Antitumor efficacy of DHA on HCC.

<p>The cytotoxicity of DHA was assessed by MTT assay after cells (<b>a</b>-HepG2, <b>b</b>-7402, <b>c</b>-LM3, <b>d</b>-ECV304) were treated with DHA at a serial doses as indicated for 48 h. Means ± SD (n = 6) are shown. Similar results were obtained in two independent experiments. ^# not significant, ** P < 0.01, *** P < 0.001.</p

968 and DHA cooperatively increase intracellular ROS leading to enhanced cytotoxicity in LM3 cells.

<p><b>a</b> & <b>b,</b> The intracellular ROS level was assessed after cells were treated with 968 and/or DHA for 12 or 24 h. The results are shown as the means ± SD of three independent experiments. *P < 0.05 and **P < 0.01 compared with the intracellular ROS level in cells treated with 968 or DHA only. Peak values associated with treatment for 12 h (left) and 24 h (right) are representative of three independent experiments. <b>c,</b> Cells were pretreated with NAC (3 mM) for 2 h and then treated with 968 and DHA for 48 h. The results are shown as the means ± SD of three independent experiments. ** P < 0.01 compared with cells without NAC pretreatment (UT group).</p

968 blocks the activity of glutaminase and decreases GSH/GSSG ratio in HCC cells.

<p>a, The activity of glutaminase in LM3 cells was tested after cells were treated with 968 at 20 μM for 24 h. Means ± SD of triplicates are shown. Similar results were obtained in two independent experiments. ** P < 0.01 compared with untreated controls. b, LM3 cells were treated with 968 at 20 μM for 36 h, then GSH/GSSG ratio was measured. The results are shown as the means ± SD of two independent experiments. ** P < 0.01 compared with untreated controls.</p

The mRNA expression of C10orf10 is frequently decreased in human BC tissues.

<p>(A) RT-PCR analysis of C10orf10 mRNA levels in normal breast and BC tissues. The β-actin was used as an internal control. (B) C10orf10 mRNA levels was also detected by qRT-PCR analysis in normal breast and BC tissues. The β-actin was used as an internal control. Error bars indicate s.d. (n = 3).</p

Low expression of C10orf10 is correlated with shorter survival in patients from KM plotter with luminal A, luminal B, Her2+ and grade 2 of the breast cancer.

<p>(A) Kaplan-Meir survival analysis of C10orf10 in 504 patients with luminal A tumors. Auto select best cutoff was chosen in the analysis; Cutoff value used was 1117; Expression range of the probe was 186–3881. (B) Kaplan-Meir survival analysis of C10orf10 in 319 patients with luminal B tumors. Auto select best cutoff was chosen in the analysis; Cutoff value used was 1052; Expression range of the probe was 181–3158. (C) Kaplan-Meir survival analysis of C10orf10 in 88 patients with Her2+ tumors. Auto select best cutoff was chosen in the analysis; Cutoff value used was 655; Expression range of the probe was 153–3475. (D) Survival analysis of C10orf10 expression in 286 patients with grade 2 of the breast cancer. Auto select best cutoff was chosen in the analysis; Cutoff value used was 1117; Expression range of the probe was 143–3859.</p

The combination of 968 with DHA exerts synergistic cytotoxic effect in HCC cells.

<p>a-d, The combinatory cytotoxicity of 968 and DHA (a-HepG2, b-7402, c-LM3, d-ECV304) was assessed by MTT assay after cells were treated with 968 and DHA at a serial of doses as indicated for 48 h. The molar ratio of 968 to DHA was 1:1. Data are presented as means ± SD (n = 6). Similar results were obtained in two independent experiments. ^# not significant, * P < 0.05, ** P < 0.01, and *** P < 0.001. e, The combinatory effect of 968 and DHA was assessed by trypan blue exclusion using a cell counter after LM3 cells were treated with 968 and DHA at 20 μM for 48 or 72 h. Data shown are means ± SD of triplicates. Similar results were obtained in three independent experiments. ** P < 0.01 and *** P < 0.001.f, LM3 cells were treated with 968 (20 μM) and/or DHA (20 μM) for 48 h, then cell viabilities were assessed by crystal violet staining assay. The representative plate of three independent experiments is shown.</p

The representative protein expression of C10orf10 in pairs of BC and corresponding noncancerous breast tissues.

<p>Immunohistochemistry analysis of C10orf10 expression in 75 tumor and corresponding noncancerous samples, and C10orf10 is expressed in epithelial cells of corresponding noncancerous breast tissues (mainly expressed in the cytoplasm, and also mildly expressed in some part of the nucleus). C10orf10 expression is decreased in most BC tissues compared to corresponding noncancerous breast tissues. Immunohistological staining was performed with an anti-C10orf10 antibody, and scale bars are 100 µm.</p

Low C10orf10 expression is correlated with shorter survival in BC patients.

<p>(A) Survival analysis of C10orf10 protein expression in 100 BC patients by Kaplan-Meier survival curve. Tissue array analysis was performed in 100 cases of patients with the survival information. The patients with low expression had a poorer overall survival than those with high expression; HR, hazard ratio; low, staining negative and weak; high, staining moderate and strong. (B) Survival analysis of C10orf10 expression in 100 BC patients by multivariate Cox regression. The C10orf10 expression is an independent prognostic factor. (C) Kaplan-Meir survival analysis of C10orf10 mRNA in 1115 BC patients with Kaplan-Meier Plotter (<a href="http://kmplot.com/analysis/index" target="_blank">http://kmplot.com/analysis/index</a>). The overall survival was longer in the <i>C10orf10</i> high expression group than in the <i>C10orf10</i> low expression group. Auto select best cutoff was chosen in the analysis; Cutoff value used was 1086; Expression range of the probe was 143–3881. (D) Kaplan-Meir survival analysis of C10orf10 mRNA in 3455 BC patients with Kaplan-Meier Plotter. The relapse-free survival was longer in the <i>C10orf10</i> high expression group than in the <i>C10orf10</i> low expression group. Auto select best cutoff was chosen in the analysis; Cutoff value used was 1088; Expression range of the probe was 32–5515.</p

Cytotoxic effect of 968 on HCC and human endothelial cells.

<p>a-d, The inhibitory effect on cell proliferation was detected by the MTT assay after cells were treated with 968 at depicted concentrations for 48 h. Four cell lines HepG2 (a), 7402 (b), LM3 (c), and ECV304 (d) were used. Means ± SD are shown (n = 6). ^# not significant, *P < 0.05, **P < 0.01, and ***P < 0.001. e, LM3 cells were treated with 20 μM 968 and the viability was measured by trypan blue exclusion on 1, 2, 3 and 4, respectively. Untreated cells were used as control. Data are presented as the means ± SD of triplicates. Similar results were obtained in two independent experiments. ^# not significant, *P < 0.05, **P < 0.01, and ***P < 0.001.</p

Low expression of C10orf10 is correlated with shorter survival in BC patients from KM plotter with ER positive and lymph node negative.

<p>(A) Survival analysis of C10orf10 expression in 377 BC patients with ER positive. Auto select best cutoff was chosen in the analysis; Cutoff value used was 1117; Expression range of the probe was 143–3356. (B) Survival analysis of C10orf10 expression in 424 BC patients with lymph node negative. Auto select best cutoff was chosen in the analysis; Cutoff value used was 923; Expression range of the probe was 143–3356.</p