, b , c , green), and DAPI nuclear counterstain ( b , c , blue; boxed areas shown at higher magnification in the lower panel). Dot plot summarize data for p66 Shc -MOMA-2 ( b ) and 8-Oxo-dG-MOMA-2 co-localization ( c , both I corr , correlation index). Cont: normoglycaemic ApoE −/− mice with normal chow diet; DM: hyperglycaemic ApoE −/− mice; DM-NG-PBS: ApoE −/− DM mice receiving the SGLT2 inhibitor and PBS from week 16 to week 22; DM-NG-aPC: ApoE −/− DM mice with concomitant SGLT2 inhibitor and aPC treatment from week 16 to week 22; DM-NG-aPC-Aza: DM-NG mice with concomitant SGLT2 inhibitor, aPC, and 5-azacytidine. Data shown as dot plots represent mean ± SEM of 8–10 mice per group ( a , c ); size bars: a – c : 20 µm; ** P < 0.01 (one-way ANOVA with Bonferroni-adjusted post hoc comparison of DM and DM-NG-aPC versus control and DM-NG-PBS and DM-NG-aPC-Aza versus DM-NG-aPC)

Article Snippet: The following antibodies were used in the current study: rabbit anti-p66 Shc (Merck, Millipore, United States); rabbit anti-CD36, mouse anti-nitrotyrosine, rabbit anti-PAR4 (Santa Cruz, Germany); rabbit anti-DNMT1, rabbit anti-NF-κB p65, rabbit anti-DNMT3a rabbit anti-DNMT3b, rabbit anti-β-actin, goat anti-rabbit IgG HRP (Cell Signaling Technology, Germany); mouse anti-8-hydroxy-2’-deoxyguanosine (Trevigen, United States); rat anti-MOMA-2, rabbit anti-mouse IgG HRP (abcam, Germany); rat anti-endothelial cell protein C receptor (EPCR), rabbit anti-GAPDH (Sigma-Aldrich, Germany).

Techniques: Expressing, Staining, Immunofluorescence, Comparison, Control

Journal: Communications Biology

Article Title: Activated protein C reverses epigenetically sustained p66 Shc expression in plaque-associated macrophages in diabetes

doi: 10.1038/s42003-018-0108-5

Figure Lengend Snippet: aPC-mediated reversal of hyperglycaemia-induced persistent p66 Shc expression and plaque instability depends on DNMT1. a Experimental design. b Representative images of Oil Red O-stained aortic root lesions (left panel) and dot plot summarizing data (right panel). c Dot plots summarizing morphometric analyses of necrotic core area (left panel), fibrous cap thickness (middle panel), and frequency of ruptured shoulders (right panel). d Representative images showing immunofluorescence staining of macrophages within lesions (MOMA-2, green; DAPI nuclear counterstain, blue; left panel) and dot plot summarizing data (right panel). e Representative co-immunofluorescence images for 8-Oxo-dG (red), macrophages (MOMA-2, green), and DAPI nuclear counterstain (blue) within aortic root lesions (left, ×10 magnified images shown in lower panel) and dot plot summarizing data ( I corr , correlation index, right). Cont: normoglycaemic ApoE −/− mice with normal chow diet; DM-NG: ApoE −/− DM mice receiving the SGLT2 inhibitor and PBS from week 16 to week 22; DM-NG-aPC-DNMT1-MO: DM-NG mice with concomitant SGLT2 inhibitor, aPC, and DNMT1-MO treatment; DM-NG-aPC-Cont-MO: DM-NG mice with concomitant SGLT2 inhibitor, aPC, and control morpholino treatment. Data shown as dot plots represent mean ± SEM of 8–10 mice per group; size bars: b , d , e 20 µm; ** P < 0.01; b – e one-way ANOVA with Bonferroni-adjusted post hoc comparison of DM-NG and DM-NG-aPC-DNMT1-MO versus DM-NG-aPC-Cont-MO)

Article Snippet: The following antibodies were used in the current study: rabbit anti-p66 Shc (Merck, Millipore, United States); rabbit anti-CD36, mouse anti-nitrotyrosine, rabbit anti-PAR4 (Santa Cruz, Germany); rabbit anti-DNMT1, rabbit anti-NF-κB p65, rabbit anti-DNMT3a rabbit anti-DNMT3b, rabbit anti-β-actin, goat anti-rabbit IgG HRP (Cell Signaling Technology, Germany); mouse anti-8-hydroxy-2’-deoxyguanosine (Trevigen, United States); rat anti-MOMA-2, rabbit anti-mouse IgG HRP (abcam, Germany); rat anti-endothelial cell protein C receptor (EPCR), rabbit anti-GAPDH (Sigma-Aldrich, Germany).

Techniques: Expressing, Staining, Immunofluorescence, Control, Comparison