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FIGURE and TABLE LEGENDS
Figure 1. In vitro properties of UD-017 as a CDK7 inhibitor.
(A) UD-017 selectivity for various kinases. Specificity of UD-017 (1000 nM) was tested against a
panel of 313 kinases. IC50 values were calculated for kinases that showed more than 50% inhibition at
1000 nM of UD-017. (B) Selectivity of UD-017 for various CDK subtypes. (C) GI50 values of UD017 against colorectal cancer cell lines.
Figure 2. Mechanism underlying the antiproliferative effects of UD-017 in HCT-116 cells.
(A) Effect of UD-017 on the phosphorylation of CDKs and Rb protein in HCT-116 cells at 8 and 24
hrs after treatment. CDK1, pCDK1, CDK2, pCDK2: 34 kDa. Rb, pRb: 110kDa. GAPDH: 37kDa. (B)
Effect of UD-017 on cell cycle progression in HCT-116 cells at 8 hrs after treatment (n = 2). Band
densities were quantified and normalized to DMSO control.
Figure 3. Induction of apoptosis by UD-017 in HCT-116 cells.
(A) Effect of UD-017 on phosphorylation of RNAPII in HCT-116 cells at 1 to 72 hrs after treatment.
RNAPII, pRNAPII: 240 kDa. (B) Effect of UD-017 on Myc expression in HCT-116 cells at 1 to 72
hrs after treatment. c-Myc: 57-65 kDa. (C) Expression of apoptosis-related proteins at 72 hrs after
treatment with UD-017 in HCT-116 cells. XIAP: 53 kDa, PARP: 116 kDa, cleaved PARP: 89 kDa.
(D) Apoptosis induction in HCT-116 cells at 72 hrs after treatment with UD-017 (n = 3–6). Band
densities were quantified and normalized to DMSO control. Data are presented as mean ± SD.
Dunnett’s multiple test using EXSUS, ***: P < 0.001.
Figure 4. Anticancer activity of UD-017 in HCT-116 xenograft mouse model.
(A) In vivo anticancer activity of UD-017 after 14 days of treatment. (B) Body weight change after
UD-017 treatment. Data are presented as mean ± SE (n = 6). Dunnett’s multiple test using EXSUS,
*p < 0.05, **p < 0.01, ***p < 0.001 versus vehicle group.
Figure 5. Mechanism underlying anticancer activity of UD-017 in HCT-116 xenograft mouse model.
(A) Phosphorylation of Rb protein at 2 hrs after administration of a single dose of UD-017 (100
mg/kg). Rb, pRb: 110kDa. NC: not calculated. (B) Phosphorylation of RNAPII protein, expression
of c-Myc, and PARP cleavage at 2 hrs after administration of a daily single dose of UD-017 (25, 50,
100 mg/kg). RNAPII, pRNAPII: 240kDa. c-Myc: 57-65kDa. PARP: 116kDa, cleaved PARP: 89kDa.
Band densities were quantified and ratios were calculated.
Figure 6. Combination treatment of HCT-116 xenograft mouse model with UD-017 and 5-FU.
(A) In vivo anticancer activity of UD-017 with 5-FU after 14 days of treatment. (B) Body weight
change after UD-017 with 5-FU treatment. Data are presented as mean ± SE (n = 6). Dunnett’s
multiple test using EXSUS, *p < 0.05, UD-017 50 mg/kg + 5-FU 15 mg/kg group versus UD-017 50
mg/kg group.
Figure 7. Schematic of proposed mechanism of action of UD-017 in HCT-116 cells.
UD-017 suppresses phosphorylation of CDK1, 2, and 4/6, which is followed by cell cycle arrest.
UD-017 also prevents phosphorylation of RNAPII and reduces the transcription of c-Myc, which
plays an important role in cell survival. Apoptosis is induced by the activation of apoptosis-related
proteins.
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