WDR36 inhibits the osteogenic differentiation and migration of periodontal ligament stem cells

Figure 1 Expression of WD repeat-containing protein 36 in periodontal ligament stem cells with 0. 1 μg/mL icariin. Western blot analysis showed that treatment with 0.1 μg/mL icariin (ICA) for 48 hours led to significantly increased expression of WD repeat-containing protein 36 (WDR36) in periodontal ligament stem cells (PDLSCs). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as an internal control.

Figure 2 Validation of transfection efficiency of WD repeat-containing protein 36 overexpression. A: Reverse transcriptase-reverse polymerase chain reaction results showed the overexpression efficiency of hemagglutinin-WD repeat-containing protein 36 (HA-WDR36) in periodontal ligament stem cells (PDLSCs). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as an internal control; B: Western blot analysis showed the overexpression efficiency of HA-WDR36 in PDLSCs. β-actin served as an internal control. The statistical significance was measured by the Student’s t-test. The error bars represent the standard deviation (n = 3). ^bP ≤ 0.01.

Figure 3 Migration and chemotaxis of WD repeat-containing protein 36 overexpression. A and B: The results for 0 hour, 24 hours, and 48 hours showed that the cell migration distance of the control group was longer than that of the hemagglutinin-WD repeat-containing protein 36 (HA-WDR36) group; C and D: Transwell results showed that the number of cells passing through the membrane in the WDR36 overexpression group was less than that in the control group. The statistical significance was measured by the Student’s t-test. The error bars represent the standard deviation (n = 3). ^bP ≤ 0.01.

Figure 4 Validation of transfection efficiency of short hairpin RNA-WD repeat-containing protein 36. A: Reverse transcriptase-reverse polymerase chain reaction results showed the knockdown efficiency of short hairpin RNA-WD repeat-containing protein 36 (sh-WDR36) in periodontal ligament stem cells (PDLSCs). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control; B: Western blot results showed the knockdown efficiency of sh-WDR36 in PDLSCs. β-actin was used as an internal control. The statistical significance was measured by the Student’s t-test. The error bars represent the standard deviation (n = 3). ^aP ≤ 0.05.

Figure 5 Migration and chemotaxis of short hairpin RNA-WD repeat-containing protein 36. A and B: The results for 0 hour and 24 hours showed that there were no significant differences between the short hairpin RNA-WD repeat-containing protein 36 (sh-WDR36) group and the control group. The 48 hours results showed that the cell migration distance of the control group was shorter than that of the sh-WDR36 group; C and D: Transwell results showed more cells passing through the membrane in the WDR36 depletion group than in the control group, and there was a statistically significant difference. The statistical significance was measured by the Student’s t-test. The error bars represent the standard deviation (n = 3). ^bP ≤ 0.01.

Figure 6 Osteogenic differentiation of WD repeat-containing protein 36 overexpression. A: Alkaline phosphatase (ALP) activity results on the third day; B: ALP activity results on the fifth day; C: Alizarin red staining; D: Calcium quantitative analysis; E: Expression of Runt-related transcription factor 2 (RUNX2) in the WD repeat-containing protein 36 (WDR36) overexpression group was significantly lower than that in the control group; F: Osterix (OSX) expression in the WDR36 overexpression group was significantly lower than that in the control group; G: Distal-less homeobox 3 (DLX3) in the WDR36 overexpression group was significantly lower than that in the control group; H: After 1 week and 2 weeks of osteogenic induction, osteopontin (OPN) expression in the WDR36 overexpression group was significantly lower than that in the control group; I: After 1 week of osteogenic induction, the expression of dentin matrix protein-1 (DMP1) in the WDR36 overexpression group was significantly lower than that in the control group. However, there were no statistically significant differences between the two groups at 0 week and 2 weeks; J: After 1 week of osteogenic induction, osteocalcin (OCN) expression in the WDR36 overexpression group was significantly lower than that in the control group. At 0 week and 2 weeks, no statistically significant differences between the two groups were observed. The statistical significance was measured by the Student’s t-test. The error bars represent the standard deviation (n = 3). ^aP ≤ 0.05; ^bP ≤ 0.01.

Figure 7 Osteogenic differentiation of short hairpin RNA-WD repeat-containing protein 36. A: Alkaline phosphatase (ALP) activity results on the third day; B: ALP activity results on the fifth day; C: Alizarin red staining; D: Calcium quantitative analysis; E: Expression of Runt-related transcription factor 2 (RUNX2) in the WD repeat-containing protein 36 (WDR36) depletion group was significantly higher than that in the control group; F: Osterix (OSX) expression in the WDR36 depletion group was significantly higher than that in the control group; G: Distal-less homeobox 3 (DLX3) in the WDR36 depletion group was significantly higher than that in the control group; H: At 0 week and 1 week of osteogenic induction, the expression of dentin matrix protein-1 (DMP1) in the WDR36 depletion group was significantly higher than that in the control group. At 2 weeks, there were no statistically significant differences between the two groups; I: The difference in osteopontin (OPN) expression between the WDR36 depletion group and the control group was not statistically significant. However, after 1 week and 2 weeks of osteogenic induction, OPN expression in the depletion group was significantly higher than that in the control group; J: At 1 week of osteogenic induction, OCN expression in the WDR36 depletion group was significantly higher than that in the control group. However, there were no statistically significant differences between the two groups at 0 week and 2 weeks. The statistical significance was measured by the Student’s t-test. The error bars represent the standard deviation (n = 3). ^aP ≤ 0.05; ^aP ≤ 0.01.

Figure 8 Senescence of 0. 1 μg/mL icariin and WD repeat-containing protein 36 overexpression. A: Senescence-associated β-galactosidase staining results; B: Senescence-associated β-galactosidase staining results showed that the number of positive cells in the icariin (ICA) group was significantly less than that in the group without ICA. By contrast, the number of positive cells in the hemagglutinin-WD repeat-containing protein 36 (HA-WDR36) group with or without ICA was significantly lower than that in the control group, which indicates that 0.1 μg/mL ICA inhibited the senescence of periodontal ligament stem cells (PDLSCs), and overexpression of WDR36 inhibited the senescence of PDLSCs. The statistical significance was measured by the Student’s t-test. The error bars represent the standard deviation (n = 3). ^aP ≤ 0.05; ^bP ≤ 0.01.

Figure 9 Senescence of 0. 1 μg/mL icariin and short hairpin RNA- WD repeat-containing protein 36. A: Senescence-associated β-galactosidase staining results; B: Senescence-associated β-galactosidase staining results showed that the number of positive cells in the icariin (ICA) group was significantly less than that in the group without ICA. By contrast, the number of positive cells in the short hairpin RNA-WD repeat-containing protein 36 (sh-WDR36) group with or without ICA was significantly higher than that in the control group, which indicates that 0.1 μg/mL ICA inhibited the senescence of periodontal ligament stem cells (PDLSCs), and WDR36 depletion promoted the senescence of PDLSCs. The statistical significance was measured by the Student’s t-test. The error bars represent the standard deviation (n = 3). ^bP ≤ 0.01.