α sma

The 4 mm paraffin-embedded tissue sections were subjected to immunohistochemical staining (IHC). IHC staining of subcutaneous graft tumors and human tissues was performed by a reputable commercial entity (Servicebio, China). For immunofluorescence, two formalin-fixed paraffin-embedded (FFPE) tissue blocks (FIGO I and FIGO III tumors) were selected from Zhongda Hospital, Southeast University. Immunofluorescence staining was also conducted by the aforementioned commercial entity. The antibodies used in the immunofluorescence panels included Vimentin (Servicebio, GB121308), FOXP3 (Servicebio, GB112325) and α-SMA (Servicebio, GB13044). Lastly, DAPI was utilized as a counterstain for nuclear labeling. Detailed methods can be found in a previously published work by the same company [3 (link), 16 (link)]. Stained slides underwent analysis using a NanoZoomer S360 slide scanner (Hamamatsu Photonics, France). For tumor area assessment, the IHC staining were quantified using Quant Center software. Staining intensities were classified into weak, moderate, or strong categories. The proportion of cells within each intensity category was calculated, and the IHC score was derived using the formula: IHC score = (percentage of weakly stained cells × 1) + (percentage of moderately stained cells × 2) + (percentage of strongly stained cells × 3).

HPMCs were seeded in 24-well dishes and treated with different interventions including HG and siRNA. Then cells were fixed with 4% Paraformaldehyde for 20 min, permeabilized in 0.2% Triton X-100 for 15 min, blocked with 5% BSA for 1 h at 37°C and incubated overnight at 4°C with primary antibodies, E-cadherin (Cell signaling technology, 3195T, 1:200) and α-SMA (Servicebio, GB111364, 1:200). Finally, the cells were incubated with the Alexa Fluor 488-conjugated secondary antibody (Invitrogen, CA, United States, 1:200) at 37°C for 1 h. After staining nuclei with DAPI (Beyotime Biotechnology), images were captured by fluorescence microscope (Leica, Germany).

The heart tissues slices were also used for immunofluorescence staining. To prepare the slices, the heart sections were dewaxed and dehydrated, followed by antigen retrieval in boiling EDTA antigen retrieval buffer. Subsequently, the slides were incubated overnight at 4 °C with primary antibodies (α-SMA, Servicebio, Cat. #GB111364; p53, Santa Cruz, c-126; γ-H2AX, Abcam, Cat# ab81299). Afterward, the slides were washed three times with PBS and incubated with secondary antibodies (FITC, EarthOx, Cat. #E031210; Cy3, EarthOx, Cat. #E031610) for 2 h at room temperature. Negative controls were included without the use of primary antibodies. The nuclei were counterstained with DAPI. Imaging was performed using a laser-scanning confocal fluorescence microscope (Nikon, Tokyo, Japan). Four random microscopic fields were selected from each slide to calculate the relative fluorescence intensity, and an average value was determined for each rat.