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1.
Figure 6

Figure 6. The effect of SPHK1 on Akt/FOXO3a/Bim pathway can be inhibited by PI3K inhibitor or SPHK1 inhibitor.. From: Sphingosine Kinase 1 Regulates the Akt/FOXO3a/Bim Pathway and Contributes to Apoptosis Resistance in Glioma Cells.

U87MG-SPHK1 and LN-382-SPHK1 cells were incubated with 50 µM LY294002 (A) or 5 µM SK-I (B) for 24 h and cell lysates were harvested and subjected to WB. phospho-Akt (p-Akt), total Akt, p-FOXO3a, total FOXO3a and Bim were detected with specific antibodies, respectively.

Hongyu Guan, et al. PLoS One. 2011;6(5):e19946.
2.
Figure 5

Figure 5. FOXO3a phosphorylation, transcriptional activity and Akt phosphorylation are regulated by SPHK1 in glioma cells.. From: Sphingosine Kinase 1 Regulates the Akt/FOXO3a/Bim Pathway and Contributes to Apoptosis Resistance in Glioma Cells.

(A) SPHK1 phosphorylates FOXO3a in glioma cells. (B) FOXO3a-dependent transcription activity is regulated by overexpression (left panel) or knockdown (right panel) of SPHK1. Error bars represent mean ± SD from three independent experiments with similar results. *, p<0.05. The GFP expression was used to indicate the transfection efficiency. (C) WB analysis of nuclear FOXO3a protein in indicated cells. (D) SPHK1 knockdown-induced upregulation of Bim could be reversed by silencing of FOXO3a in indicated glioma cells. (E) The phosphorylation status of Akt at Thr308 and Ser473 were assessed by WB in SPHK1 overexpressed and knocked-down glioma cell lines.

Hongyu Guan, et al. PLoS One. 2011;6(5):e19946.
3.
Figure 4

Figure 4. The expression of Bim is correlated with SPHK1 in vitro and in vivo.. From: Sphingosine Kinase 1 Regulates the Akt/FOXO3a/Bim Pathway and Contributes to Apoptosis Resistance in Glioma Cells.

(A) The expression of BimEL protein level was significantly decreased in SPHK1 overexpression cells (left panel) and increased in SPHK1 knocked-down cells (right panel). (B) Overexpression of SPHK1 significantly decreased mRNA level of Bim (left panel), while knockdown of SPHK1 increased transcriptional level of Bim (right panel). (C) Examples of Bim expression in correlation with SPHK1 in human primary glioma specimens. Left panel, IHC staining of SPHK1 and Bim in glioma. Right panel, correlation of Bim and SPHK1 (n = 82; p = 0.021). Each data point represents one glioma specimen, and 24 of 33 (72.7%) glioma specimens with high SPHK1 expression displayed low expression of Bim, whereas 34 of 49 (69.4%) glioma specimens that showed low SPHK1 expression exhibited high expression of Bim. 0, no staining; 1, low staining; 2, moderate staining; 3, high staining.

Hongyu Guan, et al. PLoS One. 2011;6(5):e19946.
4.
Figure 3

Figure 3. The effects of dysregulation of SPHK1 expression on epirubicin induced-apoptosis in vivo.. From: Sphingosine Kinase 1 Regulates the Akt/FOXO3a/Bim Pathway and Contributes to Apoptosis Resistance in Glioma Cells.

(A) Tumor volumes were measured on the indicated days. Left panel, indicated cells (2×106) were injected in the flank of nude mice. When the mean tumor volume reached approximately 50 mm3, the mice were injected i.p. with epirubicin (5 mg/kg), every 3 days, up to 15 days. Right panel, U87MG-vector cells (2×106) were injected in the flank of nude mice. When the mean tumor volume reached approximately 50 mm3, the mice were randomized into two groups (n = 5) and injected intraperitoneally either with epirubicin (Epi, 5 mg/kg) or epirubicin (Epi, 5 mg/kg) plus SK1-I (50 mg/kg) every 3 days, up to 15days. (B) Mean tumor weights were measured. Immunofluorescent images (C) and quantification (D) of TUNEL positive cells. For (B) and (D), error bars represent mean ± SD from three independent experiments with similar results *, p<0.05.

Hongyu Guan, et al. PLoS One. 2011;6(5):e19946.
5.
Figure 2

Figure 2. Downregulation of SPHK1 expression decreases the resistance of glioma cells to apoptosis.. From: Sphingosine Kinase 1 Regulates the Akt/FOXO3a/Bim Pathway and Contributes to Apoptosis Resistance in Glioma Cells.

(A) SPHK1 knockdown was achieved by introducing specific shRNAs in glioma cells. α-tubulin was used as a loading control. (B) Silencing of SPHK1 led to significant decrease of enzymatic activity in glioma cells. (C) Knockdown of SPHK1 enhances cell death. (D) SPHK1 knockdown enhanced the sensitivity of glioma cells to adriamycin-induced apoptosis. Quantification of TUNEL positive cells (left panel) and Annexin V+/PI cells (right panel). Results are expressed as percentages of total cells. (E) PARP cleavage and cleaved caspase 3 levels were assessed in indicated cell lines treated with UV irradiation (40 J/m2) or adriamycin (1.0 µM) for 24 h via WB. α-tubulin was used as a loading control. For (B), (C) and (D), error bars represent mean ± SD from three independent experiments with similar results. *, p<0.05.

Hongyu Guan, et al. PLoS One. 2011;6(5):e19946.
6.
Figure 1

Figure 1. Glioma cells overexpressing SPHK1 protein are less sensitive to UV- or adriamycin-induced apoptosis.. From: Sphingosine Kinase 1 Regulates the Akt/FOXO3a/Bim Pathway and Contributes to Apoptosis Resistance in Glioma Cells.

(A) Overexpression of SPHK1 in glioma cell lines analyzed by WB. α-tubulin was used as a loading control. (B) SPHK1 enzymatic activity in SPHK1-overexpressed glioma cells was markedly increased. (C) Overexpression of SPHK1 inhibited cell death induced by adriamycin (left panel) or UV irradiation (right panel). Seventy-two hours after treatment with adriamycin or UV irradiation, cell viability was assessed by the trypan blue exclusion method. (D) SPHK1 prevented adriamycin-induced apoptosis of glioma cells. Annexin-V binding and TUNEL assays of indicated cells were performed after incubation with adriamycin for 6 h and 24 h, respectively. Quantification of TUNEL positive cells (left panel) and Annexin V+/PI cells (right panel). Results are expressed as percentages of total cells. (E) PARP cleavage and cleaved caspase 3 levels were assessed in indicated cells treated with UV irradiation (40 J/m2) or adriamycin (1.0 µM) for 24 h via WB. α-tubulin was used as a loading control. For (B), (C) and (D), error bars represent mean ± SD from three independent experiments with similar results. *, p<0.05.

Hongyu Guan, et al. PLoS One. 2011;6(5):e19946.

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