CDC20 promotes bone formation via APC/C dependent ubiquitination and degradation of p65 (2021)
Figure 8

Figure 8. CDC20 regulated osteogenic differentiation of BMSCs in a p65-dependent manner

(A) Representative micro-CT images of trabecular bone from the femoral metaphysis of Cdc20f/f with negative control lentivirus, Sp7-Cre;Cdc20f/f with negative control lentivirus, and Sp7-Cre;Cdc20f/f with p65sh lentivirus. Scale bar, 500 μm.

(B) Histomorphometric analyses of Cdc20f/f with negative control lentivirus, Sp7-Cre;Cdc20f/f with negative control lentivirus, and Sp7-Cre;Cdc20f/f with p65sh lentivirus. (n=6)

(C, D) The ALP staining (C) and ALP quantification (D) of BMSCs from Cdc20f/f mice and Sp7-Cre;Cdc20f/f mice after 7 days osteogenic differentiation treated with negative control or p65si. (n=5)

(E, F) The ALP staining (E) and ALP quantification (F) of BMSCs from Cdc20f/f and Sp7-Cre;Cdc20f/f mice after 7 days osteogenic differentiation treated with BAY 11-7082 (NF-κB pathway inhibitor). (n=5)

(G) The expression of RUNX2 in BMSCs from Cdc20f/f mice and Sp7-Cre;Cdc20f/f mice after 7 days osteogenic differentiation treated with negative control or p65si by western blot analyses.

Data information: Data are displayed as mean ± SD and show one representative of n ≥ 3 independent experiments with three biological replicates. Statistical significance was calculated by one-way ANOVA followed by a Tukey's post hoc test and defined as *p<0.05; **p<0.01; ***p<0.001.

Tables

Table 1. Sequences of RNA and DNA oligonucleotides.

Sense strand/ sense primer (5'-3')

Antisense strand/ antisense primer (5'-3')

AAGGAGTAAGACCCCTGGACCA

GCAACTGTGAGCAGGGGAGATT

ACTACCAGCCACCGAGACCA

ACTGCTTGCAGCCTTAAATGACTCT

TGTGTGGGGTGAAGGCCAAT

TCGTGGTGGTCACAATGCCC

CACCATGAGAGCCCTCACACTC

CCTGCTTGGACACAAAGGCTGC

TGTGTGGCCTAGTGCTCCTG

ACACCATGCTACGGCCTTGA

GCGCATCCAGACCAACAACAA

CCCACGCTGCTCTTCTTGGAA

CGCAACAACTCATCTCATTCTGCG

CATGCTACATTTGCCGAAGAGC

CGGATAAAGGGCCAAGAGAATATCCG

TCACATTCTAGCAAACCCATTCAA

ATTCAAGCTTAGCCTGGCCG

ATTTCCGGACTGACAGGGTG

CAGGAGAGTGTTTCCTCGTCC

TGAAGGGGTCGTTGATGGCA

CTCAAAGGACACACAGCACGG

CGCCACAACCGTAGAGTCTCA

GCACACTGTGTTGCCCACTT

ACCCCTGGATGACGGTACAC

GCAAGAAGGCTCTGGCGTTT

ACTGCTTGCAGCCTTAAATATTCCT

TTCTCCGAACGTGTCACGT

CDC20-sh1

GCACTGGACAACAGTGTGTAC

GCAGAAACGGCTTCGAAATAT

GGTTCAAGCTGCTGACCTTCA

GCCAGGAATGGAAGTTCAAGG

GCCGTGGGCATGCTGCGTAAT

CTCAAGATCTGCCGAGTAAAC

GCAAAUCCAGUUCCAAGGUTT

ACCUUGGAACUGGAUUUGCTT

CCUUGUGGAUUGGAGUUCUTT

AGAACUCCAAUCCACAAGGTT

GCGACAAGGUGCAGAAAGATT

UCUUUCUGCACCUUGUCGCTT

GGAUUGAGGAGAAACGUAATT

UUACGUUUCUCCUCAAUCCTT

GCAUCUGCAGGAUGGCAUUTT

AAUGCCAUCCUGCAGAUGCTT

CCUUGGUGCCUUGACCAUUTT

AAUGGUCAAGGCACCAAGGTT

GCUGCUGCAGGUUGACUAUTT

AUAGUCAACCUGCAGCAGCTT

CCAGCAUCCUUCGGAACUUTT

AAGUUCCGAAGGAUGCUGGTT

UUCUCCGAACGUGUCACGUTT

ACGUGACACGUUCGGAGAATT

Abbreviations: shRNA, short-hairpin RNA; si-RNA, short interfering RNA.

Table 2. sgRNA sequences and genotyping primers.

Cdc20 sgRNA1

GGTGGGATTACGTAAAATTAAGG

Validation

Cdc20 sgRNA2

AAAGAAATTGGGTCCGAATGAGG

Cdc20 sgRNA3

CCTAAACTATGTGGAGTTCAAGG

Cdc20 sgRNA4

AGGTAGGGCTATGTAATAGAGGG

Cdc20 sgRNA5

AACAGTTTACGTACATATTTTGG

Cdc20 sgRNA6

CCTTGAACTCCACATAGTTTAGG

Cdc20 sgRNA7

CAAGGTCTTCCTAAACTATGTGG

Cdc20 sgRNA8

AGGTGACTCCCATTTAGTGCAGG

Validation and injection

Cdc20 sgRNA9

CTGACATCTCCGTCTCGCCCTGG

Cdc20 sgRNA10

CAGGGCGAGACGGAGATGTCAGG

Cdc20 sgRNA11

TTTTCAAACAACTCCAAGACAGG

Cdc20 sgRNA12

GAGGAGCAGCCAGGGCGAGACGG

Cdc20 sgRNA13

GACGGAGATGTCAGGTTACAGGG

Cdc20 sgRNA14

AGACGGAGATGTCAGGTTACAGG

Cdc20 sgRNA15

ATCTCCTGGAGACTCCCTCCTGG

Cdc20 sgRNA16

GTCACCAGGAGGGAGTCTCCAGG

Genotyping

Cdc20 5'loxP-F

CCTAAACTATGTGGAGTTCAAGGCCA

WT:202bp

Cdc20 5'loxP-R

AGGATCTAGGATCTAGGTGACTCCC

Mut:321bp

Cdc20 3'loxP-F

GAAGCAGCTCCTGTCTTGGAGTTGT

WT:405bp

Cdc20 3'loxP-R

CCACAGCCTGGGTGGAATGGATAAA

Mut:490bp

Sp7-Cre WT-F

TACCAGAAGCGACCACTTGAGC

263bp

Sp7-Cre WT-R

CGCCAAGAGAGCCTGGCAAG

Sp7-Cre Mut-F

445bp

Sp7-Cre Mut-R

GCACACAGACAGGAGCATCTTC

Expanded View Figure Legends