Constitutive aryl hydrocarbon receptor facilitates the regenerative potential of mouse bone marrow mesenchymal stromal cells

Figure 1 The expression of aryl hydrocarbon receptor in bone marrow tissue of mouse femur and isolated bone marrow mesenchymal stromal cells. A-C: Immunochemistry staining showed positive aryl hydrocarbon receptor (AhR) expression in bone marrow tissue [bar: 200 mm (left), 100 mm (middle) and 50 mm (right)]. D-I: Immunofluorescence staining of AhR in mouse bone marrow mesenchymal stromal cells via confocal microscopy (red: AhR; blue: 4',6-diamidino-2-phenylindole; pink: Merge) [bar: 25mm (D-F) and 10mm (G-I)].

Figure 2 Overexpression or knockdown of aryl hydrocarbon receptor in mouse bone marrow mesenchymal stromal cells. A: Quantitative polymerase chain reaction data of aryl hydrocarbon receptor (AhR) mRNA level of overexpression-AhR (oe-AhR) or knockdown-AhR (sh-AhR) and their negative controls; B: Representative images of western blot of AhR in oe-AhR or sh-AhR; C: Semi-quantitation of lanes in western blot. AhR: Aryl hydrocarbon receptor; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; oe-NC: Overexpression-negative control; oe-AhR: Overexpression-AhR; sh-NC: Knockdown-negative control; sh-AhR: Knockdown-AhR.

Figure 3 The effect of aryl hydrocarbon receptor overexpression or knockdown on the osteogenic differentiation in mouse bone marrow mesenchymal stromal cells. A: Alkaline phosphatase (ALP) staining (upper) at 7^th day and Alizarin red staining staining (lower) at 14^th day of osteogenic induction; B: Relative mRNA expression of ALP, biomineralization associated [tissue-nonspecific alkaline phosphatase (ALPL)] and runt-related transcription factor 2 (RUNX2) of overexpression-negative control and overexpression-aryl hydrocarbon receptor (AhR) mouse bone marrow mesenchymal stromal cells (mBMSCs); C: Relative ALPL and RUNX2 mRNA expression of knockdown-negative control and knockdown-AhR mBMSCs at 7^th day of osteogenic induction; D: Representative images of western blot of ALPL and RUNX2 in mBMSCs of different groups at 7^th day of osteogenic induction. oe-NC: Overexpression-negative control; oe-AhR: Overexpression-aryl hydrocarbon receptor; sh-NC: Knockdown-negative control; sh-AhR: Knockdown-aryl hydrocarbon receptor; ALPL: Tissue-nonspecific alkaline phosphatase; RUNX2: Runt-related transcription factor 2; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.

Figure 4 The cluster of differentiation (CD) 86 and CD206 expressions of RAW 264. 7 cells that directly co-cultured with different mouse bone marrow mesenchymal stromal cells. A: The brightfield images of the coculture system (10 objective lens); B: The typical four-quadrant images of flow cytometry after direct coculture of RAW 264.7 cells and mBMSCs (X-axis: F4/80; Y-axis: CD86); C: The typical four-quadrant images of flow cytometry after direct coculture of RAW 264.7 cells and mBMSCs (X-axis: F4/80; Y-axis: CD206); D: Quantitative analysis of CD86 + (Q1-UR)/F4/80 + (Q1-UR + Q1-LR) and CD206 + (Q2-UR)/F4/80 + (Q2-UR + Q2-LR) ratios in direct coculture system. oe-NC: Overexpression-negative control; oe-AhR: Overexpression-aryl hydrocarbon receptor; sh-NC: Knockdown-negative control; sh-AhR: Knockdown-aryl hydrocarbon receptor; NS: Not significant.

Figure 5 The cluster of differentiation (CD) 86 and CD206 expressions of RAW 264. 7 cells that cocultured with conditioned medium from different mouse bone marrow mesenchymal stromal cells. A: The typical histograms of CD86 expressions of RAW 264.7 cells; B: The typical histograms of CD206 expressions of RAW 264.7 cells; C: Quantitative analysis of ratios of CD86+ and CD206+ cells. oe-NC: Overexpression-negative control; oe-AhR: Overexpression-aryl hydrocarbon receptor; sh-NC: Knockdown-negative control; sh-AhR: Knockdown-aryl hydrocarbon receptor; NS: Not significant.

Figure 6 The molecular mechanism of the role of aryl hydrocarbon receptor in osteogenic differentiation and macrophage-modulating in mouse bone marrow mesenchymal stromal cells. A: Co-immunoprecipitation assay showed that aryl hydrocarbon receptor (AhR) and signal transducer and activator of transcription 3 (STAT3) directly interacted in mouse bone marrow mesenchymal stromal cells (mBMSCs); B: Western blot lanes demonstrated that AhR overexpression promoted phosphorylation of STAT3 compared to negative control, while AhR knockdown suppressed it; C: The specific STAT3 inhibitor stattic (2 mmol/L) partially alleviated the promoted STAT3 phosphorylation by AhR overexpression; D: Alkaline phosphatase (upper) and alizarin red staining (lower) staining indicated that 2 mmol/L stattic partially inhibited the elevated osteogenic potential by AhR overexpression; E and F: The histograms of flow cytometry and quantitative analysis manifested that 2 mmol/L stattic partially reversed the CD86 inhibition and CD206 promotion in RAW 264.7 by conditioned medium from overexpression-AhR mBMSCs. AhR: Aryl hydrocarbon receptor; oe-NC: Overexpression-negative control; oe-AhR: Overexpression-AhR; sh-NC: Knockdown-negative control; sh-AhR: Knockdown-AhR; STAT3: Signal transducer and activator of transcription 3; p-STAT3: Phosphorylated STAT3; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.