1. Volume 27, Issue 8, Pages 2304-2312.e6 (May 2019)
c-Myb Exacerbates Atherosclerosis through Regulation of Protective IgM-Producing Antibody-Secreting Cells 
Eric A. Shikatani, Rickvinder Besla, Sherine Ensan, Aditi Upadhye, Nadiya Khyzha, Angela Li, Takuo Emoto, Felix Chiu, Norbert Degousee, Joshua M. Moreau, Heather M. Perry, Danya Thayaparan, Henry S. Cheng, Shaun Pacheco, David Smyth, Hossein Noyan, Caleb C.J. Zavitz, Carla M.T. Bauer, Ingo Hilgendorf, Peter Libby, Filip K. Swirski, Jennifer L. Gommerman, Jason E. Fish, Martin R. Stampfli, Myron I. Cybulsky, Barry B. Rubin, Christopher J. Paige, Timothy P. Bender, Coleen A. McNamara, Mansoor Husain, Clinton S. Robbins 
Cell Reports 
Volume 27, Issue 8, Pages e6 (May 2019)
DOI: /j.celrep
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2. Cell Reports 2019 27, 2304-2312.e6DOI: (10.1016/j.celrep.2019.04.090)
Copyright © 2019 The Authors Terms and Conditions

3. Figure 1 Hematopoietic Cell-Derived c-Myb Promotes Atherosclerosis
(A) RT-PCR detection of c-Myb expression conducted on leukocytes from bone marrow of Ldlr−/− mice fed either normal chow or a diet high in cholesterol (HCD) for 12 weeks (mean ± SEM, n = 9 per group). ∗p < 0.05.
(B) Total cholesterol and triglycerides in plasma from Ldlr−/− and Ldlr−/−c-mybh/h mice (mean ± SEM, n = 7–8).
(C) Quantification of lesion size in aortic roots of Ldlr−/− and Ldlr−/−c-mybh/h mice (mean ± SEM, n = 14–15).
(D) Atherosclerotic lesion volume estimated by calculating the area under the curves in (C) (mean ± SEM, n = 14–15). ∗p < 0.05.
(E) En face lipid oil red O staining of aortic arches from Ldlr−/− and Ldlr−/−c-mybh/h mice. Data show percent of lesser curvature of ascending aorta staining positive for oil red O (mean ± SEM, n = 7–8). ∗p < 0.05.
(F) Masson trichrome staining of aortic root sections of Ldlr−/− and Ldlr−/−c-mybh/h mice (mean ± SEM, n = 11–12). ∗p < 0.05.
(G) Flow cytometry analysis of inflammatory cells in atherosclerotic aortic tissue from Ldlr−/− and Ldlr−/−c-mybh/h mice (mean ± SEM, n = 8–10). ∗p < 0.05.
(H) Quantification of aortic root lesion size in bone marrow chimeras generated by reconstituting lethally irradiated Ldlr−/− mice with either wild-type (WT) or c-mybh/h bone marrow cells (mean ± SEM, n = 9).
(I) Lesion volume estimated by calculating the area under the curves in (H) (mean ± SEM, n = 9). ∗p < Statistical testing performed using unpaired Student’s t tests.
See also Figure S1.
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4. Figure 2
c-Myb Potentiates Atherosclerosis Directly through Its Effects on B Cells
(A) Peripheral blood monocytes, neutrophils, and T and B lymphocytes in Ldlr−/−c-mybWT and Ldlr−/−c-mybh/h mice fed either chow or HCD for 12 weeks (mean ± SEM, n = 13–19). ∗p < 0.05.
(B) Enumeration of B cells in the spleen, bone marrow, para-aortic lymph nodes, and peritoneum in Ldlr−/−c-mybWT and Ldlr−/−c-mybh/h mice fed either chow or HCD (mean ± SEM, n = 13–19). ∗p < 0.05.
(C) c-Myb mRNA expression in B220+ and B220− cells isolated from the bone marrow of HCD-fed Ldlr−/− mice (mean ± SEM, n = 5). ∗p < 0.05.
(D) Total leukocytes, monocytes, neutrophils, T cells, and B cells from peripheral blood of HCD-fed mixed chimeric mice generated by reconstituting irradiated Ldlr−/− mice with a 50:50 mixture of either c-mybh/h and μMT bone marrow cells (c-mybh/h:μMT chimeras) or c-mybh/h and wild-type (WT) bone marrow cells (c-mybh/h:WT chimeras) (mean ± SEM, n = 6). ∗p < 0.05.
(E) Quantification of lesion size in aortic roots from mice in (D) (mean ± SEM, n = 6). ∗p < 0.05.
(F) Lesion volume estimated by calculating the area under the curves in (E) (mean ± SEM, n = 6). ∗p < 0.05.
(G) Quantification of lesion size in aortic roots obtained from Apoe−/−Cd19Cre/+c-mybf/f and Apoe−/−Cd19+/+c-mybf/f control mice fed HCD for 12 weeks (mean ± SEM, n = 5–6).
(H) Lesion volume estimated by calculating the area under the curves in (G) (mean ± SEM, n = 5–6). ∗p < Statistical testing was performed using unpaired Student’s t tests.
See also Figure S1.
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5. Figure 3
c-Myb Regulates B Cell Development and Survival in Atherosclerosis
(A) Flow cytometry-based profiling of B lineage restricted progenitors and mature B cells from bone marrow of Ldlr−/− and Ldlr−/−c-mybh/h mice (mean ± SEM, n = 7–8). ∗p < Fr ‘A’, fraction A; Fr ‘E’, fraction E.
