Fig 2: Genome-scale screening of the ubiquitin-specific protease subfamily for PAX9 and MSX1 proteins using CRISPR-Cas9-based DUB-knockout library kit.A Screening for USPs regulating PAX9 and MSX1 proteins using the CRISPR-Cas9-based DUB KO library kit. Equal protein concentrations from DUB KO HEK293 cell lysates were subjected to Western blotting to determine endogenous PAX9 and MSX1 protein levels. GAPDH was used as a loading control for each blot. Asterisk represents the selected DUB, which regulates both PAX9 and MSX1 proteins. The presented immunoblots are representative of three independent experiments (n = 3). B Effects of putative DUB candidates (USP11, USP13, and USP49) on PAX9 and MSX1 protein levels in HEK293 cells were estimated by Western blotting. The presented immunoblots are representative of three independent experiments (n = 3). C Effects of putative DUB candidates (USP11, USP13, and USP49) on PAX9 and MSX1 protein levels in hDPSCs were estimated by Western blotting. The presented immunoblots are representative of three independent experiments (n = 3).

Fig 3: Odontogenic differentiation is regulated by USP49.A Schematic representation of full length USP49 (1-640 aa) encoding ZnF-UBP and UCH domains (represented as USP49-FL), N-terminus USP49 (1-250 aa) encoding ZnF-UBP domain (represented as USP49-N), and C-terminus USP49 (251-640 aa) encoding UCH domain (represented as USP49-C). B Interactions between USP49-FL and PAX9; USP49-N and PAX9; USP49-C and PAX9 by IP and immunoblotting with the indicated antibodies. The presented immunoblots are representative of three independent experiments (n = 3). C Interaction between USP49-FL and MSX1; USP49-N and MSX1; USP49-C and MSX1 by IP followed by immunoblotting with the indicated antibodies. The presented immunoblots are representative of three independent experiments (n = 3). D The deubiquitination of PAX9 was analyzed by transfecting HEK293 cells with USP49-FL, USP49-N, and USP49-C, and determined by IP followed by immunoblotting with the indicated antibodies. The presented immunoblots are representative of three independent experiments (n = 3). E The deubiquitination of MSX1 was analyzed by transfecting HEK293 cells with USP49-FL, USP49-N, and USP49-C, and determined by IP followed by immunoblotting with the indicated antibodies. The presented immunoblots are representative of three independent experiments (n = 3). F Representative images of alizarin red staining performed in hDPSCs (Mock, Flag-USP49, sgRNA1 targeting USP49 and shRNA1 targeting USP49) group at the indicated time points (days 3, 7, and 14). Scale bar: 200 µm. The presented microscopic images are representative of three independent experiments (n = 3). G To quantify the alizarin red staining, the calcium content was quantified using the CPC method and normalized to the DNA content per well as measured on days 3, 7, and 14. The calcium content was calculated based on the mean of three technical replicates for each individual experiment. The data presented here represent the mean ± standard deviation (SD) of three independent experiments (n = 3) (**P < 0.001 and ***P < 0.0001) by analysis of variance (ANOVA) followed by Tukey’s post hoc test. (H) Representative images of hDPSCs (Mock, Flag-USP49, sgRNA1 targeting USP49 and shRNA1 targeting USP49) group stained with alkaline phosphatase (ALP) at the time points indicated (days 3, 7, and 14). Scale bar: 200 µm. The presented microscopic images are representative of three independent experiments (n = 3). I ALP activity was quantified and normalized to the DNA content obtained from each individual dataset on days 3, 7 and 14. The ALP activity was calculated based on the mean of three technical replicates for each individual experiment. The data presented here are the mean ± SD of three independent experiments (n = 3) (*P < 0.05, **P < 0.001 and ***P < 0.0001), by ANOVA followed by Tukey’s post hoc test. J Representative images from ALZ staining in hDPSCs (Mock, USP49-N, USP49-C) on days 3, 7, and 14. Scale bar: 200 µm. The presented microscopic images are representative of three independent experiments (n = 3). K To quantify the ALZ staining, the calcium content was quantified using the CPC method and normalized to the DNA content per well as measured on days 3, 7, and 14. The calcium content was calculated based on the mean of three technical replicates for each individual experiment. The data presented here represent the mean ± SD of three independent experiments (n = 3) (ns = non-significant, **P < 0.001) by ANOVA followed by Tukey’s post hoc test. L Representative images of hDPSCs (Mock, USP49-N, USP49-C) group stained with ALP on days 3, 7, and 14. Scale bar: 200 µm. The presented microscopic images are representative of three independent experiments (n = 3). M ALP activity was quantified and normalized to the DNA content obtained from each individual dataset on days 3, 7 and 14. The ALP activity was calculated based on the mean of three technical replicates for each individual experiment. The data presented here represent the mean ± SD of three independent experiments (n = 3) (ns = non-significant, **P < 0.001) by ANOVA followed by Tukey’s post hoc test.

