Journal: Science signaling

Article Title: Translocation of TRPV4-PI3Kγ complexes to the plasma membrane drives myofibroblast transdifferentiation

doi: 10.1126/scisignal.aau1533

Figure Lengend Snippet: (A) Representative Western blot showing TRPV4, phosphorylated AKT [p-AKT (Ser473)], total AKT (t-AKT), and GAPDH (loading control) in lysates of human lung fibroblast (HLF) treated with TRPV4 or scrambled siRNA ±TGF-β as indicated. (B) Quantification of p-AKT (Ser473) relative to total AKT as in (A) by densitometry. *P<0.05 difference between untreated and +TGF-β conditions. Band densities were measured with respect to the untreated scrambled siRNA condition. (C) Representative Western blot showing p-AKT (Ser473) and total AKT (t-AKT) in lysates from HLFs treated with the TRPV4 antagonist RN-1734 ± TGF-β as indicated. (D) Quantification of p-AKT (Ser473) relative to total AKT as in (C). **P<0.01 difference between untreated and +TGF-β conditions. Band densities were measured with respect to the untreated condition. (E) Lipid kinase activity of PI3Kγ and PI3Kα immunoprecipitated from plasma membrane fractions of HLFs treated with the TRPV4 antagonist RN-1734 ±TGF-β as indicated. PI3Kγ and PI3Kα were assayed for their capacity to generate PIP by thin layer chromatography. The line between the untreated and +TGF-β conditions indicates non-contiguous lanes from the same blot. (F) Quantification of PIP densities as in (E). *P<0.05 as indicated. PIP densities were measured with respect to the untreated condition for each PI3K isoform. All graphs show means ± SEM from N = 3 independent experiments (*P≤0.05, **P≤0.01, using ANOVA followed by Student-Newman-Keuls multiple comparisons test).

Article Snippet: Lentiviral Constructs Lentiviral constructs for HA-tagged WT PI3Kγ (full length), HA-N-terminal PI3Kγ (N-term, contains only AA 1-142), HA-N-terminal deleted PI3Kγ (N-del, missing the N-terminal (ΔAA 1-142) and the ATP binding site (ΔAA 948-981)), and GFP-tagged full length TRPV4 were produced by Cyagen Biosciences.

Techniques: Western Blot, Activity Assay, Immunoprecipitation, Thin Layer Chromatography

Journal: Science signaling

Article Title: Translocation of TRPV4-PI3Kγ complexes to the plasma membrane drives myofibroblast transdifferentiation

doi: 10.1126/scisignal.aau1533

Figure Lengend Snippet: (A) Representative fluorescence images showing α-SMA (green) and F-actin (red) in TRPV4 KO and PI3Kγ KO mouse lung fibroblasts (MLFs) treated with empty vector (control) lentivirus (LV), TRPV4 LV, or PI3Kγ LV, then stimulated with TGF-β. Non-transfected WT MLF were used as a positive control. Scale bar, 100 μm. (B) Quantification of cells with α-SMA–positive stress fibers in (A). Data are presented as % myofibroblasts (means ± SEM). N = 3 independent experiments with at least 30 cells per condition. *P<0.05 and ***P<0.005 as indicated. (C) Representative Western blot showing TRPV4, PI3Kγ, and α-SMA in TRPV4 KO MLFs transfected with PI3Kγ LV or TRPV4 LV then stimulated with TGF-β. (D) Quantification α-SMA:GAPDH band density ratios (means ± SEM) in (C). N=3 independent experiments. **P<0.01 difference between untreated and +TGF-β conditions with TRPV4 LV. (E) Representative Western blot showing TRPV4, PI3Kγ, and α-SMA in PI3Kγ KO MLFs transfected with PI3Kγ LV or TRPV4 LV then stimulated with TGF-β. GAPDH is a loading control. (F) Quantification α-SMA:GAPDH band density ratios (means ± SEM) in (E). N=3 independent experiments. *P<0.05 difference between untreated and +TGF-β conditions with PI3Kγ LV. All graphs show means ± SEM from N = 3 independent experiments (*P≤0.05, **P≤0.01, ***P≤0.005, using ANOVA followed by Student-Newman-Keuls multiple comparisons test).

