Journal: Oncology Letters

Article Title: PAK6 promotes cervical cancer progression through activation of the Wnt/β-catenin signaling pathway

doi: 10.3892/ol.2020.11797

Figure Lengend Snippet: Primers for amplifying PAK6 and GSK3β.

Article Snippet: Following centrifugation (12,000 × g; 4°C; 5 min), the lysates were incubated with 2 μg anti-PAK6 rabbit polyclonal antibody (cat. no. 13539-1-AP; ProteinTech Group, Inc.) or negative control rabbit IgG (cat. no. A7016; Beyotime Institute of Biotechnology) at 4°C overnight and then rotated at 4°C with a mixture of protein A/G sepharose beads (20 μl/ml) for 4 h. The beads were then washed 3 times with RIPA buffer, and the bound proteins were boiled in 2X Laemmli buffer and further analyzed using western blotting.

Techniques: Sequencing

Journal: Oncology Letters

Article Title: PAK6 promotes cervical cancer progression through activation of the Wnt/β-catenin signaling pathway

doi: 10.3892/ol.2020.11797

Figure Lengend Snippet: Expression levels of PAK6 in cervical carcinoma and paracarcinoma tissues.

Article Snippet: Following centrifugation (12,000 × g; 4°C; 5 min), the lysates were incubated with 2 μg anti-PAK6 rabbit polyclonal antibody (cat. no. 13539-1-AP; ProteinTech Group, Inc.) or negative control rabbit IgG (cat. no. A7016; Beyotime Institute of Biotechnology) at 4°C overnight and then rotated at 4°C with a mixture of protein A/G sepharose beads (20 μl/ml) for 4 h. The beads were then washed 3 times with RIPA buffer, and the bound proteins were boiled in 2X Laemmli buffer and further analyzed using western blotting.

Techniques: Expressing

Journal: Oncology Letters

Article Title: PAK6 promotes cervical cancer progression through activation of the Wnt/β-catenin signaling pathway

doi: 10.3892/ol.2020.11797

Figure Lengend Snippet: Association between PAK6 expression levels and clinicopathological parameters in cervical cancer.

Article Snippet: Following centrifugation (12,000 × g; 4°C; 5 min), the lysates were incubated with 2 μg anti-PAK6 rabbit polyclonal antibody (cat. no. 13539-1-AP; ProteinTech Group, Inc.) or negative control rabbit IgG (cat. no. A7016; Beyotime Institute of Biotechnology) at 4°C overnight and then rotated at 4°C with a mixture of protein A/G sepharose beads (20 μl/ml) for 4 h. The beads were then washed 3 times with RIPA buffer, and the bound proteins were boiled in 2X Laemmli buffer and further analyzed using western blotting.

Techniques: Expressing

Journal: Oncology Letters

Article Title: PAK6 promotes cervical cancer progression through activation of the Wnt/β-catenin signaling pathway

doi: 10.3892/ol.2020.11797

Figure Lengend Snippet: PAK6 expression in cervical cancer tissues and in C33A and HeLa cells. (A) Immunohistochemistry was used to analyze the expression levels of PAK6 in cervical cancer tissues. Scale bars, 200 µm. (B) Expression levels of PAK6 in C33A and HeLa cells were analyzed using western blotting. (C) Semi-quantification of the PAK6 protein expression levels presented in part (B) using ImageJ software. **P<0.01 vs. HeLa cells. PAK6, p21-activated kinase 6.

Article Snippet: Following centrifugation (12,000 × g; 4°C; 5 min), the lysates were incubated with 2 μg anti-PAK6 rabbit polyclonal antibody (cat. no. 13539-1-AP; ProteinTech Group, Inc.) or negative control rabbit IgG (cat. no. A7016; Beyotime Institute of Biotechnology) at 4°C overnight and then rotated at 4°C with a mixture of protein A/G sepharose beads (20 μl/ml) for 4 h. The beads were then washed 3 times with RIPA buffer, and the bound proteins were boiled in 2X Laemmli buffer and further analyzed using western blotting.

