Immunofluorescence microscope

TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) assay was performed to detect apoptotic cells in HCEC treated with rapamycin or control, after two weeks using ApopTagPlusFluoresceinInSituKit (Millipore, Billerica, MA, USA). Briefly, the media was removed and cells were fixed in 1% PFAinPBS (pH 7.4) for 10 minutesatroom temperature. Permeabilized cells in were cooled in ethanol after washing them with PBS, equilibration buffer applied and then TdT enzyme added and incubated at 37 °C for one hour. The cells were agitated for 15 second in wash buffer then washed with PBS. Digoxigenin-fluorescein conjugate was added and incubated at room temperature for 30 minutes in the dark. The cells were washed with PBS, mounted with DAPI and visualized with immunofluorescence microscope (Carl Zeiss, Thornwood, NY), and photographed with an AxioCam (Carl Zeiss) camera.

Apoptotic cell death was assessed with an In-Situ Cell Death Detection Kit, Fluorescein (Sigma-Aldrich) according to the manufacturer’s procedure. Nuclei were stained with the DAPI. The TUNEL-positive apoptotic cell nuclei appeared as green under an immunofluorescence microscope (Zeiss, Beijing, China). The percentage of apoptotic cells was calculated and compared.

As described previously (Che et al., 2020 (link)), apoptotic cell death was assessed with the Fluorescein In-Situ Cell Death Detection Kit (Sigma-Aldrich) according to the manufacturer's procedure. Nuclei were stained with the 4′,6-diamidino-2-phenylindole (DAPI). The TUNEL-positive nuclei appeared as green under an immunofluorescence microscope (Zeiss, Beijing, China). The percentage of TUNEL-positive cells was calculated and compared.

Immunofluorescence staining against α-SMA and SM-MHC was used to determine the purity of the PASMCs. The cells were fixed with 4% paraformaldehyde and permeabilized in 0.2% Triton X-100. After blocking with 1% BSA, the cells were incubated with primary antibodies, including rabbit monoclonal antibody against α-SMA (Abcam, Cambridge, MA, USA) and mouse monoclonal antibody against SM-MHC (Abcam) at 4 °C overnight. The cells were then incubated with Alexa Fluor 488-conjugated donkey anti-rabbit secondary IgG (Invitrogen, Carlsbad, CA, USA) and Alexa Fluor 568-conjugated donkey anti-mouse secondary IgG (Invitrogen). The nuclei were stained with DAPI (Sigma Aldrich). Fluorescence was observed with an immunofluorescence microscope (Carl Zeiss, Jena, Germany).

Immunofluorescence analysis was performed using antibodies against Lamin B1 (Abcam, ab16048, 1:1000 dilution), dsDNA marker (Santa Cruz, sc-58749, 1:250 dilution), γ-H2AX (Millipore, 05-636, 1:2000 dilution), phosphor-(Ser/Thr) ATM/ATR substrate (Cell Signaling Technology, 2851, 1:500 dilution), p16 (Santa Cruz, sc-468, 1:500 dilution), p21 (Abcam, ab2961, 1:50 dilution), 53BP1 (Santa Cruz, sc-22760; Abcam, ab36823, 1:500 dilution), IL-1β (Proteintech, 16806-1-AP, 1:300 dilution), IL-6 (Abcam, ab6672, 1:400 dilution), STING (Abcam, ab92605, 1:200 dilution), phospho-TBK1 (Bioss antibodies, bs-3440R, 1:100 dilution), IFN-β (Abcam, ab140211, 1:100 dilution), DNase2 (Bioss antibodies, bs-7652, 1:100 dilution), TREX1 (Novus Biologicals, NBP1-76977, 1:100 dilution) and Desmin (Thermo Fisher Scientific, MA5-13259, 1:100 dilution). DNA was stained with DAPI (Dojindo). Fluorescence images were observed and photographed using an immunofluorescence microscope (Carl Zeiss AG)^{11 (link),43 (link)}.

After metformin treatment for 72 h, cells were seeded in 24-well BD cell culture inserts and metformin treatment was continued for a further 48 h. 20% FBS or 10% bovine serum (FBS) was used as chemo-attractants in the lower chamber for LNCaP or PC-3 cells, respectively. After 48 h, cells on the upper side of the membrane were removed by scraping with cotton swabs while cells on the lower side were fixed with methanol and stained with the nuclear stain DAPI. Cells that had migrated through the membrane were viewed with an immunofluorescence microscope (Carl Zeiss GMBH, Oberkochen, Germany) and quantified with TissueFAXs software (TissueGnostics, Vienna; Austria).