Journal: Acta Biochimica et Biophysica Sinica

Article Title: A positive feedback loop of ARF6 activates ERK1/2 signaling pathway via DUSP6 silencing to promote pancreatic cancer progression

doi: 10.3724/abbs.2022111

Figure Lengend Snippet: Table 1 Sequences of primes used in this study

Article Snippet: Anti-ARF6 antibody (ab77581; Abcam) and anti-DUSP6 antibody (A3171; Abclonal) were used in a dilution of 1:1000.

Techniques:

Journal: Acta Biochimica et Biophysica Sinica

Article Title: A positive feedback loop of ARF6 activates ERK1/2 signaling pathway via DUSP6 silencing to promote pancreatic cancer progression

doi: 10.3724/abbs.2022111

Figure Lengend Snippet: RNA sequencing and real-time PCR are used to identify the altered gene expression of DUSP6 in ARF6-knockdown cells (A) RNA sequencing was carried out to explore the differential gene expression and shown as heat map. (B) The number of upregulated and downregulated genes are shown. (C) KEGG analysis was performed to identify the significant signaling pathway. (D) Both the upregulated and downregulated genes were analyzed for GO term enrichment. (E−H) Validation of part of the RNA sequencing results by quantitative real-time PCR. * P<0.05, ** P<0.01, *** P<0.001, and **** P<0.0001.

Article Snippet: Anti-ARF6 antibody (ab77581; Abcam) and anti-DUSP6 antibody (A3171; Abclonal) were used in a dilution of 1:1000.

Techniques: RNA Sequencing Assay, Real-time Polymerase Chain Reaction, Expressing

Journal: Acta Biochimica et Biophysica Sinica

Article Title: A positive feedback loop of ARF6 activates ERK1/2 signaling pathway via DUSP6 silencing to promote pancreatic cancer progression

doi: 10.3724/abbs.2022111

Figure Lengend Snippet: DUSP6 is negatively regulated by ARF6 (A) Knockdown of ARF6 in PANC-1 and SW1990 cells resulted in an increase of DUSP6 protein level. (B) Immunofluorescence microscopy analysis of DUSP6 and ARF6 expressions in PANC-1 and SW1990 pancreatic cancer cell lines. (C,D) Representative images showing proportion score and intensity score of ARF6 and DUSP6. (E) ARF6 is negatively correlated with DUSP6 expression in pancreatic cancer patients as demonstrated by immunohistochemical staining and scoring. (F) The correlation between ARF6 and DUSP6 expressions derived from clinical specimens. Patients with higher level of ARF6 displayed lower DUSP6 expression.

Article Snippet: Anti-ARF6 antibody (ab77581; Abcam) and anti-DUSP6 antibody (A3171; Abclonal) were used in a dilution of 1:1000.

Techniques: Immunofluorescence, Microscopy, Expressing, Immunohistochemical staining, Staining, Derivative Assay

Journal: Acta Biochimica et Biophysica Sinica

Article Title: A positive feedback loop of ARF6 activates ERK1/2 signaling pathway via DUSP6 silencing to promote pancreatic cancer progression

doi: 10.3724/abbs.2022111

Figure Lengend Snippet: DUSP6 silencing promotes cell growth and cell proliferation in human pancreatic cancer cells (A,B) The shRNAs against DUSP6 plasmid were transfected into PANC-1 and SW1990 cells to examine the decrease of DUSP6 mRNA and protein levels, respectively. (C) Western blot analysis of apoptotic proteins Caspase-3, BCL2, PARP and cleaved PARP. (D) Silencing DUSP6 expression, as indicated in (A,B), decreased the proliferation of PANC-1 and SW1990 cells, as determined by CCK-8 assay. (E,F) Decrease of DUSP6 expression increased the colony formation ability of PANC-1 and SW1990 cells. * P<0.05, ** P<0.01, *** P<0.001, and **** P<0.0001.

Article Snippet: Anti-ARF6 antibody (ab77581; Abcam) and anti-DUSP6 antibody (A3171; Abclonal) were used in a dilution of 1:1000.

Techniques: Plasmid Preparation, Transfection, Western Blot, Expressing, CCK-8 Assay

Journal: Acta Biochimica et Biophysica Sinica

Article Title: A positive feedback loop of ARF6 activates ERK1/2 signaling pathway via DUSP6 silencing to promote pancreatic cancer progression

doi: 10.3724/abbs.2022111

Figure Lengend Snippet: Upregulated expression of DUSP6 reduces cell growth and induces apoptosis of pancreatic cancer cells (A,B) qPCR and western blot analysis confirmed the overexpression (OE) efficiency of DUSP6 in PANC-1 and SW1990 cells. (C) Western blot analysis of apoptotic proteins PARP, Caspase-3 and BCL2. (D) Overexpression of DUSP6, as indicated in (A,B), increased the proliferation of PANC-1 and SW1990 cells, as determined by CCK-8 assay. (E,F) Overexpression of DUSP6 weakened the colony formation ability of PANC-1 and SW1990 cells. * P<0.05, ** P<0.01, and **** P<0.0001.

Article Snippet: Anti-ARF6 antibody (ab77581; Abcam) and anti-DUSP6 antibody (A3171; Abclonal) were used in a dilution of 1:1000.

Techniques: Expressing, Western Blot, Over Expression, CCK-8 Assay

Journal: Acta Biochimica et Biophysica Sinica

Article Title: A positive feedback loop of ARF6 activates ERK1/2 signaling pathway via DUSP6 silencing to promote pancreatic cancer progression

doi: 10.3724/abbs.2022111

Figure Lengend Snippet: ARF6 regulates ERK activation and pancreatic cancer cell proliferation via DUSP6 (A) Overexpression (OE) of DUSP6 suppressed ERK activation at protein levels. (B) The siRNA against DUSP6 was transfected into the PANC-1 and SW1990 cells to examine the decrease of DUSP6 protein level. (C,E) ERK and p-ERK expression levels were examined by western blot analysis in ARF6- and/or DUSP6-knockdown cells. (D,F) Knockdown of ARF6 and/or DUSP6 manifested different proliferation of PANC-1 and SW1990 cells, as determined by CCK-8 assay. * P<0.05, and ** P<0.01.

Article Snippet: Anti-ARF6 antibody (ab77581; Abcam) and anti-DUSP6 antibody (A3171; Abclonal) were used in a dilution of 1:1000.

Techniques: Activation Assay, Over Expression, Transfection, Expressing, Western Blot, CCK-8 Assay

Journal: Acta Biochimica et Biophysica Sinica

Article Title: A positive feedback loop of ARF6 activates ERK1/2 signaling pathway via DUSP6 silencing to promote pancreatic cancer progression

doi: 10.3724/abbs.2022111

Figure Lengend Snippet: Schematic representation of the model of the positive feedback loop of ARF6 KRAS activates ARF6 via ERK1/2, which in turn leads to the inhibition of DUSP6, which causes the attenuation of ERK1/2 inhibition in the positive feedback loop. DUSP6 acts both as a tumor suppressor in pancreatic cancer and as a vital intermediate molecule in the positive feedback loop.

Article Snippet: Anti-ARF6 antibody (ab77581; Abcam) and anti-DUSP6 antibody (A3171; Abclonal) were used in a dilution of 1:1000.

Techniques: Inhibition