Cell lines and culture
Hepatoma carcinoma cell lines Hep3B and HepG2 were cultivated in Dulbecco`s modified Eagle medium (DMEM) (Life Technologies Corporation, California, United States) containing 4,500 mg/L glucose supplemented with 10% fetal bovine serum (FBS). Culture flasks were kept at 37℃ in a humid incubator with 5% CO2.
LED irradiation
Cells are irradiation by blue (peaked at 470 nm) light at room temperature for 0 J/cm2, 72 J/cm2, 144 J/cm2, 216 J/cm2 and 288 J/cm2 respectively.
Trypan blue dyeing assay
Preparing 0.4% Trypan blue dye, the adherent cells were digested by trypsin to prepare single cell suspension. The cell suspension was mixed with 0.4% trypan blue solution at 9:1, and the living and dead cells were counted respectively in 3 min. Microscopically, dead cells are stained with a distinctive blue color, while living cells are colorless and transparent. Living cell rate (%) = total number of living cells/ (total number of living cells + total number of dead cells) × 100%.
Ethynyl-2-deoxyuridine (EdU) cell proliferation assay
Using of EdU Apollo DNA in vitro kit (Ribobio, Guangzhou, China) for dyeing. The cells grew uniformly adherent to the wall at the bottom of the six-well plate, with a density of 2.0 × 105. Using EdU reagent incubation cells after 90 min, with 4% paraformaldehyde fixed cells at 37℃ for 15 min, with 0.5% of Triton X-100 after infiltration, will join the rest of the cell cultures of Apollo dyeing liquid dark stain 30 min, and then put cells at 20 µg/mL 4', 6 − 2 amino − 2 - phenyl indole (DAPI) incubation in 20 min, PBS elution 3 times (10 min/time) in the end, under the confocal laser scanning microscope (FV10i) pictures. EdU index (%) is the average ratio of EdU positive cells to total cells in 5 randomly selected regions.
Cellular wound healing assays
Cells were plated into 6-well culture plates at the density of 2.5 × 105 cells/mL. When the confluence of cells reached to 70%, dividing three wounds evenly vertically with a 200 µL pipette tip. The cells were rinsed with PBS for twice and then the fresh medium was changed. Cells were kept at 37℃ in a humid incubator with 5% CO2 for 24 hrs. After that, cells were treated with blue LED irradiation. Wound healing was monitored at 0/6/12/24 hrs with a standard light microscopy (ECLIPSE TS100, Nikon, Japan). The wound area was measured using ImageJ software (National Institutes of Health (NIH), United States).
Propidium iodide (PI)/Hoechst 33342 staining
PI/Hoechst 33342 staining was performed using Hoechst 33342/PI Double Staining Kit (Solarbio Science, Beijing China). The cells were evenly grown on the bottom of the culture dish, fixed with 4% paraformaldehyde at 37℃ for 15 min, and stained with the dye solution at room temperature and away from light for 30 min. Finally, images of the staining were captured by the confocal laser scanning microscope (FV10i, Olympus, Tokyo, Japan).
Invasion assays
A 24 mm Transwell® chambers was used to detect cell invasive abilities according to the manufacturer’s protocol. Matrigel was diluted with PBS 1:8 and coated on the upper surface of the membrane at the bottom of the chamber. After 12 hrs of cell starvation, the cells were digested with trypsin and resuspended in serums free medium with cell density of 5 × 104 cells/mL, 200 µL of cell suspension was taken to the upper compartment, and 300 µL of 20% serum DMEM medium was added to the lower compartment, with blue light irradiation of 0 J/cm2 and 144 J/cm2. After 24 hrs, cells migrated through the membrane were stained with 0.1% crystal violet (Beyotime Biotechnology, China) for 15 min and counted using a light microscopy (ECLIPSE TS100, Nikon).
Spheroid formation assay
A total of 1000 hepatoma cells were plated in ultralow attachment plates. The cells were cultured for 10 days in DMEM/F12 medium (Invitrogen, Shanghai, China) supplemented with 4 mg/mL insulin (Sigma, Shanghai, China), B27 (1:50, GIBCO, Shanghai, China), 20 ng/mL EGF (Sigma, Shanghai, China) and 20 ng/mL basic FGF (Sigma, Shanghai, China). Liver cancer cells in good condition were digested with trypsin, centrifuged, washed twice with PBS, and resuspended with the prepared stem cell culture medium. The cells were cultured in six-well plates with ultra-low adsorption cells for 10 days, during which the cells were irradiation with blue light 0 J/cm2 and 144 J/cm2. Finally, counted using a light microscopy (ECLIPSE TS100, Nikon).
Immunofluorescence experiment
After cells irradiation by blue light of 0 J/cm2 and 144 J/cm2, were fixed with 4% paraformaldehyde for 15 min at 37℃, followed by permeability of 0.1% Triton X-100 at room temperature for 15 min, and sealed with goat serum albumin for 60 min. The cells were incubated with primary antibody at 4℃ overnight, washed with PBST for three times, then incubated with secondary antibody at room temperature in dark for 1 h, and stained with DAPI in dark for 20 min. under the confocal laser scanning microscope (FV10i) pictures. It was quantified using Image-Pro Plus software (Media Cybernetics, Silver Spring, MD). The following primary antibodies and dilutions were used for immunofluorescence microscopy experiments: γ-H2AX (Abcam, ab26350). The following secondary antibody mouse IgG (Abcam. ab150113) were used.
Western blotting
After cells irradiation by blue light of 0 J/cm2 and 144 J/cm2, the protein was extracted with RIPA buffer (Beyotime Biotechnology). Protein fractions were collected by centrifugation at 13,500 rpm for 15 min, and then supernatants were heated with SDS buffer at 100℃ for 4 min. Proteins separated by 10% SDS-PAGE and transferred to PVDF membrane. The blots were then blocked with 5% non-fat milk powder in PBST buffer for 1 h, and incubated at 4 ℃ overnight with the appropriate primary antibodies at appropriate dilutions. After washing, the bolts were incubated with HRP-conjugated secondary antibody for 1 h at room temperature. The following primary antibodies was used: γ-H2AX (Abcam, ab26350). The following secondary antibodies were used: β-actin (Absin, abs830031).