(B) Enumeration of B cell progenitors in (A) (mean ± SEM, n = 7–8). ∗p < FO, follicular; MZ, marginal zone; T1/T2/T3, transitional.
(C) Phenotype of B cell subsets in the spleen of the same animals as in (A) (mean ± SEM, n = 7–8). ∗p < 0.05.
(D) Enumeration of B cells in (C) (mean ± SEM, n = 7–8). ∗p < 0.05.
(E and F) Percentage (E) and number (F) of BrdU+B220+ cells in spleens of WT, c-mybh/h, Ldlr−/−, and Ldlr−/−c-mybh/h mice (mean ± SEM, n = 6–10). ∗p < 0.05.
(G) Flow cytometry analysis of Annexin V staining of B cells from spleen and bone marrow of Ldlr−/− HCD and Ldlr−/−c-mybh/h HCD mice (mean ± SEM, n = 7–8). ∗p < 0.05.
(H) Wild-type and GFP-UBC mice were joined in parabiosis for 5 weeks. Data show percentages of FO B cells in the spleens of individual mice and parabionts following surgical joining (mean ± SEM, n = 8 for individual mice and n = 4 for parabionts).
(I) FO B cell chimerism in the spleen (mean ± SEM, n = 4). ∗p < 0.05.
(J) Volcano plot demonstrating gene expression changes in B220+ cells of Ldlr−/− and Ldlr−/−c-mybh/h HCD mice. Of 21,610 genes analyzed, only 32 genes were differentially expressed (black circles); 22 genes were upregulated and 10 genes downregulated in B220+ cells of Ldlr−/−c-mybh/h HCD mice (n = 3 independently collected samples per experimental group).
(K) mRNA microarray analysis of Fas expression conducted on sorted FO B cells from spleens of Ldlr−/− and Ldlr−/−c-mybh/h mice fed HCD for 12 weeks (mean ± SEM, n = 3). ∗p < 0.05.
(L) B220+ cells were isolated by magnetic cell sorting (MACS) and cultured for 3 days in the absence or presence of LPS. IgM levels were measured by ELISA (mean ± SEM, n = 7).
(M) Frequency of IgM-ASCs in the peritoneum of RAG1 knockout (RAG1KO) mice receiving peritoneal cells from either WT or c-mybh/h mice (mean ± SEM; RAG1KO mice non-injected control, n = 1; RAG1KO LPS-injected mice, n = 2; RAG1KO mice injected with WT cells and LPS-stimulated, n = 6; RAG1KO mice injected with c-mybh/h cells and LPS-stimulated, n = 5).
Statistical testing was performed using unpaired Student’s t tests (A–D, G, H, and J) and ordinary one-way ANOVA with Newman-Keuls multiple comparisons test (E and F).
See also Figures S1 and S3.
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6. Figure 4 c-Myb Limits Protective IgM Responses in Atherosclerosis
(A) Total serum IgM (mean ± SEM, n = 5–12). ∗p < 0.05.
(B) Serum anti-copper-oxidized LDL (CuOxLDL) IgM levels assessed by ELISA (mean ± SEM, n = 4–9). ∗p < 0.05.
(C) Frequency of IgM antibody-secreting cells (IgM-ASCs) in the spleen and bone marrow of WT, c-mybh/h, Ldlr−/− HCD, and Ldlr−/−c-mybh/h HCD mice assessed by ELISPOT (mean ± SEM, n = 7–8). ∗p < 0.05.
(D) Absolute number of IgM-ASCs in the spleen and bone marrow of Ldlr−/− HCD and Ldlr−/−c-mybh/h HCD mice (mean ± SEM, n = 7–8). ∗p < 0.05.
(E) Surface area of IgM+ spots from (D) (mean ± SEM, n = 7–8). ∗p < 0.05.
(F and G) Percentage (F) and number (G) of syndecan-1(CD138)+IgM(i)+ cells in spleens of Ldlr−/− HCD and Ldlr−/−c-mybh/h HCD mice (mean ± SEM, n = 16). ∗p < 0.05.
(H) Immunofluorescence showing IgM staining in atherosclerotic plaques of Ldlr−/− and Ldlr−/−c-mybh/h mice. Shown are representative images from one of four to six animals examined (mean ± SEM, n = 4–6). ∗p < 0.05.
(I) IgM staining in spleens of Ldlr−/− and Ldlr−/−c-mybh/h HCD mice. Shown are representative images from one of four animals examined (mean ± SD, n = 4). ∗p < 0.05.
(J) Phenotypic characterization of splenic CD138+IgM(i)+ cells in Ldlr−/−c-mybh/h HCD mice by flow cytometry.
(K) mRNA microarray analysis of Igh-V11 expression conducted on sorted FO B cells and IgM-ASCs from spleens of Ldlr−/−c-mybh/h mice fed HCD for 12 weeks (mean ± SEM, n = 3). ∗p < 0.05.
(L) Quantification of aortic root lesion size in bone marrow chimeras generated by reconstituting lethally irradiated Ldlr−/− mice with either μMT, wild-type (WT), or c-mybh/h bone marrow cells (mean ± SEM; μMT: n = 10; WT: n = 6; c-mybh/h: n = 7). Statistical testing was performed using unpaired Student’s t tests.
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