Senescence β gal staining kit

Cells were fixed with 4% formaldehyde and stained for β-gal activity using a Senescence β-Gal Staining Kit (Cell Signaling, Danvers, MA, United States). Positive cells were counted from three random fields for each group, and total cell number was also determined.

Cells were seeded in 1.9 cm²/well and cytochemically stained at pH 6.0 for senescence-associated β-galactosidase with a Senescence-β-Gal Staining Kit (Cell Signaling Technology, Massachusetts, USA) according to the manufacturer’s instructions. As a positive control, the cells were treated with etoposide (25 µM, 48 h) and allowed to recover. Respective microscopically images were performed with Zeiss Axiovert 200. Furthermore, Western blot analysis for detection of β-galactosidase were performed (description further below). In short proteins at P3 and P10 from DSC, ASC and FB were collected, 40 µg/lane were applicated and incubated with α-β-galactosidase antibody (abcam ab616, 1:2000) and respective protein expression was normalized on total protein^{86 (link)}.

Cells were cultured on 10-cm-diameter plates up to a confluence of 90% and then were either treated with 1.5 μM doxorubicin for 1 h or left untreated. Treated and untreated cells were seeded on 6-well plates (40–50% confluency) and were stained for senescence-associated β-galactosidase activity after 24, 48, and 72 h using the Senescence-βGal Staining Kit (Cell Signaling Technology) following the manufacturer’s instructions. All the experiments were repeated three times, and one of the representative results is shown.

PFT-α was obtained from Sigma-Aldrich China (Shanghai, China; P4236). The TBK inhibitor BX-795 and the Akt inhibitor MK-2206 were purchased from Selleck chemicals (Houston, TX, USA) (s1274 and s1078). Senescence β-gal staining kit (Cell Signaling Technology, Beverly, MA, USA; #9860).
The following antibodies were used in western blot: anti-TBK1 (Abcam, Cambridge, MA, USA, ab-40676, 1:1000), anti-p16 (Abcam, ab-51243, 1:1000), and anti-cyclin A (Santa Cruz, Dallas, TX, USA; sc-596, 1:1000), anti-pAkt 473 (Cell Signaling Technology; #4060, 1:2000), anti-Bmi (Cell Signaling Technology; #648, 1:1000), anti-phosphorylation serine (Boster, China, BM1622), anti-p53 (Santa Cruz; sc-126, 1:1000), anti-β-actin (Santa Cruz; sc-47778, 1:1000). The following antibodies were used in immunofluorescence: anti-NeuN (Abcam, ab-177487, 1:200), anti-Brn-3a (Santa Cruz; sc-8429, 1:200), anti-p16 (Abcam, ab-51243, 1:100), anti-TBK1 (Abcam, ab-40676, 1:200). anti-pAkt 473 (Cell Signaling Technology; #4060, 1:200) was used in immunohistochemistry.