Additional file 2 of The crucial role of HFM1 in regulating FUS ubiquitination and localization for oocyte meiosis prophase I progression in mice

Additional file 2: Figure S1. Ad-Hfm1i significantly reduced the protein level of HFM1 in the prenatal embryonic mouse ovary. (A) 14.5 dpc ovaries were cultured with adenovirus for 4 days. Immunofluorescence staining showed adenovirus could successfully transfect into ovaries. DIC indicated differential interference contrast, and GFP indicated cells in ovaries were infected with adenovirus. Scale bars: 10μm. (B and C) Immunoblotting analysis of HFM1 protein level following HFM1 inhibition. β-Actin was used as a loading control. Quantification of HFM1 gray value. *p<0.05 (t-test), n=3. Figure S2. Knockdown of Hfm1 expression inhibited meiotic process and cell survival in mouse ovaries. (A and B) Inhibition of Hfm1 by AD-Hfm1i led to dramatic oocyte loss in fetal ovaries. 14.5 dpc embryonic mice ovaries were cultured with AD-Hfm1i in vitro for 4 days and the oocytes were counted. Oocytes were stained with DDX4 (green) and the nucleus was stained with DAPI (blue). Statistical analysis showed that the total number of oocytes decreased following the decrease of HFM1 expression compared to the control group. **p<0.01 (t-test), n=6. Scale bars: 50μm. (C and D) Inhibition of Hfm1 caused severe cellular apoptosis in the fetal ovaries. TUNEL signals (green) corresponded to apoptotic cells. The nucleus was stained by DAPI (blue). Statistical analysis showed a significant increase in apoptosis, *p<0.05 (t-test), n=3. Scale bars: 50μm. (E) Immunoblotting analysis of Cleaved-CASPASE3, P63, and P53 proteins level following HFM1 reduction. β-Actin or GAPDH were used as loading control. (F and G) Oocytes at the diplotene stage were reduced following HFM1 inhibition. MSY2(green) marked oocytes entering the diplotene stage and DDX4 (red) marked germ cells. The nucleus was immunostained by DAPI (blue). The encircled areas by dotted line were MSY2-/DDX4+ germ cells. Statistical analysis showed that the percentage of MSY2+ oocytes (number of cells both MSY2+ and DDX4+/number of cells DDX4+) per section decreased significantly following HFM1 inhibition. **p<0.01 (t-test), n=4. Scale bars: 50μm. Figure S3. Hfm1-KO mouse model was successfully obtained. (A and B) Immunoblotting demonstrated little to no expression of HFM1 protein in the KO mice model. β-Actin was used as a loading control. Quantification of HFM1 gray value. **p<0.01 (t-test), n=3. (C) Representative examples of the phases of meiosis prophase I in mouse oocytes. chromosome spreads were immunolabeled for SYCP3 (green) and nucleus stained with the nuclear marker DAPI (blue). The prophase stages were defined as: leptotene, SYCP3 was distributed in a punctate and discontinuous way; zygotene, shorter and thicker chromosomes and classical tripartite synaptonemal complex structure at homologous pairing site; pachytene, maximal shortening and thickening of the paired homologous chromosomes; diplotene, separation of homologous chromosomes. Scale bars: 10 μm. Figure S4. Knockout of Hfm1 had no impact on the proliferation of germ cells in embryonic ovaries. (A) Proliferation of germ cells showed no difference in KO ovaries compared with the control ovaries as the oocyte developed. PCNA signals (green) marked proliferating cells while the nucleus was stained by DAPI (blue). Scale bars: 50μm. (B) Statistical analysis showed that the percentage of PCNA+ germ cells (number of cells both PCNA+ and DDX4+/number of cells DDX4+) per section showed no difference between the control and KO group. p>0.05 (t-test), n=3. Figure S5. HFM1 did not affect the interaction between FUS and MDM2. (A) Endogenous protein interactions of MDM2 and FUS were assessed in embryonic mouse ovary lysates by immunoprecipitation with anti-MDM2 or anti-FUS and evaluated using immunoblotting with indicated antibodies. IgG was used as a negative control. (B) Lysates from ovaries transfected with control or AD-Hfm1i were collected for Co-IP. The binding of FUS and MDM2 was measured with the knockdown of Hfm1. (C) HFM1 co-localized with SYCP3 on chromosome axes in a dotted way. SYCP3 was stained with green, and HFM1 was stained with red. The nucleus was stained using DAPI (blue). Scale bars: 10 μm.