Journal: The Journal of Biological Chemistry

Article Title: Sea Anemone Peptide with Uncommon β-Hairpin Structure Inhibits Acid-sensing Ion Channel 3 (ASIC3) and Reveals Analgesic Activity *

doi: 10.1074/jbc.M113.485516

Figure Lengend Snippet: Isolation of the active peptide from the venom of U. grebelnyi. Fractions marked with a black box exercised an inhibitory activity on the ASIC3 channel in all separation steps. A, the first separation stage of the crude peptide fraction by size exclusion chromatography on a TSK-2000SW (7.5 × 600 mm) column in 10% acetonitrile solution with 0.1% trifluoroacetic acid and a flow rate of 0.5 ml/min. B, second separation step on a reverse-phase column Jupiter C5 column (4.6 × 150 mm) in 0.1% trifluoroacetic acid with a flow rate of 1 ml/min using a linear gradient of acetonitrile concentration. C, final separation step on a Vydac C18 column (250 × 4.6 mm) using a linear gradient of acetonitrile concentration in 0.1% trifluoroacetic acid with a flow rate 1 ml/min.

Article Snippet: Electrophysiology Xenopus laevis oocytes were removed surgically, defolliculated, and injected with 2.5 to 10 ng of human ASIC3 cRNA ( {"type":"entrez-nucleotide","attrs":{"text":"AF057711.1","term_id":"3702835","term_text":"AF057711.1"}} AF057711.1 ). cRNA transcripts were synthesized from NaeI-linearized ASIC3 cDNA template (pcDNA3.1 + human ASIC3 subcloned from clone EX-Q0260-B02 (GeneCopoeia, Inc.)), using a RiboMAX TM large-scale RNA production system T7 (Promega) according to the manufacturer's protocol for capped transcripts.

Techniques: Isolation, Activity Assay, Size-exclusion Chromatography, Concentration Assay

Journal: The Journal of Biological Chemistry

Article Title: Sea Anemone Peptide with Uncommon β-Hairpin Structure Inhibits Acid-sensing Ion Channel 3 (ASIC3) and Reveals Analgesic Activity *

doi: 10.1074/jbc.M113.485516

Figure Lengend Snippet: Electrophysiological characterization of Ugr 9-1 action on human ASIC3 expressed in the X. laevis oocyte. Acid-induced currents were evoked by pH drop; the time intervals between the recordings were about 1 to 2 min, and the holding potential was −50 mV. A, effect on both current components by application of Ugr 9-1 at a concentration of 40 μm; B, effect on the transient current by application of Ugr 9-1 at a concentration of 20 μm; C, effect on the sustained current by application of Ugr 9-1 at a concentration of 10 μm; D and E, dose-response curves for the Ugr 9-1 inhibitory activity separately calculated for the transient (D) and sustained (E) components. Each point is the mean ± S.D. of 3–7 measurements. Data were fitted by the logistic equation.

Article Snippet: Electrophysiology Xenopus laevis oocytes were removed surgically, defolliculated, and injected with 2.5 to 10 ng of human ASIC3 cRNA ( {"type":"entrez-nucleotide","attrs":{"text":"AF057711.1","term_id":"3702835","term_text":"AF057711.1"}} AF057711.1 ). cRNA transcripts were synthesized from NaeI-linearized ASIC3 cDNA template (pcDNA3.1 + human ASIC3 subcloned from clone EX-Q0260-B02 (GeneCopoeia, Inc.)), using a RiboMAX TM large-scale RNA production system T7 (Promega) according to the manufacturer's protocol for capped transcripts.

Techniques: Concentration Assay, Activity Assay

Journal: Cell Death & Disease

Article Title: ASIC1 and ASIC3 contribute to acidity-induced EMT of pancreatic cancer through activating Ca 2+ /RhoA pathway

doi: 10.1038/cddis.2017.189

Figure Lengend Snippet: ASIC1 and ASIC3 are functionally expressed in pancreatic cancer. ( a and b ) The mRNA and protein expression of ASIC1 and ASIC3 in four pancreatic cancer cell lines (PANC-1, SW1990, BxPC-3 and AsPC-1) and normal pancreatic ductal cells (HPDE) were measured by RT-PCR, qRT-PCR and western blot. Values were normalized against HPDE from three experiments performed in triplicate and were presented as means±S.D. (* P <0.05; ** P <0.01). ( c ) Representative immunofluorescence of ASIC1 and ASIC3 in PANC-1 and BxPC-3 cells. ( d ) Whole-cell current of PANC-1 and BxPC-3 incubated with acidic (pH 6.4) extracellular solution was recorded by whole-cell voltage-clamp recording. After cells were treated with amiloride, whole-cell current was recorded again. ( e ) Representative immunofluorescence of ASIC1 and ASIC3 in pancreatic cancer (PC) tissues and its paired adjacent noncancerous pancreatic (NP) tissues from patients

