Bradford s reagent

Caspase 3 activity was determined using fluorometric assay kits (ThermoFischer Scientific, Hanover Park, IL, USA), according to the manufacturer’s protocol. HCT116 or MDA-MB-231-Luc cells were cultured for 24 h for 80–90% confluence and then treated with DOX (100 nM), COS (30 μM), COS + DOX, COS-NPs (30 μM), or COS-NPs + DOX, in each flask for proliferative cells. The treated cells and respective controls were trypsinized and the cell pellet was lysed with a cell culture lysis reagent. The lysed pellet was then centrifuged at 10,000× g for 30 min, and protein concentrations were measured with Bradford’s reagent (Bio-Rad Laboratories, Hercules, CA, USA), using albumin as a standard. A total of 5 μL cell lysates (0.5 mg/mL) were added to 195 μL of buffer containing an Ac-DEVD-7-amino-4-methyl coumarin (AMC)-conjugated substrate for caspase 3 and followed by 30-min incubation at 370C in the dark. The concentration of the released AMC was calculated from the fluorescence intensity, which was read using a fluorescence plate reader with the excitation and emission wavelengths of 342 and 441 nm, respectively, and using AMC standard to calculate caspase 3 activity. Data were adjusted according to the protein content.

Cultured cells or tissues were harvested and lysed with RIPA lysis buffer (Beyotime, China) for 30 min on ice. After centrifugation at 12,000 g for 20 min, the concentration of proteins was measured using Bradford's reagent (Bio-Rad laboratories, USA). The protein samples were denatured by boiling for 10 min and loaded onto SDS–PAGE gel for electrophoresis. The proteins were transferred onto PVDF membrane (Millipore, USA) which was then incubated in the blocking solution (5% FBS) at room temperature for 1 h. The anti-REPS2 antibody (Abcam, UK) or anti-RalBP1 antibody (Santa Cruz, USA), anti-RAC1 antibody (GeneTex, USA), anti-CDC42 antibody (GeneTex, USA), anti-MMP9 antibody (Proteintech, USA), anti-MMP2 antibody (Proteintech, USA), anti-cyclinD1 antibody (Abcam, UK), anti-EGFR antibody (Abcam, UK), anti-pEGFR antibody (Abcam, UK), anti-AKT antibody (Abcam, UK), anti-pAKT antibody (Abcam, UK), anti-Erk antibody (Abcam, UK) and anti-pErk antibody (Abcam, UK) was added into blocking solution and incubated at 4°C overnight. The membranes were subsequently incubated with HRP-labeled goat anti-rabbit IgG for 1.5 h at room temperature. Protein expression was normalized against GAPDH expression (RD, USA) or β-actin expression (Bioworld, USA). Bands were visualized by employing the BeyoECL Plus DetectionSystem (Beyotime, China) and Bio-Rad Image Lab Software (CA, USA).

The cytokine levels were measured by ELISA assay in skin tissue samples. Briefly, skin tissues were homogenized with Tris–HCl buffer with protease and phosphatase inhibitors and centrifuged at 12,000 rpm at 4℃ for 10 min. The supernatant from all tissues were collected and protein estimation was done by Bradford’s reagent (Biorad, USA). Polystyrene 96 well plates were coated with TGF-β, IL-17A, IL-22, IL-1β, and IL-6 along with 100 µl of antibodies diluted with coating buffer and incubated at 4℃ overnight. Later excessive and unbound antibody was removed by washing buffer and further blocked with BSA. Further 100 µl of the tissue supernatant was incubated followed by the addition of detection antibody for 1 h at room temperature. Later, 100 µl of Avidin-HRP solution was added per well and incubated for 30 min. Then 100 µl of trimethylbenzidine solution per well for 15 min and measured absorbance at 450 and 570 nm, reaction was terminated by 1 M H₃PO₄. The difference between absorption was calculated and protein estimated in pg/mg.