Additional file 1: of CD317 Promotes the survival of cancer cells through apoptosis-inducing factor

Supporting Online Material for “CD317 Promotes the survival of cancer cells through apoptosis-inducing factor”. (DOCX 550 kb

Delayed post-ischemic treatment with salvianolic acid B improved cognitive impairment in Morris water maze task.

<p>(A) Place learning with multiple trials. There was a decrease in escape latencies with training in all three groups. (B) In the transfer task, the escape latencies (mean ± S.D.) are compared among sham-operated, untreated ischemic, and Sal B treated groups (n = 8). And the animals from the Sal B-treated group spent more time in the quadrant that contained the escape platform during the place learning than untreated group. *<i>P</i><0.05 as compared with sham group, #<i>P</i><0.05 as compared with model group.</p

Salvianolic acid B up-regulated the self-renew genes of NSPCs.

<p>(A) The mRNA levels of NSPCs self-renew markers were analyzed by RT-PCR. (B) The expression levels were semi-quantified by densitometric measurements, normalized with GAPDH internal control, compared with control, and expressed as means ± S.D from three independent experiments. *Significant difference from the control group at <i>P</i><0.05. **Significant difference from the control group at <i>P</i><0.01.</p

Salvianolic acid B promoted NSPCs proliferation in a PI3K/Akt -independent pathway.

<p>NSPCs were cultured in the proliferation medium containing Sal B (20 µM) in the presence and absence of the PI3K inhibitor Ly294002 (20 µM), MEK inhibitor U0126 (10 µM) or Notch inhibitor DAPT (10 µM) for 2 days. Cell survival was assessed by MTS assay. Data represent the mean ± S.D. from three independent experiments. **<i>P</i><0.01 as compared with control,##<i>P</i><0.01 as compared with Sal B-treated cells.</p

Screening for NSPCs proliferation-inducing natural materials.

<p>The proliferation-inducing activities on NSPCs of a total of 45 natural compounds, which were from medicinal materials extensively used in China to treat stroke clinically, were tested using a MTS assay, and the results are expressed in fold change relative to the corresponding controls. The proliferation-inducing effect of the most potent compound, Sal B (A) and berberine (B), were indicated by the arrow and its structure is shown in the inset. Data represent the mean ± S.D. from three independent experiments. **Significant difference from the control group at <i>P</i><0.01.</p

The promotive effect of salvianolic acid B on NSPCs proliferation.

<p>(A) Sal B dose-dependently promoted NSPCs proliferation. (B) Sal B time-dependently promoted NSPCs proliferation. Data represent mean ± S.D. from three independent experiments. **Significant difference from the control group at <i>P</i><0.01. (C–D) Representative microphotographs of formed neurospheres in the absence (C) or presence (D) of Sal B (20 µM). Sal B increased the size of formed neurospheres. Scale bars: 200 µm.</p

Salvianolic acid B increased the number of BrdU-positive cells <i>in vivo</i>.

<p>(A) Representative photomicrographs showing BrdU-positive cells in the hippocampus of sham, ischemia and ischemia+Sal B (50 mg/kg)-treated rats. BrdU-labeled cells were indicated by arrows. Scale bar: 200 µm. (B) Quantification of BrdU-positive cells in the hippocampus. Each column represents the represent the mean ± S.D. (n = 10). **Significant difference from the sham group at <i>P</i><0.01.</p

The effect of Salvianolic acid B on BrdU-incorporation in cultured NSPCs.

<p>Dissociated NSPCs were cultured on poly-L-lysine-coated 12-well chamber slides with or without Sal B for 12 h and were subsequently incubated with BrdU (10 µg/ml) for other 12 h. After treatment, cells were fixed, immunostained with anti BrdU antibody. (A) Visualization of BrdU-positive cells by the Immunofluorescence staining assay on NSPCs. Scale bar: 100 µm. (B) BrdU-positive cells were counted in 10 randomly selected fields from three different chambers. Data represent the mean ± S.D. from three independent experiments. *Significant difference from the control group at <i>P</i><0.05. **Significant difference from the control group at <i>P</i><0.01.</p

Salvianolic acid B activated PI3K/Akt in NSPCs.

<p>Cell lysates from NSPCs treated or untreated with Sal B (20 µM) were subjected to Western blot analysis with antibodies against both total and phosphorylated forms of ERK1/2, and Akt. Actin was used as loading control. (A) Sal B increased the phosphorylation of Akt. Akt phosphorylation in Sal B-treated runners (15 min, 30 min, 60 min, 120 min) were significantly greater than control runners. Histograms represent the change in the phosphorylation of Akt normalized to anti-actin antibodies (n = 4). **Significant difference from the control group at <i>P</i><0.01. (B) PI3K/Akt inhibitors Ly294002 regulated the Sal B-mediated phosphorylation of Akt. Ly294002 abolished the phosphorylation of Akt induced by Sal B. Histograms represent the change in the phosphorylation of Akt normalized to anti-actin antibodies (n = 4). *<i>P</i><0.05 as compared with control, **<i>P</i><0.01 as compared with control, ##<i>P</i><0.01 as compared with Sal B-treated cells. No change in total Akt was observed.</p