Article Snippet: Lentiviral Constructs Lentiviral constructs for HA-tagged WT PI3Kγ (full length), HA-N-terminal PI3Kγ (N-term, contains only AA 1-142), HA-N-terminal deleted PI3Kγ (N-del, missing the N-terminal (ΔAA 1-142) and the ATP binding site (ΔAA 948-981)), and GFP-tagged full length TRPV4 were produced by Cyagen Biosciences.

Techniques: Fluorescence, Plasmid Preparation, Transfection, Positive Control, Western Blot

Journal: Science signaling

Article Title: Translocation of TRPV4-PI3Kγ complexes to the plasma membrane drives myofibroblast transdifferentiation

doi: 10.1126/scisignal.aau1533

Figure Lengend Snippet: (A) Representative immunoblots showing collagen-1 in WT, TRPV4 KO, and PI3Kγ KO mouse lung fibroblasts (MLFs) treated ±TGF-β. Black lines indicate non-contiguous lanes from the same blot. GAPDH is a loading control. (B) Quantification of collagen-1:GAPDH band density ratios (means ± SEM) from (A). N=3 independent experiments. *P<0.05 compared with untreated WT MLF, TRPV4 KO MLF ±TGF-β, or PI3Kγ KO MLF ±TGF-β. (C) Representative traction force intensity images and corresponding phase images with the region of interest outlined in red for WT, TRPV4 KO, and PI3Kγ KO MLFs treated ±TGF-β. Red and orange, areas of high contraction; blue, areas of low contraction. Scale bar, 50 μm. (D) Quantification of root mean square (RMS) traction force (means ± SEM) results from (C). N=3 independent experiments with at least 30 cells per condition. ***P<0.005 compared with untreated WT MLF, TRPV4 KO MLF ±TGF-β, or PI3Kγ KO MLF ±TGF-β. (E) Quantification of WT and PI3Kγ KO MLFs that differentiated into myofibroblasts when plated on polyacrylamide gels of varying stiffness and treated with TGF-β, as measured by α-smooth muscle actin (α-SMA) in stress fibers. Data shown as % of WT MLF on glass. N=3 independent experiments with at least 30 cells per condition *P<0.05 between WT and PI3Kγ KO MLF at 8 kPa, ***P<0.005 between WT and PI3Kγ KO MLF at 25kPa and glass. (F) Quantification of WT and PI3Kγ KO MLFs plated on polyacrylamide gels of varying stiffness ad treated with TGF-β that showed a TRPV4 plasma membrane:cytoplasm ratio >1 as measured by TRPV4 immunofluorescence. Data shown as % of WT MLF on glass. N=3 independent experiments with at least 30 cells per condition. ***P<0.005 between WT and PI3Kγ KO MLF at 8 kPa, 25 kPa, and glass. All graphs show means ± SEM from N = 3 independent experiments (*P≤0.05, ***P≤0.005, using ANOVA followed by Student-Newman-Keuls multiple comparisons test).

Article Snippet: Lentiviral Constructs Lentiviral constructs for HA-tagged WT PI3Kγ (full length), HA-N-terminal PI3Kγ (N-term, contains only AA 1-142), HA-N-terminal deleted PI3Kγ (N-del, missing the N-terminal (ΔAA 1-142) and the ATP binding site (ΔAA 948-981)), and GFP-tagged full length TRPV4 were produced by Cyagen Biosciences.

Techniques: Western Blot, Immunofluorescence

Journal: Science signaling

Article Title: Translocation of TRPV4-PI3Kγ complexes to the plasma membrane drives myofibroblast transdifferentiation