Techniques: Expressing, Immunohistochemistry, Western Blot, Software

Journal: Oncology Letters

Article Title: PAK6 promotes cervical cancer progression through activation of the Wnt/β-catenin signaling pathway

doi: 10.3892/ol.2020.11797

Figure Lengend Snippet: Effect of the knockdown of PAK6 expression levels on the proliferation, migration and invasion of HeLa cells. (A) PAK6 mRNA expression levels were analyzed in stably shPAK6-transfected HeLa cells. (B) PAK6 protein expression levels were analyzed in stably shPAK6-transfected HeLa cells using western blotting. (C) Semi-quantification of PAK6 expression levels presented in part (B). (D) Cell Counting Kit-8 assays and (E) colony formation assays were used to analyze the proliferative rate of shPAK6-transfected HeLa cells. (F) Semi-quantification of the number of colonies formed from part (E). (G) Cell migration and invasion were determined in stably shPAK6-transfected HeLa cells, (magnification ×200). (H) Semi-quantification of the number of invasive cells from part (G). (I) Semi-quantification of the number of migratory cells from part (G). **P<0.01 vs. shPAK6 NC. PAK6, p21-activated kinase 6; sh, short hairpin RNA; NC, negative control.

Article Snippet: Following centrifugation (12,000 × g; 4°C; 5 min), the lysates were incubated with 2 μg anti-PAK6 rabbit polyclonal antibody (cat. no. 13539-1-AP; ProteinTech Group, Inc.) or negative control rabbit IgG (cat. no. A7016; Beyotime Institute of Biotechnology) at 4°C overnight and then rotated at 4°C with a mixture of protein A/G sepharose beads (20 μl/ml) for 4 h. The beads were then washed 3 times with RIPA buffer, and the bound proteins were boiled in 2X Laemmli buffer and further analyzed using western blotting.

Techniques: Knockdown, Expressing, Migration, Stable Transfection, Transfection, Western Blot, Cell Counting, shRNA, Negative Control

Journal: Oncology Letters

Article Title: PAK6 promotes cervical cancer progression through activation of the Wnt/β-catenin signaling pathway

doi: 10.3892/ol.2020.11797

Figure Lengend Snippet: Effects of the overexpression of PAK6 on the proliferation, migration and invasion of HeLa cells. (A) PAK6 mRNA expression levels in stable PAK6 overexpressing HeLa cells were analyzed. (B) PAK6 protein expression levels were analyzed in stable PAK6 overexpressing HeLa cells using western blotting. (C) Semi-quantification of PAK6 expression levels presented in part (B). (D) Cell Counting Kit-8 assays and (E) colony formation assays were used to analyze the proliferative rate of stable PAK6 overexpressing HeLa cells. (F) Semi-quantification of the number of colonies formed from part (E). (G) Cell migration and invasion were determined in stable overexpressing PAK6 HeLa cells, (magnification ×200). (H) Semi-quantification of the number of invasive cells from part (G). (I) Semi-quantification of the migratory cell number from part (G). *P<0.05, **P<0.01 vs. PAK6 NC. PAK6, p21-activated kinase 6; NC, negative control.

Article Snippet: Following centrifugation (12,000 × g; 4°C; 5 min), the lysates were incubated with 2 μg anti-PAK6 rabbit polyclonal antibody (cat. no. 13539-1-AP; ProteinTech Group, Inc.) or negative control rabbit IgG (cat. no. A7016; Beyotime Institute of Biotechnology) at 4°C overnight and then rotated at 4°C with a mixture of protein A/G sepharose beads (20 μl/ml) for 4 h. The beads were then washed 3 times with RIPA buffer, and the bound proteins were boiled in 2X Laemmli buffer and further analyzed using western blotting.

Techniques: Over Expression, Migration, Expressing, Western Blot, Cell Counting, Negative Control

Journal: Oncology Letters

Article Title: PAK6 promotes cervical cancer progression through activation of the Wnt/β-catenin signaling pathway

doi: 10.3892/ol.2020.11797

Figure Lengend Snippet: Effect of PAK6 knockdown or overexpression on the Wnt/β-catenin signaling pathway in HeLa cells. Western blotting was used to analyze the expression levels of (A) β-catenin, p-β-catenin, GSK3β and p-GSK3β, and (B) E-cadherin and Cyclin D1 in stably shPAK6-transfected HeLa cells. (C) Semi-quantification of the expression levels of proteins in parts (A) and (B). *P<0.05, **P<0.01 vs. shPAK6 NC. Western blotting was used to analyze the expression levels of (D) β-catenin, p-β-catenin, GSK3β and p-GSK3β, and (E) E-cadherin and cyclin D1 in stable PAK6 overexpressing HeLa cells. (F) Semi-quantification of the expression levels of proteins in parts (D) and (E). *P<0.05, **P<0.01 vs. PAK6 NC. (G) Immunofluorescence was used to demonstrate the co-localization of PAK6 and GSK3β. Scale bars, 10 µm. (H) Co-IP was used to analyze the interaction between PAK6 and GSK3β. PAK6, p21-activated kinase 6; sh, short hairpin RNA; NC, negative control; p-, phosphorylated; GSK3β, glycogen synthase kinase 3β; IP, immunoprecipitation.

