Fig 2: Evaluation of CDK9 expression and staining in an osteosarcoma TMA by immunohistochemistry. (A) Representative images of different immunohistochemistry staining intensities of CDK9 and HE staining are shown in osteosarcoma tissues. According to the CDK9 staining in the tumor samples, the staining patterns were divided into 6 groups: no positive staining (0); <10% positive cells (1+); 10–25% positive cells (2+); 26–50% positive cells (3+); 51–75% positive cells (4+); >75% positive cells (5+). (Original magnification, 400×; Scale bar, 50 μm). (B) Pie chart representing relative frequency of different CDK9 expression levels in osteosarcoma tissue microarrays. (C) Tumors with the staining score of ≤2+ were defined as low CDK9 expression group (blue), ≥3+ were defined as the high CDK9 expression group (green). Pie chart representing relative frequency of the two groups in osteosarcoma tissue microarrays.

Fig 3: Effects of the CDK9 inhibitor LDC067 on the expression of CDK9 and cell growth in osteosarcoma cell lines. LDC067, at the indicated concentrations, inhibited osteosarcoma cell proliferation in (A) U2OS, and (B) KHOS cell lines, which was determined by MTT assays. The data represent the mean ± SE of 2 experiments carried out in triplicate. (C) Microscopy images of morphologic changes and a reduction in cells number after 72 h of LDC067 treatment (Scale bar, 100 μm). (D) The expression of proteins involved in the CDK9-signaling pathway in the U2OS osteosarcoma cell line was examined by Western blotting after 48 h of LDC067 treatment. (E) Semiquantitative analysis of Fig. 6D densitometry relative to tubulin. The data represent the mean ± SE of the experiment carried out in triplicate. (F) The expression of proteins involved in the CDK9-signaling pathway in the KHOS osteosarcoma cell line was examined by Western blotting after 48 h of LDC067 treatment. (G) Semiquantitative analysis of Fig. 6F densitometry relative to tubulin. The data are mean ± SE of the experiment carried out in triplicate.

Fig 4: CDK9 inhibition reduced osteosarcoma cell clonogenicity and migration in vitro and decreased the spheroid diameter of osteosarcoma cell lines in a 3D cell culture. (A) Representative results of colony formation in U2OS and KHOS. The numbers of colonies and their sizes were markedly decreased in cells treated with LDC067. Representative images of (B) U2OS, and (C) KHOS cell migration after LDC067 treatment for 24 h, 48 h, and 72 h. Cell migration distance of osteosarcoma cell lines (D) U2OS, and (E) KHOS were measured after 10 μM of LDC067 treatment. Spheroid formation of (F) U2OS, and (G) KHOS were significantly smaller than untreated cells at all observation points. Cells fluorescence images of spheroid formation were taken after 12 days of cultivation. The curves of relative spheroid diameter of (H) U2OS, and (I) KHOS treated with CDK9 inhibitor compared with untreated cells at all observation points. The data are mean ± SE of the 2 experiments carried out in triplicate (Scale bar, 100 μm). (** indicates P < .01).

Fig 5: Inactivation of p-CDK9 by CDK9 inhibitor or CDK9-si prevented ox-LDL-induced inflammation and phenotype switch of VSMCs in vitro. (A) LDC000067 suppressed ox-LDL-induced activation of p-CDK9. VSMCs were pretreated with LDC000067 (indicated concentrations) for 1 h and then incubated with ox-LDL (50 μg/mL) for 2 h. The levels of p-CDK9 and CDK9 were detected by western blot. (B–D) VSMCs were treated with LDC000067 (2.5 or 5 μM) for 1 hour and then exposed to ox-LDL (50 μg/mL) for 6 h (in panels B), 24 h (in panels C) or 12 h ((in panels D). (B) The levels of TNF-α and IL-6 were detected using real-time qPCR assay. (C) Expressions of α-SMA, Vimentin, OPN and PCNA in the cultural medium were detected by western blot. (D) The mRNA levels of α-SMA, Vimentin, OPN were detected using real-time qPCR assay (n = 3 ##P < 0.01, ###P < 0.001, compared to control; *P < 0.05, **P < 0.01, ***P < 0.001, compared to ox-LDL). (E–H) VSMCs were transfected with siRNA against CDK9 and then incubated with ox-LDL (50 μg/mL) for 6 h (in panels G), 24 h (in panels H) or 12 h ((in panels H). (E) VSMCs were transfected with siRNA against CDK9 for 6 h and then detected expression of CDK9 by western blot. (F) The level of TNF-α and IL-6 were detected using real-time qPCR assay. (G) Expressions of α-SMA, Vimentin, OPN and PCNA in the cultural medium were detected by western blot. (H) The mRNA level of α-SMA, Vimentin, OPN were detected using real-time qPCR assay (n = 3; ##P < 0.01, ###P < 0.001 compared to control; *P < 0.05, **P < 0.01, compared to ox-LDL).