Ab108937

The transplanted tumors were surgically removed until the tumor volume reached 100 mm³, and then fixed in 10% formalin. Then, the tumors derived from the donor’s pelvic lymph node and the transplanted tumors were subjected to routine hematoxylin and eosin (HE) staining and staining for other markers, including P16 (1:50, ab51243, Abcam, Cambridge, UK), P53 (1:2000, ab32389, Abcam), ER (1:800, 21244-1-AP, Proteintech), PR (1:200, 25871-1-AP, Proteintech, Chicago, IL, USA), PAX8 (1:200, 10336-1-AP, Proteintech), and WT1 (1:500, ab89901, Abcam). Microscopic slides were reviewed by a senior gynecology-dedicated pathologist (Prof. Yang).
Similarly, immunohistochemistry (IHC) was conducted to assess the expression of Ki67 (1:200, AF0198, Affinity, San Francisco, CA, USA), CyclinD1 (1:250, ab134175, Abcam), Hes1 (1:200, ab108937, Abcam), N-cadherin (1:200, #13116, Cell Signaling Technology, Danvers, MA, USA), vimentin (1:200, #5741, Cell Signaling Technology) and CSF3 (1:200, 17185-1-AP, Proteintech) in the tumors extracted from mice. The kidney and liver were also removed for HE staining to evaluate the toxicity of viscera.
Finally, the expression of CSF3 in the corresponding lymph nodes samples from 10 ovarian cancer patients was evaluated by IHC. The scoring criteria for staining intensity were described in our previous research [33 (link)].

The liver tissue was lysed in RIPA buffer (CatNo: R0010, Solarbio), and the total protein concentration was determined using a BCA kit (CatNo. 23225, Thermo Scientific). 30 μg protein per lane were separated from a 6-15% sodium dodecyl sulfate-polyacrylamide gel (CatNo: 28312, Thermo Scientific) and further transferred to a polyvinylidene fluoride (PVDF) membrane (CatNo: 24585 Thermo Scientific). The membrane was blocked with 5% BSA for one hour. The membrane was then incubated overnight at 4°C with primary antibodies against MMP9 (1:500, CatNo: ab76003, Abcam), NOTCH 1 1 (1:1000, CatNo: ab52627, Abcam), Hes1 (1:1500, CatNo: ab108937, Abcam), Math1 (1:1000, CatNo, ab249467, Abcam), and GAPDH (1:10000, CatNo: 14C10, Cell Signaling Technology). After three washes with TBS buffer containing 0.1% Tween-20 (TBST), the membrane was incubated with secondary antibodies conjugated horseradish peroxidase (CatNo: ab2116, Abcam) for one hour. After three times washing with TBST, a HyGLO-HRP kit (CatNo: 32106, Thermo Scientific) was used to visualize the bands.

For the determination of transcription factor protein abundance, protein components of lysates (100 µg) were separated by SDS-polyacrylamide gel electrophoresis on 12% (w/v) polyacrylamide mini gels, containing 0.1% (w/v) SDS, whilst for the determination of SGLT1 protein abundance, protein contents of BBMV (20 µg) were separated using 8% (w/v) polyacrylamide mini gels. Membranes were incubated with either our custom-made antibody to SGLT1^{40 (link), 41 (link)} or with antibodies to Neurogenin 3 (ab38548, Abcam, Cambridge, UK), NeuroD1 (ab109224), HATH1 (ab168374) and Hes1 (ab108937). Immunoreactive bands were detected by affinity purified horseradish peroxidase-linked anti-rabbit secondary antibody (DAKO Ltd, Cambridge, UK) and visualised using Immobilon Western Chemiluminescent HRP Substrate (Millipore, Hertfordshire, UK) and Bio-Max Light Chemiluminescence Film (Sigma-Aldrich, Poole, Dorset, UK). The intensity of the immunoreactive bands was quantified using scanning densitometry (Total Lab, Newcastle-upon-Tyne, UK). β-actin protein abundance was used as a loading control.