Article Snippet: Following centrifugation (12,000 × g; 4°C; 5 min), the lysates were incubated with 2 μg anti-PAK6 rabbit polyclonal antibody (cat. no. 13539-1-AP; ProteinTech Group, Inc.) or negative control rabbit IgG (cat. no. A7016; Beyotime Institute of Biotechnology) at 4°C overnight and then rotated at 4°C with a mixture of protein A/G sepharose beads (20 μl/ml) for 4 h. The beads were then washed 3 times with RIPA buffer, and the bound proteins were boiled in 2X Laemmli buffer and further analyzed using western blotting.

Techniques: Knockdown, Over Expression, Western Blot, Expressing, Stable Transfection, Transfection, Immunofluorescence, Co-Immunoprecipitation Assay, shRNA, Negative Control, Immunoprecipitation

Journal: BMC Cancer

Article Title: Docosahexaenoic acid reduces sterol regulatory element binding protein-1 and fatty acid synthase expression and inhibits cell proliferation by inhibiting pAkt signaling in a human breast cancer MCF-7 cell line

doi: 10.1186/s12885-017-3936-7

Figure Lengend Snippet: Effect of DHA or E 2 on the pERK1/2/ERK1/2, pAkt/Akt, and pS6/S6 ratios and p-SREBP-1, m-SREBP-1, and FASN expression in MCF-7 cells. Cells were pretreated with BSA or 60 μM BSA-bound DHA for 48 h in DMEM containing 5% CD-FBS, then the same medium alone or 10 nM E 2 was added. Western blots were then used to measure the pERK1/2/ERK1/2, pAkt/Akt, and pS6/S6 ratios after 1 h and p-SREBP-1, m-SREBP-1, and FASN expression after 24 h ( a ). Total ERK1/2, Akt, or S6 was used as the loading control for pERK1/2 ( b ), pAkt ( c ), or pS6 ( d ), respectively, while GAPDH was used as the loading control for p-SREBP-1 ( e ), m-SREBP-1 ( f ), and FASN ( g ).The levels are expressed as a fold value compared to the BSA-treated control with no E 2 stimulation. Two-way ANOVA followed by the Bonferroni posttest was used to compare DHA and E 2 effects. The data are presented as the mean ± S.E.M for 5–6 independent experiments

Article Snippet: The membranes were immunoblotted overnight at 4 °C with primary antibodies diluted in TBST; the antibodies used were rabbit monoclonal antibody against pAkt (1:1000), S6 (1:1000), or GAPDH (1:2000), rabbit polyclonal antibodies against Akt (1:1000), pS6 (1:1000), pERK1/2 (1:1000), or ERK1/2 (1:1000) (all from Cell Signaling), rabbit polyclonal anti-FASN antibodies (1:1000), or mouse monoclonal anti-SREBP1 antibody (1:200) (both from Santa Cruz).

Techniques: Expressing, Western Blot, Control

Journal: BMC Cancer

Article Title: Docosahexaenoic acid reduces sterol regulatory element binding protein-1 and fatty acid synthase expression and inhibits cell proliferation by inhibiting pAkt signaling in a human breast cancer MCF-7 cell line

doi: 10.1186/s12885-017-3936-7

Figure Lengend Snippet: Effect of DHA or insulin on the pERK1/2/ERK1/2, pAkt/Akt, and pS6/S6 ratios and p-SREBP-1, m-SREBP-1, and FASN expression in MCF-7 cells. Cells were pretreated with BSA or 60 μM BSA-bound DHA for 48 h in DMEM containing 5% FBS, then the same medium alone or 1 μg/ml of insulin was added. Western blots were then used to measure the pERK1/2/ERK1/2, pAkt/Akt, and pS6/S6 ratios after 1 h and p-SREBP-1, m-SREBP-1, and FASN expression after 24 h ( a ). Total ERK1/2, Akt, or S6 was used as the loading control for pERK1/2 ( b ), pAkt ( c ), or pS6 ( d ), respectively, while GAPDH was used as the loading control for p-SREBP-1 ( e ), m-SREBP-1 ( f ), and FASN ( g ).The levels are expressed as a fold value compared to the BSA-treated control with no insulin stimulation. Two-way ANOVA followed by the Bonferroni posttest was used to compare DHA and insulin effects. The data are presented as the mean ± S.E.M for 5–6 independent experiments

Article Snippet: The membranes were immunoblotted overnight at 4 °C with primary antibodies diluted in TBST; the antibodies used were rabbit monoclonal antibody against pAkt (1:1000), S6 (1:1000), or GAPDH (1:2000), rabbit polyclonal antibodies against Akt (1:1000), pS6 (1:1000), pERK1/2 (1:1000), or ERK1/2 (1:1000) (all from Cell Signaling), rabbit polyclonal anti-FASN antibodies (1:1000), or mouse monoclonal anti-SREBP1 antibody (1:200) (both from Santa Cruz).

