Fig 2: CB1 upregulated the osteo/dentinogenic differentiation potential of PDLSCs under TNF‐α and INF‐γ stimulation. A‐D, 10 ng/mL TNF‐α was used to treat PDLSCs. A, Real‐time RT‐PCR results showed the expression of CB1 at 1, 2, 4 and 8 h after 10 ng/mL TNF‐α treatment in PDLSCs. B, ALP activity assay. C, Alizarin Red staining. D, Calcium quantitative analysis. E‐H, 100 ng/mL INF‐γ was used to treat PDLSCs. E, Real‐time RT‐PCR results showed the expression of CB1 at 1, 2, 4 and 8 h after 100 ng/mL INF‐γ treatment in PDLSCs. F, ALP activity assay. G, Alizarin Red staining. H, Calcium quantitative analysis. GAPDH was used as an internal control. One‐way ANOVA was performed to determine statistical significance. Error bars represent the SD (n = 3). *P ≤ .05; **P ≤ .01

Fig 3: Overexpression of CB1 enhanced the osteo/dentinogenic differentiation of PDLSCs. (A) Western blot results showed the overexpression efficiency of HA‐CB1 in PDLSCs. β‐actin was used as an internal control. (B) ALP activity assay. (C) Alizarin Red staining. (D) Calcium quantitative analysis. (E‐H) Real‐time RT‐PCR results of the ON (E), DSPP (F), DMP1 (G) and BSP (H) expression levels during PDLSC osteo/dentinogenic differentiation. (I‐L) Real‐time RT‐PCR results of OSX (I), DLX2 (J), DLX3 (K) and DLX5 (L) expression levels in PDLSCs. GAPDH was used as an internal control. Student's t test was performed to determine statistical significance. Error bars represent the SD (n = 3). *P ≤ .05; **P ≤ .01

Fig 4: CB1 promoted the osteogenic differentiation of hBMSCs. A Western blot results showed the knockdown efficiency of CB1 shRNA1 in BMSCs. β-actin was used as an internal control. B The ALP activity assay in CB1 depleted hBMSCs. C Alizarin red staining in CB1 depleted hBMSCs. D OD values of the alizarin red staining in CB1 depleted hBMSCs. E Calcium quantitative analysis in CB1 depleted hBMSCs. F Western blot results showed the over-expression efficiency of HA-CB1 in BMSCs. β-actin was used as an internal control. G The ALP activity assay in CB1 over-expressing hBMSCs. H Alizarin red staining in CB1 over-expressing hBMSCs. I OD values of the alizarin red staining in CB1 over-expressing hBMSCs. J Calcium quantitative analysis in CB1 over-expressing hBMSCs. Student’s t test was performed to determine statistical significance. Error bars represent SD (n = 3). *P ≤ 0.05; **P ≤ 0.01

Fig 5: Periodic CB1 hotspots display stable dynamics revealed by live SIM imaging.a Representative live image of transfected CB1-RFP in the primary neuron (DIV 9–12) of the SD rat acquired by SIM. Individual CB1 hotspots are marked with purple balls, and locations of each individual time points are connected with lines. Line colors indicate trace indexes. N = 3 biological replicates. b Five CB1 hotspots shown in (a) with their relative locations. c Displacement changes from (b) are around 60–70 nm between neighboring time points. d Averaged autocorrelation analysis of CB1 distributions at different time points with the histogram of the autocorrelation amplitude. p = 0.99 (no significance), one-way ANOVA. Actual autocorrelation from left to right, 0.47 ± 0.02, 0.47 ± 0.03, 0.47 ± 0.02. e Averaged cross-correlation analysis between the neighboring frames (0 vs 60 s, 0 vs 120 s) showed similar distribution properties and histogram showing amplitude of average cross-correlation. p = 0.386 (no significance), statistical analysis was performed by unpaired two-tailed Student’s t test. Actual cross-correlation amplitude (from left to right), 0.45 ± 0.05; 0.51 ± 0.02. f The histogram of CB1 spacing across time points. p = 0.69 (no significance, one-way ANOVA). Actual spacing (from left to right), 192 ± 0.6, 192 ± 0.7, 191 ± 0.6 nm. Data in (d−f) are mean ± s.e.m. (N = 3 biological replicates; 70–120 axonal regions were examined per condition). g Traces of the individual CB1 hotspots over time. Source data are provided as a Source Data file.