Reagents and antibodies
The specific primary antibodies for cleaved caspase-3, ERK, phospho-ERK (Thr202/Tyr204), Beclin-1, Smad2, Smad3, phospho-Smad2 (Ser465/467) and phospho-Smad3 (Ser423/425) were purchased from Cell Signaling Technology (Boston, MA, USA); antibody against LC3B, Rapacymin, 3-methyladeine (3-MA), Mitochondrial Membrane Potential Assay Kit (II), Autophagy assay kit (Sigma-Aldrich, St. Louis, MO, USA); anti-p62/SQSTM1 MBL International (Woburn, MA, USA); β-actin antibody and peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology, USA); pre-stained protein standards (Fermentas, Lithuania); CCK-8 assay kit (Dojindo, Kumamoto, Japan); Click-iT™ EdU Alexa Fluor Imaging kit was bought from Invitrogen (Carlsbad, CA, USA); transfection reagents Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA); Human AnnexinV-FITC (AV) and propidium iodide (PI) detection kit and Cycle test Plus DNA Reagent kit were from BD Bioscience (San Jose, CA, USA); Reverse transcription kit and SYBR Green I Master mix and Chemiluminescence (ECL) detection kit were purchased from Thermo Fisher Scientific Inc (MA, USA); HA was purchased from the Institute of Chemistry, Chinese Academy of Sciences, China. Hypocrellin (HA) was dissolved with dimethyl sulfoxide (DMSO) and stored at the temperature of −20 °C. The real-time fluorescence quantitative PCR was from ABI 7500 (USA). Micro-plate reader was Spectra Max 190 produced by Molecular Devices (Sunnyvale, CA), Flow cytometer was from Becton Dickinson (CA, USA) and fluorescence Microscope was from Zeiss (Carl Zeiss AG, Germany).
LED source and irradiation
Considering the skin penetration of visible light and HA absorption, we selected LED red light at a wavelength of 630 nm. The output power of LED light source was inspected by the Chinese National Institute of Metrology (Supplementary Fig. 1). The irradiation intensity is 5.68 mW/cm2. Irradiation dose = irradiation intensity × time. In our experiment, the irradiation dosage of 3J/cm2 was selected based on pre-tests. KFs were pre-treated with HA for 4 hours followed by irradiation with LED red light at a distance about 20 mm. Prior to irradiation, the medium was replaced by PBS to avoid the formation of photochemical compounds. Controls groups were incubated in PBS, placed outside incubator, and kept light-protected for an equivalent time to ensure the consistency of the experiments. All the tests were classified into four groups: non-treated control group, HA treated alone, LED red light irradiation alone and HA-R-PDT group, where the HA concentration used in our study is 0.25 μM.
Cell culture
Primary human KFs were excised from chest keloid tissues of a male patient after having obtained informed consent. In brief, tissues were made a 5-mintuts wash in PBS for 3 times, then the epidermis was eliminated by scalpels and the rest dermis was cut into minor blocks followed by 0.25% trypsinase-EDTA digestion at 37°C for 4 hours. After digestion, the tissue/cell suspension was passed through a 100 μm filter to get rid of the remaining tissue residuals. Then collected cells were washed with PBS and centrifuged at 1000 rpm for 5 min. Finally, re-suspended cells in FibroLife Basel medium supplemented with 2.5μg rh Insulin, 7.5mM L-Glutamine, 25μg Ascorbic Acid, 5ng rh FGF-β, 2% FBS (LifeFactor), 5μg Hydrocortisone Hemisuccinate and 100 U/ml penicillin (Gibco). KFs were maintained at 37 °C in a saturated humidity atmosphere with 5% CO2. Cells of third and sixth passages were utilized in the present research.
Cell viability assay
The KFs were seeded in 96-well plates at 6 × 103 cells/well and cultured for 20 hours. Then replaced with fresh medium with or without Hypocrellin (0–1 μM) and continually cultured for 4 hours. After pre-procession, KFs were irradiated with red light for 9 min. Twenty hours later, cell growth was detected by the CCK-8 assay kit. In brief, aspirated the medium from the plates and added 10 μL/well CCK-8 (5 mg/ml) solution in medium to plated at 100 μl/well and incubated at 37 °C for 2 hours. The optical density was surveyed using a microplate reader at 450 nm (Spectra Max 190; Molecular Devices, Sunnyvale, CA). All the assays were performed in triplicate and three replicate wells were performed for each group.
