Fig 2: F2RL1/PAR2 regulates the transcription of ATP6V0E1 via YAP1-FOXA2. (A) The overlap between four genes downregulated with F2RL1 knockdown in HT29 cells and transcription factors predicted for ATP6V0E1. (B) Relative mRNA levels of FOXA2 following F2RL1 knockdown in HT29, RKO, and SW620 cells. (C) Representative western blots of FOXA2 proteins in HT29, RKO, and SW620 cells following F2RL1 knockdown. (D) Representative western blots of FOXA2 in the cytoplasm and nuclei of HT29 cells following F2RL1 knockdown. (E) Representative western blots of autophagy-related proteins (MAP1LC3/LC3-I/II and SQSTM1/p62) and ATP6V0E1 following FOXA2 silencing or overexpression in F2RL1-knockout HT29 and RKO cells. (F) Representative images of CTSB activity (upper panel) and autophagosome-lysosome fusion based on the colocalization of MAP1LC3/LC3-positive autophagosomes (green) with LAMP1-positive lysosomes (red) in HT29 cells after FOXA2 silencing or overexpression along with F2RL1 knockdown. Scale bar: 5 µm. (G) The bioinformatics analysis of ATP6V0E1 promoter region binding to FOXA2. (H) The luciferase activity of vectors containing ATP6V0E1 or a mutant ATP6V0E1 promoter, which were transfected into HEK-293T cells expressing FOXA2, showing a dose-dependent response. (I) FOXA2 enrichment in promoter regions of ATP6V0E1 (compared with control IgG binding). (J) Representative western blots of FOXA2, ATP6V0E1, and autophagy-related proteins in HT29 cells treated with ELANE (1 μg/mL, 24 h), CTSG (2 μg/mL, 24 h), and PRTN3 (1 μg/mL, 24 h) with or without overexpression of YAP1 when F2RL1 was knocked down. Results are presented as mean ± SD. Statistical significance was determined using a two-tailed unpaired Student’s t-test or a two-way ANOVA with Tukey’s post-hoc test. *p < 0.05, **p < 0.01, ****p < 0.0001.

Fig 3: F2RL1 deficiency impairs autophagic flux and lysosome acidification in intestinal epithelial cells (IECs). (A) Representative western blots of the autophagy-related proteins (MAP1LC3/LC3-I/II and SQSTM1/p62) in HT29, RKO, and SW620 cells following F2RL1 knockdown. (B) Representative TEM images (left panel) and numbers (right panel) of HT29 cells with F2RL1 knockdown showing intercellular autophagosomes. Red arrows indicate autophagosomes enclosing intact undigested cargo. A minimum of 10 images from each group were randomly selected for quantification using the ImageJ software. Scale bar: 2 µm. (C) Representative images (left panel) and statistics (right panel) of autophagosome-lysosome fusion based on the colocalization between LC3-positive autophagosomes (green) and LAMP1-positive lysosomes (red) in F2RL1-knockout HT29, RKO, and SW620 cells. Red arrows denote autophagosomes that did not undergo fusion with lysosomes. A minimum of 10 images from each group were randomly selected for quantification using the ImageJ software. Scale bar: 5 µm. (D) Representative images (left panel) and statistics (right panel) of acidified (digestive) and non-acidified (non-digestive) MAP1LC3/LC3-positive autophagosomes visualized under a confocal microscope after F2RL1 knockdown in HT29 cells transfected with the mCherry-EGFP-MAP1LC3/LC3 plasmid. BafA1 was employed as a control to disrupt lysosomal acidification and autophagic flux. A minimum of 10 images from each group were randomly selected for quantification using the ImageJ software. Scale bar: 5 µm. (E) Representative images (left panel) and fluorescence intensities detected via FACS (right panel) of acidic lysosomes in HT29 cells following F2RL1 knockdown. BafA1 (100 nM, 12 h) was employed as a control to disrupt lysosomal acidification. Scale bar: 5 µm. (F) The acid phosphatase activity detected in HT29 cells after F2RL1 knockdown. BafA1 (100 nM, 12 h) was employed as a control to disrupt lysosomal acidification. (G) Representative images of intracellular CTSB activity in HT29 cells after F2RL1 knockdown. BafA1 was employed as a control to disrupt lysosomal acidification. Scale bar: 5 μm. Results are presented as mean ± SD. Statistical significance was determined using a two-tailed unpaired Student’s t-test or a two-way ANOVA with Tukey’s post-hoc test. *p < 0.05, ***p < 0.001, ****p < 0.0001.

