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. 2023 Aug 11;12:e85647. doi: 10.7554/eLife.85647

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© 2023, Jackson, Giacomassi et al

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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Figure 4. — (A) BALB/c mice underwent splenectomy or sham surgery and were left to recover for 7 wk. Among the splenectomised mice, a group was treated 6× with topical R848 (n = 4, blue line) and the remaining mice with acetone (n = 3, solid black line). The sham surgery group was treated with R848 (n = 4, red line). Blood counts are shown for total monocytes, Ly6C-high monocytes, Ly6C-low monocytes, and the monocyte subpopulation ratio at baseline and 24 hr after the last treatments. (B, C) HSC-SCL-Cre-ERT;R26R-EYFP mice (n = 4 per group) received tamoxifen (4 mg/100 µl) by oral gavage for five consecutive days. Three days later, the left ears were treated topically 4× with R848 (blue squares) or left untreated (open circles). Shown are the total blood monocyte counts and the monocyte subpopulation ratio (B); the total blood EYFP+ monocyte counts and the subpopulation ratio among the EYFP-expressing monocytes at baseline and 24 hr after the last treatment (C). (D) Diagram illustrating BM myeloid progenitor differentiation: haematopoietic stem and progenitor cells (HSPC); common myeloid progenitor (CMP); monocyte-dendritic precursor (MDP); granulocyte-monocyte progenitor (GMP); common monocyte progenitor (cMoP); common dendritic progenitor (CDP); monocyte-committed progenitor (MP); granulocyte-committed progenitor (GP). (E–G) C57BL/6 mice were injected with 2 mg BRDU intraperitoneally (IP), either naïve (n = 3, white bars) or after two topical R848 treatments (n = 5, grey bars). Mice culled at 16 hr after the BRDU injection and BM harvested. (E) Percentage of BRDU positivity in HSPC, MDP, cMoP, GMP, total monocytes, Ly6C-high monocytes, and Ly6C-low monocytes. (F) Representative histogram of BRDU expression in HSPC at baseline (blue) or after 2× topical R848 (magenta). (G) Percentage of Sca1 positivity in total monocytes, Ly6C-high monocytes and Ly6C-low monocytes in the BM. (H) BALB/c mice (n = 3) received four treatments with topical R848. Blood counts for Ly6C-high monocytes (white bars), Ly6C-low monocytes (grey bars), and lymphocytes were monitored at the indicated time points after the cessation of treatment. Data representative of two independent experiments (except A and H). Two-way ANOVA with Tukey’s multiple comparison for time-course experiments (A–C, H); unpaired t-test (E, G). Data are the mean ± SEM; only significant p-values are indicated; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

Figure 4—source data 1. FACS raw data.

elife-85647-fig4-data1.xlsx^{(18.8KB, xlsx)}

Figure 4—figure supplement 1. — (A) BALB/c mice underwent splenectomy or sham surgery and were left to recover for 7 wk. Among the splenectomised mice, a group was treated 6× with topical R848 (n = 4, blue line) and the remaining mice with acetone (n = 3, solid black line). The sham surgery group was treated with R848 (n = 4, red line). Blood counts are shown for total monocytes, Ly6C-high monocytes, Ly6C-low monocytes, and the monocyte subpopulation ratio at baseline and 24 hr after the last treatments. (B, C) HSC-SCL-Cre-ERT;R26R-EYFP mice (n = 4 per group) received tamoxifen (4 mg/100 µl) by oral gavage for five consecutive days. Three days later, the left ears were treated topically 4× with R848 (blue squares) or left untreated (open circles). Shown are the total blood monocyte counts and the monocyte subpopulation ratio (B); the total blood EYFP+ monocyte counts and the subpopulation ratio among the EYFP-expressing monocytes at baseline and 24 hr after the last treatment (C). (D) Diagram illustrating BM myeloid progenitor differentiation: haematopoietic stem and progenitor cells (HSPC); common myeloid progenitor (CMP); monocyte-dendritic precursor (MDP); granulocyte-monocyte progenitor (GMP); common monocyte progenitor (cMoP); common dendritic progenitor (CDP); monocyte-committed progenitor (MP); granulocyte-committed progenitor (GP). (E–G) C57BL/6 mice were injected with 2 mg BRDU intraperitoneally (IP), either naïve (n = 3, white bars) or after two topical R848 treatments (n = 5, grey bars). Mice culled at 16 hr after the BRDU injection and BM harvested. (E) Percentage of BRDU positivity in HSPC, MDP, cMoP, GMP, total monocytes, Ly6C-high monocytes, and Ly6C-low monocytes. (F) Representative histogram of BRDU expression in HSPC at baseline (blue) or after 2× topical R848 (magenta). (G) Percentage of Sca1 positivity in total monocytes, Ly6C-high monocytes and Ly6C-low monocytes in the BM. (H) BALB/c mice (n = 3) received four treatments with topical R848. Blood counts for Ly6C-high monocytes (white bars), Ly6C-low monocytes (grey bars), and lymphocytes were monitored at the indicated time points after the cessation of treatment. Data representative of two independent experiments (except A and H). Two-way ANOVA with Tukey’s multiple comparison for time-course experiments (A–C, H); unpaired t-test (E, G). Data are the mean ± SEM; only significant p-values are indicated; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

Figure 4—source data 1. FACS raw data.

elife-85647-fig4-data1.xlsx^{(18.8KB, xlsx)}