Fig 1: Anticarin-β inhibits STAT3 expression and activation. (A) Pearson correlation analysis of anticarin-β efficiency with CLEC11A expression in cancer cell lines. The coefficients (r-value) and P values were calculated for the correlations between genome-wide gene expression profiles and the IC50 of anticarin-β (in Fig. 3A) across all overlapping cell lines (n = 24). The correlation coefficients are shown on the x-axis, and the –log (P values) are shown on the y-axis. PharmacoDB was then used to query the correlation between the expression of CLEC11A and the sensitivities of cancer cell lines to various compounds. (B) Antibody array analysis of AKT and ERK1/2 activation upon anticarin-β treatment. The data are represented as mean ± SD, n = 3; ∗∗P < 0.01 versus the control group, two-tailed t-test. (C) MG-63 cells were treated with anticarin-β and subjected to Western analysis for STATs. Anticarin-β inhibits STATs activation. MG-63 cells were exposed various hours to 0.5 μmol/L anticarin-β. Lysates were analyzed by Western blotting using the indicated antibodies. One representative experiment of three is shown. (D) Overexpression of CCT4 could delay the inactivation of p-STAT3 induced by anticarin-β in MG-63 cells. MG-63 cells were transfected with plasmids as indicated in the overexpression of CCT4 or GFP control plasmids. The levels of p-STAT3 were analyzed by Western blotting. One representative experiment of three is shown. The data are represented as mean ± SD, n = 3, ∗∗P < 0.01 versus the control group, two-tailed t-test. (E) Heatmap showing the expression of STAT3-regulated genes in MG-63 cells upon either anticarin-β (1 μmol/L) treatment or CCT4 knockdown. siCon, siRNA control (GSE166143). (F) MG-63 cells were treated with anticarin-β and before subjected to quantitative reverse transcription PCR analysis of the expression of STAT3-repressed pro-apoptotic genes. The data are represented as mean ± SD, n = 4; ns, not significant; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001 versus the control group, two-tailed t-test. (G) MG-63 cells were introduced with the indicated form of STAT3 isoform, treated with anticarin-β, and cell viability was measured. STAT3 CA: constitutively active STAT3, A662C, and N664C; STAT3 DN: dominant-negative STAT3, Y705F; STAT3 WT: wild-type STAT3. The data are represented as mean ± SD, n = 4; ns, not significant; ∗∗P < 0.01, two-tailed t-test.
Fig 2: The expression of TRiC subunits is increased in human osteosarcoma (OS). (A) Quantitative reverse transcription PCR analysis of the expression of TRiC subunits in OS tissues and matched para-cancerous bone tissues (n = 11). Relative quantification (RQ) for each subunit is presented as a dot-box plot. (B) TRiC expression was compared in patients with or without metastasis in the GSE21257 dataset. (C) Association between TRiC activity score and overall survival of the patients was estimated by Kaplan–Meier analysis. (D) Immunohistochemical analysis of CCT4 in OS tissues (17 cases in TMA (Tissue microarray)) and NBT (normal bone tissue, n = 6). Scale bar, 2 mm. (E) CCT4 knockout MG-63 cells were generated, and then the cells were plated and cultured for colony formation assays. The data are presented as the mean ± SD, n = 3. ∗P < 0.05; ∗∗P < 0.01, two-tailed t-test.
