ANKRD2 Knockdown as a Therapeutic Strategy in Osteosarcoma: Effects on Proliferation and Drug Response in U2OS and HOS Cells

Figures

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  Figure 1. Ankrd2 silencing attenuates cell cycle progression. (A,B) Representative flow cytometric analysis performed on control (mock-transfected) and Ankrd2-knockdown (Ankrd2 KD) U2OS cells. Cells were synchronized by serum and glucose deprivation and then induced to cycle by mitogenic stimulation for 16 (T16) and 24 (T24) hours. As control, cells were harvested at T0; the percentages of cells in G1, S, and G2/M phases are indicated, and highlighted in the graphs by red, green and purple colors respectively. (C) Stacked bar graphs showing the percentage of cells in G1, S, and G2/M phases. Results represent mean values from four independent experiments. Statistical significance of the effects obtained after Ankrd2 silencing was calculated vs. those obtained in control cells. Following the p values for Ankrd2 KD vs. control: at T0, G2/M p < 0.05; at T16, G1 p < 0.05, G2/M p < 0.001; at T24, G1 p < 0.05; S p < 0.05; G2/M p < 0.05. (D) Cell counts of control (gray line) and Ankrd2-knockdown (Ankrd2 KD, red line) U2OS cells collected at the end of serum deprivation (T0) and after 24 and 48 h of full medium replacement. (D) Doubling time (expressed in hours) of cells collected as in (E)…

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  Figure 2. Ankrd2 reduction impairs the response to mitogenic stimuli. (A,B) Level of expression of Ankrd2 (protein) and (C) transcript (ANKRD2) in mock-transfected (control) and Ankrd2-knockdown (Ankrd2 KD) cells used for RPPA analysis. *** p ≤ 0.001. (D) RPPA analysis performed on total lysates from control and Ankrd2-knockdown cells (control and Ankrd2 KD, respectively) stimulated with FCS for 30 min. Values are reported as the mean of signal intensities, with n = 3. (E) Bar charts displaying optical density values of phosphorylated proteins quantified by RPPA for control (gray) and Ankrd2-knockdown (red) samples, and categorized within five sub-groups of signaling pathways: RTKs, Akt/PDK1, mTOR, MAPK/STAT/JAKs, and FOXOs/NFKB. Mean is only shown. Statistical significance was calculated with an unpaired t-test (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001). The complete list of antibodies used is reported on Supplementary Table S1.

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  Figure 3. Ankrd2 reduction triggers lamin A reduction and nuclear aberrations in U2OS and HOS cells. (A) Immunostaining of Ankrd2 (green) and lamin A/C (red). (B) In situ proximity ligation assay (PLA) showing lamin A/C and Ankrd2 interaction (red dots), *** p < 0.0001. DNA was counterstained with DAPI. On the right, graphical presentation of PLA signals obtained following incubation with lamin A and Ankrd2 antibodies (purple bar), or as control, with Ankrd2 alone (black bar). (C) Western blot analysis showing the level of expression of lamin A, lamin A/C, lamin B, and γH2AX in lysates from control (control) and Ankrd2-knockdown (KD) U2OS and HOS cells. As controls, blots were also incubated with antibodies against Ankrd2 and β-tubulin (loading control). (D) Densitometric analysis showing the relative level of expression of lamin A (lam A), lamin C (lam C), and lamin B (lam B) normalized to β-tubulin. (E) The real-time PCR analysis of transcripts specific for lamin A or for lamin A and C, which are alternatively spliced products of the same gene (LMNA), was performed on RNA isolated from control and Ankrd2-knockdown U2OS cells (control and Ankrd2 KD, respectively). Histograms represent the…

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  Figure 4. Ankrd2 reduction strengthens the cytotoxic effect of doxorubicin and cisplatin. (A,B) Viability assays performed on mock-transfected and Ankrd2-knockdown U2OS cells (control and Ankrd2 KD, respectively) treated for 48 and 72 h (48 h and 72 h) with concentrations of doxorubicin (A) and cisplatin (B) (30–1000 nM and 1–50 µM, respectively). IC50 values were calculated by GraphPad Prism 6 software. (C,D) The same analyses were performed on HOS cells. Three replicates per tested concentration, and at least three independent experiments were performed. Statistical analysis was performed by two-way ANOVA; *** p ≤ 0.001, **** p < 0.0001.