X gal

The mouse colon frozen blocks for section were prepared. In Situ β-galactosidase Staining Kit (Beyotime) was used. Tissues were perfused with ice-cold PBS followed by PBS containing 2% paraformaldehyde for 15 min. After fixation, samples were rinsed with PBS and stained with X-gal (Stratagene, Beyotime) overnight at 37°C in the dark. Stained specimens were rinsed with PBS and photographed under bright-field illumination using a Nikon Ti-S microscope.

Cells transfected with sh‐TS or treated with pemetrexed for 48 h were plated in 12‐well plates. The chromogenic β‐gal substrate X‐gal (C0602, Beyotime Biotechnology) was used to stain the fixed cells for 8 h at 37°C. The cells were washed three times with PBS, and microscopy images were taken by bright field microscopy at 100× magnification.

HFF were washed with PBS and fixed in 3% formaldehyde at room temperature for 15 min. After washing with PBS twice, HFF were incubated overnight at 37 °C with β-galactosidase staining reagents (1 mg/mL X-gal, 2 mM MgCl₂, 150 mM NaCl, pH 6.0, 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide and 40 nM citric acid/sodium phosphate: Beyotime, Shanghai, China). Eventually, stained HFF were imaged with a light microscope (Nikon, Tokyo, Japan) at 50× magnification, and the percentage of positive cells were analyzed with Image-Pro Plus 6.0 (Media Cybernetics, Silver Spring, MD, USA). All experiments were performed three times.

Cell cycle alteration and apoptosis induction were evaluated using Flow cytometry analysis in HPV16-positive cell lines following exposure to Z_HPV16E61235 and Z_HPV16E7384 alone and in combination. PI (MultiSciences, Hangzhou, China) was used to stain the cells for cell cycle analysis as described by the manufacturer’s instructions and analyzed by flow cytometry (BD Biosciences, SanJose, CA, United States). G0/G1, S, and G2/M phase percentages were calculated and compared using ModFit LT 3.0 software. PI and Annexin V-FITC (Invitrogen, Carlsbad, CA, United States) were used to stain the apoptotic cells as described by the manufacturer’s recommendations and quantified using flow cytometry (CytoFLEX, Beckman Coulter, United States). Cells were categorized into viable, dead, early apoptotic, and apoptotic cells and the ratio of apoptotic (including early apoptotic) cells were compared with the control for each experiment.
For senescence analysis, the cells were fixed in 2% formaldehyde/0.2% glutaraldehyde and stained using X-Gal (5-bromo-4-chloro-3-indolyl-galactopyranoside, Beyotime Biotechnology, Shanghai, China) at pH of 6.0 as described by manufacturer’s instructions. SA-β-Gal-positive cells were counted in three representative fields.

SA-β-gal was visualised using X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) (Beyotime, China) (Stenderup et al., 2003). SA-β-gal–positive cells were green/blue. The stained cells were photographed under an optical inverted microscope (Olympus cx31) and evaluated using Image-Pro Plus software (version 7.0).

A bacterial adenylate cyclase-based two-hybrid system was used to verify the interaction of Atl and vimentin in vivo, as described previously [53 (link), 54 (link)]. Briefly, Atl-pUT18 and vimentin-pKT25 plasmids or empty vector control were co-transformed into BTH101 reporter cells that lack endogenous adenylate cyclase activity. The transformants were identified on LB plates containing X-Gal (40 μg/mL, Beyotime) and IPTG (0.5 M) or MacConkey plates containing maltose (1%) and IPTG (0.5 M).

The most widely used biomarker for senescent and aging cells is senescence-associated beta-galactosidase (SA-beta-gal), which is defined as beta-galactosidase activity detectable at pH 6.0 in senescent cells. Forty eight hours after treatment, cells were fixed in 2% fomaldehyde/0.2% glutaraldehyde for 5 min at room temperature. Cells were rinsed with PBS and β-galactosidase staining solution containing 20 mg/mL X-gal (Beyotime) was added. Cells were incubated for 6-10 h at 37°C incubator without CO₂.