Fig 2: Gm364 is essential for Notch2 activation.A Immunoprecipitation with control IgG and Gm364 antibody was performed and followed by SDS-PAGE and silver staining. Then distinct bands were sent for MALDI. TTC37, MIB2, and GRAMD1A were identified as Gm364-interacting proteins. B RT-PCR showed that within oocytes, Notch2 is the most abundant among Notch family members 1–4. C Immunofluorescence showed that Notch2 was enriched on the oocyte membrane. DNA in blue, Notch2 in green. D Western blot showed that Notch2 is more abundant in oocytes than in granular cells. E Co-IP and blots showed that Gm364 interacts with Notch2 in oocytes. F. Western blot showed that NICD2 was more abundant in oocytes than in granular cells. G Blot showed that NICD2 protein levels decreased gradually during oocyte meiosis. H–J Immunofluorescence and blot showed that Gm364 knockout significantly decreased the NICD2 protein level. DNA in blue, NICD2 in green. K Blot showed that γ-secretase inhibition significantly decreased NICD2 levels. L. NICD2 reduction by γ-secretase inhibition greatly decreased the percentage of MII oocytes. M, N Immunofluorescence of in vitro fertilized oocytes and quantification showed that inhibiting γ-secretase significantly decreased the percentage of fertilized oocytes and the percentage of 2-PN (two pronucleus). PNs in the control oocyte or chromosomes in the γ-secretase-inhibited (γ-secretase(-)) oocytes were delineated with red dot-line circle; polar bodies (pbs) were labeled with arrows. DNA in blue, tubulin in green. β-actin or α-tubulin was used as a loading control. Scale bar, 20 μm. *Indicates p < 0.05.

Fig 3: Gm364 regulated the correct states of MIB2 and DLL3.A, B RT-PCR and quantification showed that Mib2 mRNA is more abundant than Mib1 mRNA within oocytes. C, D Blots and quantification showed that MIB2 is more abundant in oocytes than in granular cells. E Co-IP and blot showed that Gm364 interacts with MIB2. F Co-IP and blot showed that Notch2 interacts with MIB2. G, H Blots and quantification showed that Gm364 knockout did not affect MIB2 protein levels. I, J Immunofluorescence and quantification showed that MIB2 was enriched on the membrane in WT oocytes, whereas the membrane enrichment completely disappeared and cytoplasmic MIB2 increased in Gm364 knockout oocytes. K, L RT-PCR showed that among Dll members, Dll3 was the most abundant in oocytes. M, N Blots and quantification showed that DLL3 is more abundant in oocytes (oo) than in granular cells (GCs). O Co-IP and blot showed that Gm364 interacts with DLL3. P Co-IP and blot showed that Notch2 interacts with DLL3. Q, R DLL3 antibody IP and Ub46 blots showed that Gm364 knockout significantly decreased DLL3 ubiquitination levels. S, T Blot and quantification showed that Gm364 knockout significantly decreased DLL3 protein levels. U Immuno-EM showed that MIB2 and DLL3 localized at the oocyte membrane and were close (<20 nm) to each other. MIB2 and DLL3 primary antibodies were bound with 15-nm and 35-nm gold-conjugated secondary antibodies, respectively. V Immuno-EM showed that MIB2 and Gm364 localized at the oocyte membrane and were close (<20 nm) to each other. MIB2 and Gm364 primary antibodies were bound with 10-nm and 15-nm gold-conjugated secondary antibodies, respectively. β-Actin or GAPDH was used as a loading control. Scale bar in panel (I), 20 μm; scale bar in panels (U) and (V), 50 nm. *Indicates p < 0.05.

Fig 4: Notch expression in different histological types. The line of box indicated the median of Notch expression, and the box presented the 25th to 75th percentile of Notch expression. Patients with adenocarcinoma had a higher expression of Notch2, and patients with squamous cell carcinoma had a relatively higher expression of Notch1 and Notch3.

Fig 5: SNHG16 promoted fibrogenesis by regulating expression of Notch2. a TGF-β1 treated lung fibroblast cells which co-transfected with sh-SNHG16 or sh-NC and Notch2 or mock were used for experiments: The proliferation (a) and migration (b) abilities of TGF-β1 treated lung fibroblast cells in different groups were checked by EdU staining and wound healing assay, respectively. c The protein expression of α-SMA, E-cadherin, collagen 1 and fibronectin 1 were determined by western blot. Data were expressed as mean ± SD. **P < 0.01 vs Ad-shNC + mock; ##P < 0.01 vs Ad-sh-SNHG16 + mock, represent statistically difference