T56566

Total cellular proteins were extracted with RIPA lysis buffer (Servicebio) and measured using the BCA protein quantification kit (Beyotime). Equivalent amounts of protein samples were loaded in each lane and separated by 10% sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE). Subsequently, the samples were transferred to polyvinylidene fluoride (PVDF) membranes. After blocking with 5% skim milk, the membranes were incubated with a primary antibody overnight at 4°C. The primary antibodies were as follows: MTHFD2 (1:5000, PHF6156, Abmart); PD‐L1 (1:500, T55980, Abmart); p‐JAK1 (1:2000, TP56310, Abmart); JAK1 (1:2000, T57173, Abmart); p‐STAT3 (1:1000, T56566, Abmart); STAT3 (1:2000, T55292, Abmart); Bcl‐2 (1:500, WL01556, Wanleibio); Bax (1:1000, WL01637, Wanleibio); Cyclin D1 (1:5000, 60186‐1‐Ig, Proteintech) and β‐Tubulin (1:5000, M20005, Abmart). Endogenous β‐tubulin was performed as an internal control protein for WB analysis. After washing with tris‐buffered saline with 0.1% Tween‐20 (TBST), the membranes were incubated with secondary antibodies for 1 h at room temperature. All bands were measured using an enhanced chemiluminescence (ECL) system kit (Servicebio) and analysed using ImageJ software. The statistical graphs about WB was presented in Figure S1.

The fresh tumour tissues and adjacent nontumour tissues of eight patients with BC were obtained from Renmin Hospital of Wuhan University. This study has been approved by the Ethics Committee of Medical School of Wuhan University. All resected tissues were fixed with 4% paraformaldehyde and embedded in paraffin. Then, paraffin‐embedded tissues were sectioned into 5 μm thick. The tissue sections were dewaxed and the endogenous peroxidase was blocked by 1% hydrogen peroxide. Next, the sections were incubated with primary antibodies MTHFD2 (1:200, PHF6156, Abmart), p‐JAK1 (1:200, TP56310, Abmart), p‐STAT3 (1:100, T56566, Abmart) and PD‐L1 (1:50, T55980, Abmart) overnight at 4°C in a humidified box. After being washed, the sections were further incubated with a secondary anti‐rabbit antibody for 1 h at room temperature. Finally, the sections were reacted with 3,3‐diaminobenzidine (DAB) and counterstained with haematoxylin. The histochemistry score (H‐score) of protein expression was calculated by multiplying the staining intensity (0, negative; 1, weak; 2, moderate; and 3, strong) and the percentage of positive cells (0, 0%–10%; 1, 11%–25%; 2, 26%–50%; 3, 51%–75%; and 4, >75%).