Techniques: Expressing, Western Blot, Control

Journal: BMC Cancer

Article Title: Docosahexaenoic acid reduces sterol regulatory element binding protein-1 and fatty acid synthase expression and inhibits cell proliferation by inhibiting pAkt signaling in a human breast cancer MCF-7 cell line

doi: 10.1186/s12885-017-3936-7

Figure Lengend Snippet: Effect of inhibitors and DHA on the E 2 -stimulated increase in the pAkt/Akt and pS6/S6 ratios and p-SREBP-1, m-SREBP-1, and FASN expression in MCF-7 cells. Cells were pretreated with BSA or 60 μM BSA-bound DHA for 48 h in DMEM containing 5% CD-FBS, then the same medium alone, 20 μM LY294002 (LY), or 0.5 nM rapamycin (Rap) was added for 1 h, followed by stimulation with 10 nM E 2 , then Western blot analysis was used to measure the pAkt/Akt and pS6/S6 ratios after 1 h of incubation and p-SREBP-1, m-SREBP-1, and FASN expression after 24 h ( a ). Total Akt or total S6 was used as the loading control for pAkt ( b ) or pS6 ( c ), respectively, while GAPDH was used as the loading control for p-SREBP-1 ( d ), m-SREBP-1 ( e ), and FASN ( f ).The levels are expressed as a fold value compared to control BSA-treated cells. Two-way ANOVA followed by the Bonferroni posttest was used to compare DHA and inhibitor effects. The data are presented as the mean ± S.E.M for 4–5 independent experiments

Article Snippet: The membranes were immunoblotted overnight at 4 °C with primary antibodies diluted in TBST; the antibodies used were rabbit monoclonal antibody against pAkt (1:1000), S6 (1:1000), or GAPDH (1:2000), rabbit polyclonal antibodies against Akt (1:1000), pS6 (1:1000), pERK1/2 (1:1000), or ERK1/2 (1:1000) (all from Cell Signaling), rabbit polyclonal anti-FASN antibodies (1:1000), or mouse monoclonal anti-SREBP1 antibody (1:200) (both from Santa Cruz).

Techniques: Expressing, Western Blot, Incubation, Control

Journal: BMC Cancer

Article Title: Docosahexaenoic acid reduces sterol regulatory element binding protein-1 and fatty acid synthase expression and inhibits cell proliferation by inhibiting pAkt signaling in a human breast cancer MCF-7 cell line

doi: 10.1186/s12885-017-3936-7

Figure Lengend Snippet: Effect of inhibitors and DHA on the insulin-stimulated increase in the pAkt/Akt and pS6/S6 ratios and p-SREBP-1, m-SREBP-1, and FASN expression in MCF-7 cells. Cells were pretreated with BSA or 60 μM BSA-bound DHA for 48 h in DMEM containing 5% FBS, then the same medium, 20 μM LY294002 (LY), or 0.5 nM rapamycin (Rap) was added for 1 h following by stimulation with 1 μg/ml of insulin, then Western blot analysis was used to measure the pAkt/Akt and pS6/S6 ratios after 1 h of incubation and p-SREBP-1, m-SREBP-1, and FASN expression after 24 h ( a ). Total Akt or S6 was used as the loading control for pAkt ( b ) or pS6 ( c ), respectively, while GAPDH was used as the loading control for p-SREBP-1 ( d ), m-SREBP-1 ( e ), and FASN ( f ).The levels are expressed as a fold value compared to control BSA-treated cells. Two-way ANOVA followed by the Bonferroni posttest was used to compare DHA and inhibitor effects. The data are presented as the mean ± S.E.M for 5 independent experiments

Article Snippet: The membranes were immunoblotted overnight at 4 °C with primary antibodies diluted in TBST; the antibodies used were rabbit monoclonal antibody against pAkt (1:1000), S6 (1:1000), or GAPDH (1:2000), rabbit polyclonal antibodies against Akt (1:1000), pS6 (1:1000), pERK1/2 (1:1000), or ERK1/2 (1:1000) (all from Cell Signaling), rabbit polyclonal anti-FASN antibodies (1:1000), or mouse monoclonal anti-SREBP1 antibody (1:200) (both from Santa Cruz).

Techniques: Expressing, Western Blot, Incubation, Control