doi: 10.1126/scisignal.aau1533

Figure Lengend Snippet: (A) Domain structure of WT and mutant PI3Kγ constructs consisting of only the non-catalytic N-terminal domain (N-term) or lacking both the N-terminal domain and the ATP binding site in the catalytic domain (N-del). (B) Immunoblotting of His-tagged N-del or N-term forms of PI3Kγ coupled to Ni-NTA beads incubated with lysates of human lung fibroblasts (HLFs). Blots were probed with an antibody recognizing TRPV4, then stripped and reprobed for PI3Kγ. N=3 independent experiments. (C) Direct interaction between immobilized purified 6-His-TRPV4 and purified 6-His-N-term-PI3Kγ was measured by surface plasmon resonance. N=3 independent experiments. (D) Representative fluorescence images showing TRPV4 in PI3Kγ KO murine lung fibroblasts (MLFs) transfected with N-del or N-term PI3Kγ lentivirus (LV) and treated with TGF-β and the corresponding plot profiles of TRPV4 immunofluorescence. White arrows indicate TRPV4 at the plasma membrane; orange lines indicate regions where plot profiles were obtained; white boxes indicate higher magnification insets. Scale bar, 50 μm. (E) Quantification of results from (D). N = 3 independent experiments with at least 30 cells per condition. ***P<0.005 compared with PI3Kγ KO MLF + N-del PI3Kγ LV by Student’s t-test. (F) Western blotting of plasma membrane fractions of PI3Kγ KO MLFs transfected with lentivirus encoding WT PI3Kγ, N-term PI3Kγ, or N-del PI3Kγ, then treated with TGF-β as indicated. Blots were probed for TRPV4, PI3Kγ, and flotillin-1 (loading control). N=3 independent experiments. (G) Representative fluorescence images showing α-SMA in PI3Kγ KO MLFs transfected with control LV (empty vector), WT PI3Kγ LV, N-term PI3Kγ LV or N-del PI3Kγ LV, then stimulated with TGF-β. White boxes indicate areas enlarged in insets. Scale bar, 100 μm. (H) Quantification of results from (G). N = 3 independent experiments with at least 30 cells per condition. ***P<0.005 untreated vs +TGF-β conditions by ANOVA followed by Student-Newman-Keuls multiple comparisons test. All graphs show means ± SEM from N = 3 independent experiments.

Article Snippet: Lentiviral Constructs Lentiviral constructs for HA-tagged WT PI3Kγ (full length), HA-N-terminal PI3Kγ (N-term, contains only AA 1-142), HA-N-terminal deleted PI3Kγ (N-del, missing the N-terminal (ΔAA 1-142) and the ATP binding site (ΔAA 948-981)), and GFP-tagged full length TRPV4 were produced by Cyagen Biosciences.

Techniques: Mutagenesis, Construct, Binding Assay, Western Blot, Incubation, Purification, SPR Assay, Fluorescence, Transfection, Immunofluorescence, Plasmid Preparation

Journal: Science signaling

Article Title: Translocation of TRPV4-PI3Kγ complexes to the plasma membrane drives myofibroblast transdifferentiation

doi: 10.1126/scisignal.aau1533

Figure Lengend Snippet: (A) Representative confocal images showing TRPV4 in wild-type (WT) and PI3Kγ knockout (KO) murine lung fibroblasts (MLFs) ±TGF-β as indicated and the corresponding plot profiles of the TRPV4 immunofluorescence. Orange lines indicate regions where plot profiles were obtained. White boxes indicate areas shown in higher magnification in insets. (B) Quantification of plasma membrane:cytoplasm fluorescence for experiments in (A). N = 3 independent experiments with at least 30 cells per condition. ***P<0.005 compared with untreated WT MLF or with PI3Kγ KO MLF ± TGF-β. (C) Representative confocal images showing PI3Kγ in WT and TRPV4 KO MLFs ±TGF-β and the corresponding plot profiles of the PI3Kγ immunofluorescence. (D) Quantification of results in (C). N = 3 independent experiments with at least 30 cells per condition. ***P<0.005 compared with untreated WT MLF or with TRPV4 KO MLF ±TGF-β. (E) Representative immunoblots for TRPV4 and β1 integrin (loading control) from a surface biotinylation assay in WT and PI3Kγ KO MLF ±TGF-β. (F) Quantification of results from (E). N=3 independent experiments. **P<0.01 compared with untreated WT MLF or with PI3Kγ KO MLF ± TGF-β. (G) Representative immunoblots for PI3Kγ and flotillin-1 (loading control) from the plasma membrane fractions of WT and TRPV4 KO MLF ± TGF-β. The line between WT and TRPV4 KO MLF conditions indicates non-contiguous lanes from the same blot. (H) Quantification of results from (G). N=3 independent experiments. ***P<0.005 compared with untreated WT MLF or with TRPV4 KO ±TGF-β. All graphs show means ± SEM from N = 3 independent experiments. **P≤0.01, ***P≤0.005, using ANOVA followed by Student-Newman-Keuls multiple comparisons test. Scale bars, 50 μm.