Article Snippet: SiRNA duplexes targeting the human ASIC1 or ASIC3, and negative control were synthesized and purified by RiboBio (Ribobio Co., Guangzhou, China).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Immunofluorescence, Incubation

Journal: Cell Death & Disease

Article Title: ASIC1 and ASIC3 contribute to acidity-induced EMT of pancreatic cancer through activating Ca 2+ /RhoA pathway

doi: 10.1038/cddis.2017.189

Figure Lengend Snippet: Inhibition of ASIC1 and ASIC3 suppresses acidity-promoted invasion and migration of pancreatic cancer cells. PANC-1 and BxPC-3 were transfected with negative control (NC) or siRNA of ASIC1 and ASIC3 (siASIC1 and siASIC3) and cultured in pH 7.4 or pH 6.4 medium as indicated for 48 h. ( a ) The invasive ability was evaluated by Transwell assay. The histogram showed the percentage of invasion cells per field when compared with negative control cultured in pH 6.4 medium. ( b ) The migration ability was measured by wound-healing assay. ( c ) The cell viability was measured by MTT assay. PANC-1 and BxPC-3 were treated with amiloride and DMSO, and cultured in pH 7.4 or pH 6.4 medium as indicated for 48 h. ( d ) The invasive ability was evaluated by Transwell assay. The histogram showed the percentage of invasion cells per field when compared with negative control cultured in pH 6.4 medium. ( e ) The migration ability was measured by wound-healing assay. Experiments were performed three times in triplicate and were presented as means±S.D. (* P <0.05; ** P <0.01)

Article Snippet: SiRNA duplexes targeting the human ASIC1 or ASIC3, and negative control were synthesized and purified by RiboBio (Ribobio Co., Guangzhou, China).

Techniques: Inhibition, Migration, Transfection, Negative Control, Cell Culture, Transwell Assay, Wound Healing Assay, MTT Assay

Journal: Cell Death & Disease

Article Title: ASIC1 and ASIC3 contribute to acidity-induced EMT of pancreatic cancer through activating Ca 2+ /RhoA pathway

doi: 10.1038/cddis.2017.189

Figure Lengend Snippet: Knockdown of ASIC1 and ASIC3 suppresses acidity-induced EMT of pancreatic cancer cells. ( a and b ) PANC-1 and BxPC-3 were transfected with negative control (NC) or siRNA of ASIC1 and ASIC3 (siASIC1 and siASIC3) and cultured in pH 7.4 or pH 6.4 medium as indicated for 48 h. The mRNA and protein expression of N-cadherin, E-cadherin, Vimentin, ZEB1, Snail were measured by qRT-PCR and western blot. ( c and d ) PANC-1 and BxPC-3 were treated with amiloride and DMSO, and cultured in pH 7.4 or pH 6.4 medium as indicated for 48 h. The mRNA and protein expression of N-cadherin, E-cadherin, Vimentin, ZEB1, Snail were measured by qRT-PCR and western blot. Values were normalized against negative control in pH 6.4 medium. Experiments were performed three times in triplicate and are presented as means±S.D. (* P <0.05; ** P <0.01; *** P <0.001)

Article Snippet: SiRNA duplexes targeting the human ASIC1 or ASIC3, and negative control were synthesized and purified by RiboBio (Ribobio Co., Guangzhou, China).

Techniques: Knockdown, Transfection, Negative Control, Cell Culture, Expressing, Quantitative RT-PCR, Western Blot

Journal: Cell Death & Disease

Article Title: ASIC1 and ASIC3 contribute to acidity-induced EMT of pancreatic cancer through activating Ca 2+ /RhoA pathway

doi: 10.1038/cddis.2017.189

Figure Lengend Snippet: The acidity-induced elevation of [Ca 2+ ]i is suppressed by inhibition of ASIC1 and ASIC3. ( a ) Calcium imaging detected dynamic change in [Ca 2+ ]i when PANC-1 and BxPC-3 cells were treated with different acidic pH medium at 90 s. Pictures showed [Ca 2+ ]i indicated by Fura-2/AM at indicated time. ( b and c ) After inhibition of ASIC1 and ASIC3 by siRNA or amiloride, calcium imaging detected dynamic change in [Ca 2+ ]i of PANC-1 and BxPC-3 cells in acidic medium (pH 6.4). Pictures showed intracellular Ca 2+ concentration indicated by Fura-2/AM at indicated time. Experiments were performed three times

Article Snippet: SiRNA duplexes targeting the human ASIC1 or ASIC3, and negative control were synthesized and purified by RiboBio (Ribobio Co., Guangzhou, China).

