Bovine serum albumin bsa

For immunofluorescence staining, cells were fixed with 4% paraformaldehyde for 15 min at RT and then washed with 1× PBS. The cells were permeabilized with 0.5% Triton X-100 in 1× PBS without Ca²⁺ and Mg²⁺ for 10 min and blocked with 1× PBS with 1% bovine serum albumin (BSA; Bovogen, Australia) for 1 h at RT. The cells were incubated with specific primary antibodies, including anti-TRA-1-60 (1:200; Santa Cruz Biotechnology, Dallas, TX, USA, sc-21705) and anti-SSEA4 (1:200; Santa Cruz Biotechnology, sc-21704) at 4 °C overnight. The following day, the primary antibodies were removed and the cells were washed thrice with 1× PBS containing 0.1% Tween 20 for 10 min. Following this process, the cells were incubated with fluorescence-conjugated Alexa Fluor 488 (1:600; Invitrogen, Waltham, MA, USA, A-10680) secondary antibodies to detect the primary antibodies for 1 h at RT. Lastly, the cells were washed three times with 1× PBS containing 0.1% Tween20 and mounted using Vectashield with DAPI (Vector Laboratories, California). Fluorescent signals were observed using a confocal laser scanning microscope (Carl Zeiss, Göttingen, Germany, LSM900) and examined using Zen Blue Software 3.6. The fluorescence intensity was analyzed using ImageJ software v1.53t.

After 24 h of treatment with acetylshikonin (0, 1.25, 2.5, and 5 μM), total protein from A498 and ACHN cells was extracted using RIPA buffer (Sigma, St. Louis, MO, USA) with protease and phosphatase inhibitors, PMSF phenylmethylsulfonyl fluoride (Sigma, St. Louis, MO, USA). Proteins were loaded on SDS-PAGE gels with appropriate percentage according to protein size and blotted onto polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA). Membranes were blocked with 3% bovine serum albumin (BSA; Bovogen, Victoria, Australia) for 30 min at room temperature (25°C), and immunoblotting was performed with specific primary antibodies at 4°C overnight. Membranes were then incubated with HRP-tagged secondary antibodies for 1 h at room temperature (25°C). Protein bands were visualized by using the enhanced chemiluminescence (ECL; Gendepot, Barker, USA) and detected with Chemi-doc detection system (Bio-Rad, Hercules, CA, USA).

Protein was extracted from cultured cells using a lysis buffer (Beyotime) with phosphatase and protease inhibitors (Boster) as described previously [33 (link)]. A total of 30 μg protein was separated by SDS-PAGE and then transferred to polyvinylidene fluoride (PVDF) membranes (Immobilon-PSQ). The membranes were blocked with 5% Bovine Serum Albumin (BSA) (Bovogen), probed with primary antibodies overnight at 4 °C, and then incubated with the relevant secondary antibody for 2 h at room temperature. Electrochemiluminescence (ECL, BioRad) with a detection reagent (Advansta) was used to detect signals. The antibodies used in western blotting included anti-Akt (CST, 9271S), anti-pAkt (CST, 12694S), anti-Erk (CST, 9102S), anti-pErk (CST, 9106S), anti-cyckinD1 (Abcam, ab134175), anti-caspase-3 (Abcam, ab32351), anti-cleaved-caspase-3 (CST, 9664S), anti-P21 (Abcam, ab109520), anti- P27^kip1 (Abcam, ab32034), anti-β-actin (Boster, BM5422), anti-rabbit-IgG-HRP (CST, 7074S), and anti-mouse-IgG-HRP (CST, 7076P2).