Fig 2: The contribution of Jmjd3 to the maintenance of RGCs presumably by regulation of Eomes expression. (A, B) The Jmjd3-cKO retinas at postnatal stages were harvested, cryosectioned and immunostained with BRN3B, RBPMS, or EOMES. Cell nuclei were stained with DAPI. The averages of marker positive cells in three independent retinas are shown with standard deviations. (C) The mRNA expression level of Eomes in the postnatal Jmjd3-cKO retinas. RT-qPCR analysis using the whole retinas at indicated stages was performed. The averages of three independent retinas are shown with standard deviations. (D) The H3K27me3 level in the promoter region of Eomes in the Jmjd3-cKO retina at P15. Relative H3K27me3 level normalized by control was calculated. The averages of three independent retinas are shown with standard deviations. (E) The H3K27me3 level and JMJD3 binding in the promoter region of Eomes in RGCs- or rod-enriched fraction. Cd73-epxressing rods and Thy1.2-expressing RGCs in retinas at P3 were purified by FACS, and ChIP-qPCR analysis using H3K27me3 or JMJD3 antibody was performed. The percentage of input was calculated. The averages of three independent samples are shown with standard deviations. ONL, outer nuclear layer. **P < 0.01, *P < 0.05 was calculated by Student's t test. Scale bar: 50 µm.

Fig 3: Intestinal microbiota is required for MyD88-independent mucosal immunity against T. gondii.(A) MyD88-/- mice were treated with antibiotics two weeks prior to and throughout infection. Feces and tissues were collected at time points indicated. (B) Fecal CFU were determined weekly throughout the infection. At day 7 post-infection, tissues were harvested, and LP cells were cultured ex vivo. Supernatants were harvested after 72 hr and (C) IL-12 was quantified. (D) T cell (Lineage+, CD90+) and (E) ILC (Lineage-, CD90+) frequencies in the LP were determined by flow cytometry. (F) Within the T cell and ILC gates, IFN-γ+ frequencies were quantified. (G) IFN-γ levels from ex vivo cell cultures were determined by ELISA. Values are the means ± SEM of two independent experiments. Open and closed symbols indicate animals from independent experiments. An unpaired Student’s t test was used to analyze the data where **p<0.01 *** p<0.001.

Fig 4: Isolation of macrophage-derived fibroblast-like cells from wound-derived explants. a Primary dermal fibroblasts isolated from skin or d10 post-wounding tissue of LysMCreRosamT/mG mice. Scale bar = 50 µm. b GFP+ FSP+ fibroblasts interlinked with GFP− FSP+ fibroblast isolated from d10 wound explants. Scale bar = 20 µm. c (left) Immunostaining of GFP+ (myeloid, green) and FSP1 (fibroblast, blue) cells in d10 wound explants skin cultures n = 5, scale bar = 50 µm. Colocalization of the GFP+ cells (green) with FSP1+ (blue) demonstrating their myeloid origin. (right) Immunostaining of GFP + (myeloid, green) and Col1 (fibroblast, blue) cells in d10 wound explants skin cultures n = 5, scale bar = 50 µm. Multiple fibroblast-specific staining (FSP1/Col1) demonstrated the purity (>99%, fibroblasts) of the explant culture. d Heat map illustrating clusters of transcripts from GFP−CD90+ or GFP+CD90+ primary fibroblasts isolated from d10 wound explants. Additionally, GFP-CD90+ primary fibroblast were isolated from unwounded skin. Gene expression profiling of 70 genes relating to ECM/fibroblast was performed using NanoString nCounter system. The expression pattern of GFP−CD90+ or GFP+CD90+ primary fibroblasts from d10 wound explants were largely similar, i.e., not significantly different (t-test with Bonferroni correction) but was distinctly different from unwounded skin GFP-CD90+ primary fibroblasts

Fig 5: Majority of FSP1+ fibroblast-like cells in wound granulation tissue are of myeloid origin. a LysMCreRosamT/mG mice express cell membrane-localized td Tomato (red) fluorescence while cells of myeloid origin express GFP (green) fluorescence. b FSP1 (blue) immunostaining of LysMCreRosamT/mG mice d5 wound. Colocalization was performed using Olympus Fluoview® software. Estimated (65 ± 5%) FSP1+ cells (blue) were GFP+ demonstrating their myeloid origin, n = 5, scale bar = 10 µm. c Macrophage (F4/80, red) and fibroblast (FSP1, green) immunostaining at wound-edge tissue (d5) from C57BL/6 mice, n = 5, scale bar = 10 µm. d Laser capture micro-dissected (LCM) GFP+ cells from full thickness excisional wounds on d3 (early inflammation phase) or d7 (inflammation resolution phase) post-wounding (PW), scale bar = 10 µm. qRT-PCR of LCM-captured GFP+ cells showed increased expression of FSP1 and Col1A1 at d7, n = 5, Student’s t test p < 0.05. e, f Multicolor flow cytometry of CD11b+ of wound macrophages on d3 and d7 PW with the F4/80 (macrophage, x-axis) and CD90 (fibroblast marker, y-axis) displayed increase (68 ± 2%) in F4/80+CD90+ cells on d7 PW, n = 4, Student’s t test p < 0.05. g Chronic wound fluid derived from human subjects contain CD68+ FSP1+ cells, scale bar = 10 µm. h Fibrocyte marker CD34 (blue), immune-staining of LysMCreRosamT/mG mice d5 wound tissue. Estimated (15 ± 2%) of CD34+ cells (blue) were GFP+, n = 5, scale bar = 10 µm. Data presented as ±SD