Techniques: Inhibition, Imaging, Concentration Assay

Journal: Cell Death & Disease

Article Title: ASIC1 and ASIC3 contribute to acidity-induced EMT of pancreatic cancer through activating Ca 2+ /RhoA pathway

doi: 10.1038/cddis.2017.189

Figure Lengend Snippet: ASIC1/ASIC3-[Ca 2+ ]i signaling pathway upregulates RhoA activity of pancreatic cancer cells in acidity condition. ( a ) RhoA activity (GTP-bound RhoA) was detected in PANC-1 and BxPC-3 treated in pH 6.4 or pH 7.4 medium as indicated for 48 h. ( b ) After inhibition of ASIC1 and ASIC3 by siRNA or amiloride, the RhoA activity was detected in PANC-1 and BxPC-3 cells, which were incubated acidity medium (pH 6.4) for 48 h. ( c ) With pretreatment of BAPTA-AM or DMSO for 1 h, RhoA activity was detected in PANC-1 and BxPC-3 cells after cells were incubated with acidic medium (pH 6.4) for 48 h. PANC-1 and BxPC-3 were transfected with negative control (NC) or siRNA of RhoA (siRhoA) and cultured in pH 7.4 or pH 6.4 medium as indicated for 48 h. ( d ) The invasive ability was evaluated by Transwell assay and the histogram showed the percentage of invasion cells per field when compared with negative control cultured in pH 6.4 medium. ( e ) The migration ability was measured by wound-healing assay. ( f ) The representative pictures of cellular morphology. ( g ) The mRNA of N-cadherin, E-cadherin, Vimentin, ZEB1, Snail was measured by qRT-PCR. ( h ) The protein of N-cadherin, E-cadherin, Vimentin, ZEB1, Snail was measured by western blot. Values were normalized against negative control in pH 6.4 medium. Experiments were performed three times in triplicate and are presented as means±S.D. (* P <0.05; ** P <0.01; *** P <0.001)

Article Snippet: SiRNA duplexes targeting the human ASIC1 or ASIC3, and negative control were synthesized and purified by RiboBio (Ribobio Co., Guangzhou, China).

Techniques: Activity Assay, Inhibition, Incubation, Transfection, Negative Control, Cell Culture, Transwell Assay, Migration, Wound Healing Assay, Quantitative RT-PCR, Western Blot

Journal: Cell Death & Disease

Article Title: ASIC1 and ASIC3 contribute to acidity-induced EMT of pancreatic cancer through activating Ca 2+ /RhoA pathway

doi: 10.1038/cddis.2017.189

Figure Lengend Snippet: Knockdown of ASIC1 and ASIC3 inhibits metastasis of pancreatic cancer cells in vivo . ( a ) Photographs of nude mice and tumor showed subcutaneous xenograft of BxPC-3, which was transfected with lentivirus containing sequence of ASIC1 siRNA, ASIC3 siRNA or negative control (Lv-siASIC1, Lv-siASIC3 and Lv-NC). H&E stain confirmed tumor formation. Graph represented tumor volumes at the indicated days during the experiment and tumor weight after mice were killed. ( b ) Pictures of liver and H&E stain showed liver metastases of subcutaneous xenograft. The histogram showed average number of live metastases in each group. ( c ) H&E stain showed lung metastases of subcutaneous xenograft. The histogram showed average number of lung metastases in each mouse. The data represent the mean±S.D. (* P <0.05)

Article Snippet: SiRNA duplexes targeting the human ASIC1 or ASIC3, and negative control were synthesized and purified by RiboBio (Ribobio Co., Guangzhou, China).

Techniques: Knockdown, In Vivo, Transfection, Sequencing, Negative Control, Staining

Journal: Cell Death & Disease

Article Title: ASIC1 and ASIC3 contribute to acidity-induced EMT of pancreatic cancer through activating Ca 2+ /RhoA pathway

doi: 10.1038/cddis.2017.189

Figure Lengend Snippet: ASIC1 and ASIC3 are overexpressed and correlated to EMT marker in pancreatic cancer. ( a ) The mRNA expression of ASIC1 and ASIC3 in pancreatic cancer (PC) tissues when compared with matched adjacent noncancerous pancreatic (NP) tissues. Values were normalized against negative control in pH 6.4. Experiments were performed three times in triplicate and are presented as means±S.D. (* P <0.05). ( b ) Representative picture of immunohistochemical analysis showed the expression of ASIC1 and ASIC3 in pancreatic cancer tissues and matched adjacent noncancerous pancreatic tissues. ( c ) The representative immunofluorescence of ASIC1 (red), ASIC3 (red), E-cadherin (green), vimentin (yellow) and DAPI (blue) in pancreatic cancer tissues

Article Snippet: SiRNA duplexes targeting the human ASIC1 or ASIC3, and negative control were synthesized and purified by RiboBio (Ribobio Co., Guangzhou, China).

Techniques: Marker, Expressing, Negative Control, Immunohistochemical staining, Immunofluorescence