Jolkinolide B downregulates the protein of MSI2 and the p53 expression in HCC cells.

(A, C) Huh-7 and (B, D) SK-Hep-1 cells were treated with the indicated concentrations of Jolkinolide B for 48 h. The MSI2 and p53 protein expressions were analyzed using western blotting. GAPDH was used as an internal control. *p p p <0.001.</p

Jolkinolide B inhibits HCC cell lines migration, invasion and promotes HCC cell apoptosis.

(A) Huh-7 and SK-Hep-1 cells were seeded, scratched, and then treated with Jolkinolide B at a concentration of 10 μM for 48 h. The wound healing assays were performed to assess the migration abilities of Huh-7 and SK-Hep-1 cells. (B) Huh-7 and SK-Hep-1 cells were seeded in the upper transwell chamber and treated with DMSO or Jolkinolide B of 10 μM for 48 h to evaluate the migration and invasion abilities of HCC cells. (C) Huh-7 and SK-Hep-1 cells were treated with DMSO or Jolkinolide B at a concentration of 10 μM for 48 h; then the protein expression of E-cadherin and vimentin was analyzed using western blotting. GAPDH was used as an internal control. (D) Western blotting was used to assess Bax and BCL-2 protein expressions. GAPDH was used as an internal control. The relative protein intensities were analyzed. (E) Huh-7 and SK-Hep-1 cells were treated with DMSO or Jolkinolide B, the cells apoptosis was analyzed by flow cytometry and apoptosis rates were calculated. *p p p <0.001.</p

The effects of Jolkinolide B on the β-catenin pathway in HCC cells.

(A) Huh-7 and (B) SK-Hep-1 cells were treated with the indicated concentrations of Jolkinolide B for 48 h. The β-catenin protein expression was analyzed using western blotting. GAPDH was used as an internal control. The relative protein intensities were analyzed. (C, D) Huh-7 and SK-Hep-1 cells were treated with 10 μM Jolkinolide B for 48 h. The mRNA expression of MMP-7 and C-MYC in Huh-7 (C) and SK-Hep-1 (D) cells analyzed using RT-qPCR method. GAPDH was used as an internal control. *p p p p <0.0001.</p

MSI2 overexpression promotes β-catenin expression in HCC cells.

Huh-7 and SK-Hep-1 cells were transfected with lentivirus-mediated MSI2 overexpression plasmids for 48 h, the MSI2 protein expression (A) and MSI2 mRNA expression (B) were analyzed using western blotting and RT-qPCR methods, respectively. (C) β-catenin protein was detected in Huh-7 and SK-Hep-1 cells after MSI2 overexpression. The relative protein intensities were analyzed. *p p p <0.001.</p

S1 File -

Slide 1: Original images for Fig 2A, 2B, 2E. Slide 2: Original immunoblots for Fig 2C, 2D. Slide 3: Original immunoblots for Fig 3. Slide 4: Original immunoblots for Fig 4. Slide 5: Original immunoblots for Fig 5A, 5C. Slide 6: Original immunoblots for Fig 6A, 6B. Slide 7: Original immunoblots for Fig 6C. Sheet 1: Original CCK8 of cell viability for Fig 1A-1C. Original RT-qPCR for Figs 3C, 3D, 5B. (ZIP)</p

Jolkinolide B inhibits HCC cells proliferation.

Cell viability assays of Huh-7 (A), SK-Hep-1 (B) and L-02 cells (C) were performed using CCK-8 method after treatment with different concentrations (0, 5, 10, 25, 50, or 100 μM) of Jolkinolide B for 48 h.</p

Overexpression of MSI2 reverses Jolkinolide B-promoted apoptosis and Jolkinolide B-inhibited EMT and β-catenin signaling in HCC cells.

Huh-7 and SK-Hep-1 cells were treated with DMSO, 10 μM Jolkinolide B, or Jolkinolide B together with MSI2 plasmids. Then, the protein expressions of (A) Bax, BCL-2, (B) E-cadherin, vimentin and (C) β-catenin were analyzed using western blotting. GAPDH was used as an internal control. The relative protein intensities were analyzed. (D) Proposed working model of Jolkinolide B-induced inhibition of HCC. *p p p <0.001.</p

RecQL4 associates with survivin.

<p>(A) Endogenous survivin was immunoprecipitated with Flag-RecQL4 recognized by an anti-Flag antibody from cell extracts of 10⁶ U2OS cells, but not with Flag-GFP. The immunoprecipitated proteins were detected with antibody against survivin (Cell Signal). Five percent of the lysate was used for the loading control (Input) and the remaining 95% for co-immunoprecipitation. (B) Endogeneous RecQL4 was immunoprecipitated with Flag-survivin recognized by an anti-Flag antibody from cell extracts of 10⁶ U2OS cells, but not with Flag-GFP. The immunoprecipitated proteins were visualized by Western blot analysis with antibody against RecQL4 (Cell signal). (C) In the upper panel, schematic diagram of RecQL4 deletion constructs used for Co-IP studies is shown. In the lower panel, 293T cells were co-transfected with pRc-CMV2-survivin and one of the Flag-tagged truncated RecQL4 expressing vectors: pFlag-RecQL4-NT(−), pFlag-RecQL4-HD(−) or pFlag-RecQL4-CT(−). Survivin was immunoprecipitated with N-terminal (NT) deleted Flag-RecQL4 protein, not with helicase domain (HD) or C-terminal (CT) deleted Flag-RecQL4 protein.</p

RecQL4 protein or mRNA level in breast tumor cell lines and clinical breast cancer specimens.

<p>(A) Western blot detection of RecQL4 protein level in HMEC, MCF-10F and five breast tumor cell lines. β-Actin was used to verify equal loading of proteins. (B) Analysis of RecQL4 expression by quantitative real time PCR in normal breast tissues and breast cancer specimens with different pathological grades. TissueScan breast cancer tissue qPCR array was purchased from Origene. RecQL4 expression detected in normal breast tissues was considered as 1. The data represent mean ± SD from three independent experiments.</p

Amplification of RecQL4 genomic locus analyzed by mBAND-FISH and quantitative real time PCR.

<p>(A) Analysis of chromosome specific mBAND-FISH in the metaphase spreads of normal primary (HMEC), immortalized (MCF-10F) and tumorigenic breast cancer cell lines. Chromosomes and chromosome regions positive for the probe are shown in the inserts. (B) FISH analysis using spectrum orange labeled BAC (Bacterial Artificial Chromosome) probe proximal to 8q24.3 chromosome locus harboring the RecQL4 gene in MDA-MB453 and MDA-MB361 cells. The BAC probe was purchased from Open Biosystems (RP11–374B7, Huntsville, Alabama, USA). (C) Upper panel: Agarose gel electrophoresis showing the abundance of RecQL4 genomic DNA detected by PCR in breast cancer cell lines relative to normal primary and immortalized breast epithelial cells. GAPDH was used as an internal control; Lower panel: Abundance of RecQL4 genomic DNA detected by quantitative real time PCR in breast cancer cell lines relative to normal primary and immortalized breast epithelial cells. GAPDH was used for normalizing the values of RecQL4.</p