Hoechst staining
Cell apoptosis was evaluated by dyeing with Hoechst 33258. KFs were treated as mentioned earlier. After being fixed with 4% paraformaldehyde, cells were incubated with 10 μM Hoechst 33258 for 20 min at room temperature. Then cells were monitored using a fluorescence microscope (Zeiss, Germany). Apoptosis was stipulated as the ratio of nucleus condensation and the assessment was performed by counting about 200 cells of each group.
Mitochondrial membrane potential measurement
Mitochondrial membrane potential was detected through the Mitochondrial Membrane Potential Assay Kit (II) (Sigma-Aldrich, MO, USA) according to the product instruction. TMRE (tetramethylrhodamine ethyl ester perchlorate), a cell membrane penetrable fluorescence dye, can assemble at intact mitochondria. In normal cells, TMRE accumulates in the mitochondria and emits an orange-red fluorescence with a maximum at 575 nm; while in cells with damaged mitochondria or have lost mitochondria activity cannot accumulate TMRE. After KFs were treated as described above, discarded the medium and incubation with 200 nm TMRE in 1 X PBS at 37°C for 15 min. Then cells were washed with 1 X PBS for 3 times. In the end, fluorescence intensity was measured under the fluorescence microscope (Zeiss, Germany).
Apoptosis assay
Cell apoptosis was monitored by Human AnnexinV-FITC (AV) and propidium iodide (PI) detection kit. In brief, KFs were treated as described above. After harvested and washed with PBS, cells were re-suspended in 100 μl 1 × Annexin V-FITC binding buffer. Afterwards, cells were incubated with 5μL/tube Annexin V-FITC in the dark for 15 min at room temperature and stained with 5μL/tube propidium iodide (PI) dye. Apoptotic cells were evaluated by a fluorescence-activated cell-sorting (FACS) flow cytometer (Becton-Dickinson, CA, USA). The results were estimated by the Cell Quest software on flow cytometry. All experiments were repeated three times.
Cell cycle analysis
For cell cycle analysis, KFs were harvested after being treated as previously mentioned and washed with PBS. The quantification of cell cycle was analyzed by Cycletest Plus DNA Reagent kit according to the product's manual. In brief, cells were first incubated in solution A for 10 min at room temperature and light-protected, then sequentially added solution B and solution C. Analyses were performed using the FACS Calibur system and data were dissected with Cell Quest software (BD Biosciences, San Jose, CA, USA).
EdU incorporation assay
The EdU incorporation was performed using the ClickiT™ EdU Alexa Fluor Imaging kit according to its manufacturer's instruction. After noted treatment, KFs were incubated with 25 μM 5-ethynyl-2′deoxyuridine in the culture medium for additional 4 hours. Then cells were fixed with 4% formaldehyde at room temperature for 30 min and neutralized the residual fixing solution with Glycine for 5 min. Afterwards, cells were washed with 3% BSA for 3 times and then permeabilized via exposing to 0.5 % Triton X-100 in PBS for 20 min. Ultimately, cells were stained with reaction cocktail light-protected for 30 min, followed by dyeing with Hoechst 33342 for 20 min at the concentration of 5 μg/ml. The Hoechst positive cells (blue) and EdU positive cells (green) were photographed under a fluorescence microscope (Zeiss, Germany). These results were repeated in three independent experiments and observed in three randomly selected regions.
Transient transfection
The si-ATG5 and si-Smad 3 shRNA-expressing vector was obtained from Invitrogen (Shanghai, China). GFP-LC3 and GFP-vector were kindly donated by doctor Lin. About 5 × 104 cells were seeded in 6 well plates 20 hours before transfection. KFs were transfected with lipofectamine 3000 according to the manufacturer’s protocol.
Identification of cellular autophagy
KFs were seeded in 6 well plate at a density of 2 × 105 cells. After reaching 40-50% confluence, cells were transfected with 1 μg/μl GFP-LC3 or GFP-vector DNA with lipofectamine 3000 according to the product instructions. After that, cells were treated as stated above. Autophagy was inspected by analyzing the dispersion of fluorescent LC3 spots of autophagosome under a fluorescence microscope (Zeiss, Germany). The number of LC3 dots was counted not less than 100 cells per well.
