Actinred

Apigenin was purchased from Aladdin (Los Angeles, USA). Crystal violet and 3-(4, 5-dimethylthiazol-2-y1)-2, 5-diphenyltetrazolium bromide (MTT) were purchased from Sangon Biotech (Shanghai, China). Matrigel and transwell chambers were purchased from BD Biosciences (San Jose, CA, USA). The antibodies for claudin, N-cadherin, vimentin, and E-cadherin were purchased from Affinity Bioreagents (Colorado, USA). An immunofluorescence staining kit with FITC- and TRITC-labeled goat anti-rabbit IgG and cytoskeleton red fluorescent probe ActinRed were purchased from KeyGEN BioTECH (Nanjing, China). Fluorescein isothiocyanate AffiniPure Goat Anti-Rabbit IgG (H + L) secondary antibodies were purchased from EarthOx (San Francisco, CA, USA). A dual luminescence assay kit was purchased from GeneCopoeia (Guangzhou, China).

PTL was purchased from Aladdin (Shanghai, China). Crystal violet and 3-(4, 5-dimethylthiazol-2-y1)-2, 5-diphenyltetrazolium bromide (MTT) were purchased from Sangon Biotech (Shanghai, China). The antibodies to Occludin, Claudin3, EMA, N-cadherin, VEGFR1, CD34, and Vimentin were purchased from Affinity Bioreagents(Colorado, USA). The immunofluorescence staining kit with FITC-labeled goat anti-rabbit IgG and cytoskeleton red fluorescent probe Actin Red were purchased from KeyGENBioTECH (Nanjing, China). A dual luminescence assay kit was purchased from GeneCopoeia (Guangzhou, China).

The entire sequence of the complementary DNA (cDNA) of Twist1 was generated using the total cDNA of normal human embryo. The latter was digested with XhoI/EcoRI, and subcloned into the plasmid pcDNA3.1 (Invitrogen, Carlsbad, CA). The resulting constructs were verified by DNA sequencing. Twist1 small interfering RNA (siRNA), Opti-MEM medium, control siRNA, and siRNA transfection reagent were purchased from Santa Cruz. Crystal violet, 3-(4,5-dimethylthiazol-2-y1)-2,5-diphenyltetrazolium bromide (MTT), and Rh123 were purchased from Sangon Biotech (Shanghai, China). Matrigel and transwell chambers were purchased from Biosciences (San Jose, CA, USA). ActinRed and Annexin V-FITC Apoptosis Detection Kit were purchased from KeyGEN BioTECH (Nanjing, China). Human recombinant TGF-β1 (rhTGFβ1) and anti-TGF-β1 neutralizing antibody was purchased from R&D Systems (Minneapolis, MN). The antibodies to ABCB1, ABCC1, vimentin, E-cadherin, and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were purchased from Affinity Bioreagents (Colorado, USA). Annexin V-fluorescein isothiocyanate (FITC)-goat anti-mouse IgG and tetramethylrhodamine (TRITC)-goat anti-rabbit IgG were purchased from EarthOx (San Francisco, USA).

PCL/COL scaffolds and different BBR/PCL/COL (25, 50, 75 and 100 μg/mL) scaffolds were cut into circular plates (diameter 14 mm) and transferred to the bottom of the 24-well plate. The scaffolds were incubated in DMEM overnight before cell seeding. DPSCs with a density of 2 × 10⁴ cells were seeded in each well. For the immunocytochemical staining observation, the samples were fixed with 3.7% formaldehyde for 10 min, and permeabilized with 0.1% Triton X-100 for 5 min. The nucleus was counterstained by DAPI (Invitrogen, 0.5 mg/mL) and the cytoskeleton was stained by ActinRed (KeyGEN, 5U/mL). After rinsed, the samples were observed under the confocal microscope (Nikon A1, Japon). We further observed the morphology of DPSCs cultured on the scaffold with SEM, after coculture for 7 days, the samples were fixed with 2.5% glutaraldehyde for 30 min, progressively dehydrated in ethanol with 3 min for each step (30%, 50%, 70%, 80%, 90%, 95% and 100%). The sample was sprayed with gold on the surface, morphology of DPSCs grown on the scaffolds were observed by SEM under the voltage of 3.0 kV.

BV2 cells were cultured on 24-well plates and transfected as described above. 24 h post-transfection, cells were stimulated with 1000 ng/ml LPS for 12 h, followed by collection of bright-field images using a Nikon microscope. The cells were then rinsed with PBS, fixed in 4% paraformaldehyde in PBS at RT for 10 min, and permeabilized with 0.1% Triton X-100 in PBS for 10 min. The cells were subsequently blocked in 2% bovine serum albumin in PBS at RT for 1 h, followed by incubation with a goat anti-Iba1 monoclonal primary antibody (1:500, Abcam, United States) at 4°C overnight. The following day, the cells were washed 3 times with PBS, and incubated sequentially with a donkey anti-goat IgG secondary antibody Alexa 488 (1:1000, Abcam, United States) at RT for 2 h, the cytoskeleton red fluorescent probe ActinRed (1:50, KeyGEN BioTECH, China) at RT for 20 min, and DAPI at RT for 5 min, and finally washed 3 times with PBS. Fluorescence intensity was detected using a Zeiss LSM710 fluorescence microscope.