Fig 4: USP49 upregulates endogenous PAX9 and MSX1 proteins.A Validation of the efficiency of sgRNAs were carried out in hDPSCs by transiently transfecting sgRNA1 to sgRNA4 targeting USP49, and immunoblotting with the USP49 antibody. The presented immunoblots are representative of three independent experiments (n = 3). B Validation of the efficiency of shRNAs were carried out in hDPSCs by transiently transducing shRNA1 and shRNA2 targeting USP49, and immunoblotting with the USP49 antibody. The presented immunoblots are representative of three independent experiments (n = 3). C hDPSCs were transfected with two best sgRNAs (sgRNA1 and sgRNA3) and one shRNA (shRNA1) targeting USP49 to check the endogenous protein levels of USP49, PAX9, and MSX1 using their respective endogenous antibodies. The presented immunoblots are representative of three independent experiments (n = 3). D hDPSCs were transfected with increasing concentrations of Flag-USP49 to check the endogenous USP49, PAX9, and MSX1 proteins. The presented immunoblots are representative of three independent experiments (n = 3). E hDPSCs were transfected with increasing concentrations of Flag-USP49CA to check the endogenous USP49, PAX9, and MSX1 proteins. The presented immunoblots are representative of three independent experiments (n = 3). F Reconstitution effect of USP49 on endogenous PAX9 or MSX1 in USP49-depleted cells. Protein expression was analyzed by Western blotting using the indicated antibodies. GAPDH was used as the loading control. The presented immunoblots are representative of three independent experiments (n = 3).

Fig 5: Loss of USP49 in hESCs delays differentiation by downregulating PAX9 and MSX1.A USP49 KO in hESCs was confirmed by analyzing endogenous USP49 protein expression through Western blotting. GAPDH was used as the internal loading control. The presented immunoblots are representative of three independent experiments (n = 3). B Expression levels of endogenous pluripotency markers (SOX2, OCT4, SSEA-4 and NANOG) were confirmed by immunofluorescence staining in mock and USP49 KO-hESCs. Scale bar: 100 µm. The presented microscopic images are representative of three independent experiments (n = 3). C Endogenous expression levels of USP49, MSX1 and PAX9 were confirmed by immunofluorescence staining in mock and USP49 KO-hESCs. Scale bar: 100 µm. The presented microscopic images are representative of three independent experiments (n = 3). D Embryoid bodies (EBs) derived from mock and USP49 KO-hESCs were differentiated into NCLCs. Cell morphology during differentiation was analyzed by bright field microscopy at the indicated time points. Dotted outline indicates neural rosette formation during differentiation. Scale bar: 200 µm. The presented microscopic images are representative of three independent experiments (n = 3). E Immunofluorescence staining was performed on mock and USP49 KO-derived NCLCs on day 18 to analyze the NCLC markers (p75 and NESTIN). Scale bar: 100 µm. The presented microscopic images are representative of three independent experiments (n = 3). F Corrected total cell fluorescence (CTCF) intensity of p75 and NESTIN in mock and USP49 KO-derived NCLCs was quantified by the ImageJ software. CTCF intensities were calculated for each individual experiment based on the mean of three technical replicates The data presented here represent the mean ± SD of three independent experiments (n = 3), (*P < 0.05) by Student’s t test. G Effects of USP49 gene disruption on the endogenous expression levels of dental mesenchymal markers (MSX1, PAX9, and LHX6) were analyzed by immunofluorescence staining on day 18 of NCLC differentiation. Scale bar: 100 µm. The presented microscopic images are representative of three independent experiments (n = 3). H Corrected total cell fluorescence (CTCF) intensity of USP49, MSX1, PAX9, and LHX6 in mock and USP49 KO-derived NCLCs was quantified by the ImageJ software. CTCF intensities were calculated for each individual experiment based on the mean of three technical replicates. The data presented here represent the mean ± SD of three independent experiments (n = 3), (*P < 0.05, ***P < 0.0001 and non-significant (ns), by Student’s t test). I Effects of USP49 KO on the endogenous expression levels of dental mesenchymal markers (MSX1, PAX9, and LHX6) and a pluripotency marker (OCT4) in undifferentiated and differentiated cells were analyzed by immunoblotting with specific antibodies. GAPDH was used as the internal loading control. The presented immunoblots are representative of two independent experiments (n = 2). J Effects of USP49 KO on the mRNA expression levels of USP49, MSX1, and PAX9 in undifferentiated and differentiated cell lines were analyzed by qRT-PCR with specific primers. Relative mRNA expression levels are shown after normalization to GAPDH mRNA expression. The fold changes of mRNA levels were calculated for each individual experiment based on the mean of three technical replicates. The data presented here represent the mean ± SD of three independent experiments (n = 3). For USP49 mRNA expression: ***P < 0.0001 for mock-hESCs vs. USP49 KO-hESCs, ***P < 0.0001 for mock-NCLCs vs. USP49 KO-NCLCs. For MSX1 mRNA expression: non-significant (ns) for mock-hESCs vs. USP49 KO-hESCs, non-significant (ns) for mock-NCLCs vs. USP49 KO-NCLCs. For PAX9 mRNA expression: non-significant (ns) for mock-hESCs vs. USP49 KO-hESCs, non-significant (ns) for mock-NCLCs vs. USP49 KO-NCLCs. Statistical significance was analyzed by ANOVA followed by Tukey’s post hoc test.