Fig 4: The defective meiotic resumption and anaphase onset are fundamentally attributed to mis‐localized cathepsins. A) Western blot images illustrating protein level alterations in GV oocytes treated with Ctrl MO, Ccdc41MO, and Ccdc41 MO + Ctsb MO. B) Quantitative analysis of protein levels in Ctrl MO, Ccdc41 MO, and Ccdc41MO + Ctsb MO‐treated GV oocytes, respectively. C) Western blot images illustrating Sirt1 alterations in GV oocytes treated with Ctrl MO and Ccdc41 MO. D) Quantitative analysis of Sirt1 in Ctrl MO and Ccdc41 MO‐treated GV oocytes, respectively. E) The protein level of pro‐CTSB were assessed by western blot in Ctrl MO and Ctsb MO oocytes. Each sample contained 180 oocytes. F) Quantitative assessment of pro‐CTSB protein level across different experimental groups of oocytes. G) Representative images of oocytes in Ctrl MO, Ccdc41 MO, and Ccdc41 MO + Ctsb MO groups at 8 h after GVBD (10 h of IVM). Arrows indicate enlarged PB1 in Ccdc41 MO and Ccdc41 MO + Ctsb MO groups. Scale bar = 200 µm. H) Statistical analysis of MetII oocytes at 8 h after GVBD in Ctrl MO (n = 100), Ccdc41 MO (n = 100), and Ccdc41 MO + Ctsb MO (n = 72) groups. I) Western blots of CDC20 and Cyclin B1 were detected by Western blot in Ctrl MO, Ccdc41 MO, and Ccdc41 MO + Ctsb MO oocytes. Each sample contained 90 oocytes. J‐K) Statistical comparison of CDC20 and Cyclin B1 protein levels in Ctrl MO, Ccdc41 MO, and Ccdc41 MO + Ctsb MO groups. L) SAC activity was assessed by the localization of MAD1 at the pro‐MetI stage in Ctrl MO, Ccdc41 MO, and Ccdc41 MO + Ctsb MO oocytes. Oocytes were fixed and immunostained for MAD1, CREST, and DNA at 6 h after GVBD. White lines indicate the direction of fluorescence intensity measurement. Fluorescence intensities of CREST (green line) and MAD1 (red line) are shown in the line graph. The distance is measured in µm. Scale bar = 2.5 µm. M) The relative fluorescence intensity of MAD1/CREST was measured in Ctrl MO (n = 301), Ccdc41 MO (n = 313), and Ccdc41 MO + Ctsb MO oocytes (n = 299). The experiment was performed three times independently. P‐values were calculated using one‐way ANOVA for B, J–M, or unpaired Student's t‐tests (two‐tailed) for D, F. Statistical significance is indicated as *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Fig 5: CCDC41 depletion disrupts cathepsin delivery to lysosomes. A) Immunoblot analysis of protein levels in Ctrl and Ccdc41 MO oocytes. Each sample contained 180 oocytes. B) Quantitative analysis of active CTSB, LAMP2, and LAMP1 across Ctrl MO and Ccdc41 MO groups. C) Immunoblot analysis of protein levels in Ctrl MO, Ccdc41 MO, and Ccdc41 MO + myc‐Ccdc41 cRNA oocytes. Each sample contained 180 oocytes. D) Statistical evaluation of active CTSB, LAMP2, LAMP1, and active CTSD protein levels among Ctrl MO, Ccdc41 MO, and Ccdc41 MO + myc‐Ccdc41 cRNA groups. E) Oocytes were immunoprecipitated with anti‐CCDC41 or IgG antibody conjugated to Protein‐A/G beads, and the precipitates were subjected to immunoblotting with anti‐CCDC41 and anti‐LAMP2. IP lysate contained 800 oocytes. F) Representative images showing the relationship between CTSB (red) and LAMP1 (green) in oocytes at 8 h of IVM. LAMP1 was visualized in green, CTSB in red, and DNA in gray. The white‐boxed area is magnified to reveal details. Scale bar = 20 µm. G) Statistical analysis of Pearson's correlation coefficients between CTSB and LAMP1 in Ctrl MO (n = 103), Ccdc41 MO (n = 99), and Ccdc41 MO + myc‐Ccdc41 cRNA (n = 99) groups. H) Representative images showing the relationship between CTSB (red) and Rab7 (green) in oocytes at 8 h of IVM. Rab7 was visualized in green LINE, CTSB in red, and DNA in gray. Scale bar = 20 µm. I) Statistical analysis of Pearson's correlation coefficients between CTSB and Rab7 in Ctrl MO (n = 105) and Ccdc41 MO (n = 75) groups. J) Quantification of the number and area of RAB7 (>1 µm2) in Ctrl MO (n = 88) and Ccdc41 MO (n = 61) oocytes. The experiment was performed three times independently. P‐values were calculated using one‐way ANOVA for D, G, or unpaired Student's t‐tests (two‐tailed) for B, I, and J. Statistical significance is indicated as *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.