Autophagy level detection
The autophagy level in KFs was inspected using an autophagy assay kit (Sigma, USA). KFs were seeded in 96-well plates at 6 × 103 cells/well and cultured for 20 hours. Then replaced the culture medium with fresh medium with or without Hypocrellin (0.25 μ M) and continually cultured for 4 hours. After pre-procession, KFs were irradiated with red light for 9 min. 20 hours later, cells were incubated with 100 μl of the autophagosome detection working solution and incubated at 37 °C for 30 min. Then cells were washed with wash buffer for 3 times. The autophagy signal was detected by fluorescence microscope (Zeiss, Germany).
ELISA assay
About 8 × 103 KFs were seeded in 96-well plates in triplicate 20 hours before treatment. KFs were treated as described above. Then the supernatants were collected and measured by human TGF-β1 ELISA kit according to the manufacturer’s protocol (KeyGEN Biotech, CHN). In brief, incubated the samples in the plates coated with anti-TGF-β1 antibody for 90 min at 37 ℃. After washing with wash buffer, the plate was then incubated with antibody conjugated to horseradish peroxidase for 60 min at 37 ℃. Afterwards, added developer solution to each well and incubated for 15 min, and then added stop solution to each well to stop enzyme reaction. Ultimately, the plate was detected using a microplate reader at 450 nm (Spectra Max 190; Molecular Devices, Sunnyvale, CA). Each assay was performed in triplicate and three replicate wells were performed for each group.
In vitro migration assay
The migration potential was monitored by both wound healing assay and trans-well assay. For the wound healing assay, cells were pre-seeded in 35 mm plates. When reaching 90% confluence, the single cell layer was scratched with a sterile 200-μl pipette tip. Then we treated the KFs with HA and irradiated with red light as described above and continued to culture. The speed of scratch healing was surveyed at different time points under microscope. Results were presented as the ratio of scratching filled by the KFs. Besides, the invasion capability was tested by Trans-well assay (8.0 μm pore membranes, Corning). In short, after KFs were treated as described earlier, harvested cells and seeded 2 × 103 cells in the above compartment of the cubicle with 0.15 ml of serum-free medium in 24-well plates, and the downward well was appended 0.5 ml of medium plus 10% FBS. Then cells were incubated overnight, in 5% CO2 at 37°C. After that, discarded the medium in the chambers, and used a sterilized cotton bud to erase the non-migrated cells on the top surface of the membrane. After that, we fixed the filter membrane in 4% paraformaldehyde for 25 min and stained with 0.1% crystalviolet for 5 min. Micrographs were captured by fluorescence microscope (Zeiss, Germany). The numbers of the migrated cells were obtained from five stochastically selected fields under the microscope.
RNA reversed transcription and quantitative real-time
Total RNA extraction was performed using the Trizol RNA isolation reagent (Invitrogen) and reverse transcription were using Reverse Transcription Kit (Promega, USA). The mRNA levels were detected using SYBR Green PCR Master Mix (Promega, USA) in a real-time thermal cycler (Bio-Rad, USA). The primer sequences used in each reaction were listed in table 1. Briefly, reaction conditions include: initial denaturation at 95°C for 2 min; then 95°C for 15s, 60°C for 45s and total of 40 cycles; meltingtemperature:60degrees centigradeto90degrees centigrade consuming 30min. The relative gene expression levels were normalized to the endogenous control gene β-actin and calculated by the comparative Ct (ΔΔCt) method. Each data was presented as the mean standard deviation from three independent experiments.
Western Blotting
After having been treated as previously described, cells were washed with PBS and lysed using ice-cold RIPA buffer, and measured by BCA Protein Assay Kit (Beyotime, Nanjing, China). The equivalent proteins were separated on 12% SDS-PAGE gels and then transferred onto PVDF membranes (Millipore, Billerica, MA, USA). After that, membranes were blocked in 5% milk for 45 min at room temperature and then incubated with specific primary antibodies at 4°C overnight, followed by probed with secondary anti-rabbit horseradish peroxidase-conjugated antibody. The expression of related protein was detected by an enhanced chemiluminescent ECL reagent (Millipore, Billerica, MA, USA). Bands were quantified by Image J software (National Institutes of Health, Maryland, USA).
Statistical analysis.
Statistical analysis was carried out using the Student's paired t-test or two-way ANOVA with GraphPad Prism software (Version 8.0, La Jolla, CA, USA). All values were shown as means ± standard deviation (SD) of three individual experiments. Value of * p < 0.05 and ** p < 0.01 were considered statistically significant.