Fig 2: The expression of CBS was reduced in ovaries and oocytes of 10-month-old mice. (A) Western blot showed the level of CBS protein in ovaries of 10-month-old mice was reduced. (B) Data were expressed as mean ± SEM, 2 m: n = 9, 10 m: n = 9. *p < 0.05. (C) The expression of CBS in 10-month-old mouse oocytes of follicles was decreased, presented by immunohistochemistry. Scale bar, 50 µm. The black arrow referred to follicles, the white arrow referred to oocytes. (D) Data were expressed as mean ± SEM of at least three independent experiments. 2 m: n = 3, 10 m: n = 3. *p < 0.05. (E) Western blot showed the expression of CBS in MI oocytes of 10-month-old mice was decreased. (F) Data were expressed as mean ± SEM of at least three independent experiments. 2 m: n = 99, 10 m: n = 99. *p < 0.05. (G) MI oocytes were removed from 2- and 10-month-old mice for immunofluorescence with CBS antibody (red). Scale bar, 20 µm. (H) Data were expressed as mean ± SEM of at least three independent experiments. 2 m: n = 25, 10 m: n = 22. **p < 0.01

Fig 3: CBS promotes continual phagocytosis of erythrocytes by microglia in an H2S-dependent manner. (A) Representative fluorescence images showing the engulfment of PHK26–labeled RBCs (red fluorescence) by microglia (Iba1+) treated with RBC lysate and/or ZnCl2 for 6 h. Ctrl: control cells treated with vehicle for RBC lysate. ZnCl2 was used to block the effects of H2S. (B and C) Microglial phagocytosis of RBCs assessed by counting the number of RBCs remaining in the media (nonphagocytosed, n = 3) and measuring the red fluorescence intensity in the microglial lysate (phagocytosis index, n = 3) at 6 h after microglia were exposed to RBCs. (D and E) The H2S donor ADT rescued the impairment in the engulfment of RBCs by CBS-deficient microglia (Cx3cr1Cre: Cbsfl/fl) following RBC lysate treatment, as indicated by the number of nonphagocytosed RBCs (D, n = 3) and phagocytosis index (E, n = 3). Cbsfl/fl: control microglia. (F) Fluorescence microscopic visualization of microglial distribution (Tomato+ cells) in the perihematomal area at 3 days after ICH by using Cx3cr1CreER: Rosa26-dtTomato mice (3 independent replicates). (G) Representative images showing that microglial deletion of CBS had no effect on the first round of phagocytosis of RBCs but impaired continual phagocytosis of RBCs by microglia treated with RBC lysate, as indicated by the percentages of microglia that internalized PKH26-labeled RBCs (red fluorescence) and the percentages of microglia that internalized both PKH26-labeled and PKH67-labeled RBCs (green fluorescence). (H and I) Quantitative analysis of first-round phagocytosis (H, n = 3) and continual phagocytosis (I, n = 3) of RBCs by CBS-deleted microglia (Cx3cr1Cre: Cbsfl/fl) and control microglia (Cbsfl/fl). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig 4: CBS-derived H2S production is enhanced in microglia following ICH.(A) CBS protein levels analyzed by Western blot in the hemorrhagic striatum following collagenase-induced ICH (cICH, n = 3). (B) H2S synthesis activity in homogenates harvested from the ipsilateral (hemorrhagic, ipsil) and contralateral (Contra) striatum following cICH (n = 3 or 4). Sham: sham-operated mice. (C) Representative photomicrographs showing that CBS expression (red) was enhanced throughout the hematoma and perihematoma areas and that activated phagocytes (microglia/macrophages: Iba1+ cells) were present only in the perihematoma area at 3 days after cICH. (D) Representative fluorescence images showing the enhanced expression of CBS and the colocalization of CBS (red) and IbaI (green) signals in the perihematomal area at 3 days following cICH (3 independent experiments). (E) Western blot analysis of CBS protein levels in primary microglia cotreated with lysates of red blood cells (lysate) and intact red blood cells (RBC) for 2–8 h (n = 3). Ctrl: control microglia treated with vehicle for RBC lysate. (F) Western blot analysis of CBS protein levels at 6 h after treatment with RBC lysate in primary microglia derived from the brains of wild-type mice (WT), control mice (Cbsfl/fl) or Cx3cr1Cre: Cbsfl/fl mice (representative image from 3 independent replicates). (G and H) Representative fluorescence images and quantitative analysis of endogenous H2S production in primary microglia treated with RBCs and/or RBC lysate for 6 h using a H2S fluorescence probe (n = 9 from 3 independent experiments).

Fig 5: Mucosal biopsy specimens from patients with ulcerative colitis (UC) show reduced CSE expression. A) Representative Western blot comparing CBS protein expression in colon mucosa biopsies from patients with ulcerative colitis (UC) and healthy individuals (Normal). B) Results of densitometric analysis of CBS expression in UC (n = 8) and normal (n = 8) mucosal specimens. C) Reverse Transcription-quantitative Polymerase Chain Reaction (RT-qPCR) assay comparing CSE mRNA levels in mucosal biopsies from normal and patients with UC. D) Representative Western blot comparing CSE protein levels in colon mucosa biopsies from healthy and UC patients. E) Results of densitometric analysis of CSE expression in UC (n = 14) and normal (n = 14) mucosal specimens. Statistical analyses of data in panels B, C, and E: D'Agostino & Pearson Normality test, Unpair t-test, two-tailed, error bars represent standard deviation, p values shown. F&G) Immunohistochemical staining for CSE protein (brown) using formalin-fixed paraffin-embedded tissue sections (5 µm thickness) from representative healthy and UC biopsy specimens. The sections were counterstained with hematoxylin (purple) to reveal tissue architecture. Non-specific antibody binding was determined by immunostaining serial tissue sections with an isotype-matched rabbit immunoglobulin (IgG) at the same concentration (2 ng/µL) as the primary rabbit anti-CSE antibody. Images were taken at 40X (F) and 200× (G) magnification (EpCs: epithelial cells; LP: lamina propria). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)