Fig 3: Anticarin-β inhibits the chaperonin activity of TRiC through interaction with CCT4. (A) Workflow for identification of CCT4 binding compounds, which mainly consists of virtual screening, pharmacophore screening, and experimental validation. The box represented the 3D structural of CCT4 and binding site (red boll) for virtual screening. (B) The viability of MG-63 cells treated with the top 5 candidates on the screen. Dapagliflozin IC50 ∼200 μmol/L, n = 3. (C) Comparison of the 3D pharmacophores of anticarin-β and dapagliflozin, purple/green spheres represent electron acceptor/donor, yellow spheres represent ring aromatic, blue spheres represent hydrophobe group. (D) The luciferase refolding assay was performed to measure the inhibitory effect of anticarin-β on the chaperonin activity of purified human TRiC. Bovine albumin was used as a negative control. The mean ± SD from three representative experiments is shown. (E) The cellular thermal shift assay was performed on intact MG-63 cells with 2.5 μmol/L anticarin-β. The band intensity values were calculated from three independent experiments. The points and error bars represent the mean ± SD, n = 3; ∗P < 0.05, ∗∗P < 0.01 versus the control group, two-tailed t-test. (F) CCT4 mutant plasmids were transfected into U2OS cells followed by 1 μmol/L anticarin-β treatment for 20 h. Cell viability was analyzed by trypan blue. The points and error bars represent the mean ± SD, n = 4; ∗∗∗∗P < 0.0001 versus the WT group, two-tailed t-test. CK, blank vector; WT, wild type CCT4. (G) 3D structure of the binding site of anticarin-β with CCT4, the green dotted lines represent hydrogen bonds, and the pink dotted lines represent alkyl hydrophobic bonds. (H) Venn diagrams show the number of overlapped genes among top 100 significantly up-regulated (↑) and down-regulated (↓) genes in OS cells with anticarin-β or siCCT4 treatment. (I) Hallmark GSEA shows a significant overlap of enriched pathways in OS cells between anticarin-β and siCCT4 treatment. Bar plot shows commonly enriched Hallmark pathways with FDR < 0.25. The numbers represent the normalized enrichment scores (NES) for the corresponding gene sets.
Fig 4: Anticarin-β inhibits OS cell growth by inducing apoptosis and disrupting autophagosome degradation. (A) Heatmap showing the selective cytotoxicity of anticarin-β towards a panel of normal and cancer cell lines. The cells were treated with a serial concentration of anticarin-β for 48 h. Cell viability was measured by MTT assay, and the IC50 was determined, n = 4. (B) The real-time cell analysis data show that anticarin-β has selective cytotoxicity on osteosarcoma cells (left, anticarin-β 0.5 μmol/L) compared with non-malignant cells (right, anticarin-β 2 μmol/L). Arrows indicate the time that anticarin-β was applied. (C) OS cells were treated with 0.05 μmol/L anticarin-β for 1 day and then plated for colony formation. Anticarin-β has no effects on the cell cycle distribution of OS cells. OS cells were treated with 0.5 μmol/L anticarin-β for 12 h, and the proportion of cells in G0/G1, S, G2/M, was determined by PI (propidium iodide) staining. The points and error bars represent the mean ± SD, n = 3; ns, not significant; ∗∗∗P < 0.001 versus the phosphate buffered saline (PBS) group, two-tailed t-test. (D) MG-63 cells were treated with 0.5 μmol/L anticarin-β for the indicated durations before being analyzed by Western blot. (E) BrdU analysis of MG-63 cells treated with either PBS or anticarin-β. Scale bar, 20 μm. The data represent mean ± SD, n = 20; ∗∗∗∗P < 0.0001 versus the PBS group, two-tailed t-test. (F) MG-63 cells were loaded with caspase 3/7 green probes and then treated with 0.5 μmol/L anticarin-β or vehicle for 24 h. An increase in fluorescence indicated the occurrence of apoptosis. Scale bar, 1 μm. The data represent mean ± SD, n = 20; ∗∗∗∗P < 0.0001 versus the PBS group, two-tailed t-test. (G) After treatment with various concentrations of anticarin-β for 12 h, the apoptotic cells were stained by Annexin V and PI staining. The cells at the stage of early apoptosis (Av+/PI–) or late apoptosis (Av+/PI+) were quantified by flow cytometry. The data represent mean ± SD, n = 3; ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001 versus the control group, two-tailed t-test. (H) Ectopic expression of CCT4 protected, whereas siRNA-mediated knockdown of CCT4 promoted, cell death of MG-63 induced by anticarin-β (two-tailed t-test, n = 3). (I) Electron microscopy images showing the autophagosomes (green arrow) and lysosomes (red arrow) in MG-63 cells treated with PBS or 0.5 μmol/L anticarin-β for the indicated times. Scale bar, 1 μm. The data represent mean ± SD, n = 5; ∗∗∗∗P < 0.0001 versus the PBS group, two-tailed t-test. (J) MG-63 cells expressing mCherry-GFP-LC3 were treated with 0.25 and 0.5 μmol/L anticarin-β for 24 h. Autophagosomes (yellow) and autolysosomes (red) were counted. Scale bar, 20 μm. Data are mean ± SD; ∗∗∗P < 0.001, ∗∗P < 0.01, ∗P < 0.05; ns, not significant; two-tailed t-test, compared with the PBS group.
Supplier Page from Abcam for Anti-TCP1 delta antibody