Article Snippet: Lentiviral Constructs Lentiviral constructs for HA-tagged WT PI3Kγ (full length), HA-N-terminal PI3Kγ (N-term, contains only AA 1-142), HA-N-terminal deleted PI3Kγ (N-del, missing the N-terminal (ΔAA 1-142) and the ATP binding site (ΔAA 948-981)), and GFP-tagged full length TRPV4 were produced by Cyagen Biosciences.

Techniques: Knock-Out, Immunofluorescence, Fluorescence, Western Blot, Surface Biotinylation Assay

Journal: Science signaling

Article Title: Translocation of TRPV4-PI3Kγ complexes to the plasma membrane drives myofibroblast transdifferentiation

doi: 10.1126/scisignal.aau1533

Figure Lengend Snippet: (A) The binding of purified PI3Kγ to purified 6-His-TRPV4 coupled to Ni-NTA beads was assessed by immunoblotting with an antibody specific for PI3Kγ. The blots were stripped and reprobed with an antibody for TRPV4. Control indicates beads alone (no protein bound to beads), N=3 independent experiments. (B) The direct interaction between immobilized purified 6-His-TRPV4 and purified 6-His-PI3Kγ was measured by surface plasmon resonance. N=3 independent experiments. (C) The binding of purified 6-His-PI3Kγ coupled to Ni-NTA beads to endogenous TRPV4 from human lung fibroblast (HLF) lysates was assessed by Western blotting. Blots were stripped and reprobed with antibodies specific for PI3Kγ, GRK2 (positive control), and TRPV2 (negative control). Control indicates beads alone (no protein bound to beads), N=3 independent experiments. (D) PI3Kγ immunoprecipitates (IP) from plasma membrane and cytosolic fractions of HLFs treated with TGF-β as indicated were immunoblotted for PI3Kγ and TRPV4. The line between the plasma membrane and cytosolic fractions indicates non-contiguous lanes from the same blot. Total whole cell lysate of cells treated ±TGF-β (input) was immunoblotted for TRPV4 and PI3Kγ. GAPDH is a loading control. (E) Quantification of TRPV4 in plasma membrane and cytosolic fractions normalized to TRPV4 in whole cell lysate as in (D). Data represent means ± SEM. N=3 independent experiments. *P≤0.05 between untreated and TGF-β conditions, using ANOVA followed by Student-Newman-Keuls multiple comparisons test.

Article Snippet: Lentiviral Constructs Lentiviral constructs for HA-tagged WT PI3Kγ (full length), HA-N-terminal PI3Kγ (N-term, contains only AA 1-142), HA-N-terminal deleted PI3Kγ (N-del, missing the N-terminal (ΔAA 1-142) and the ATP binding site (ΔAA 948-981)), and GFP-tagged full length TRPV4 were produced by Cyagen Biosciences.

Techniques: Binding Assay, Purification, Western Blot, SPR Assay, Positive Control, Negative Control

Journal: Science signaling

Article Title: Translocation of TRPV4-PI3Kγ complexes to the plasma membrane drives myofibroblast transdifferentiation

doi: 10.1126/scisignal.aau1533

Figure Lengend Snippet: (A) Representative plots showing the effects of scrambled, PI3Kα, and PI3Kγ siRNA on Ca2+ influx induced by the TRPV4 agonist GSK1016790A (GSK) in human lung fibroblasts. Ca2+ influx was measured in relative fluorescence units (RFU) using Calcium 5 dye on intact human lung fibroblast (19Lu) monolayers treated with scrambled, PI3Kα, or PI3Kγ siRNA. (B) Quantification of experiments in (A). ***P<0.005 compared with PI3Kα siRNA or scrambled siRNA. (C) Representative immunoblots showing PI3Kα and PI3Kγ in 19Lu cells treated with scrambled, PI3Kα, or PI3Kγ siRNA. GAPDH is a loading control. (D) Quantification of PI3Kα and PI3Kγ band density relative to GAPDH in (C). ***P<0.005 as indicated. (E) Representative plot showing GSK1016790A-induced Ca2+ influx in WT and PI3Kγ KO mouse lung fibroblasts (MLFs) treated ±TGF-β as indicated. (F) Quantification of RFU in 300 nM GSK1016790A conditions in (E). *P<0.05 as indicated. (G) Representative immunoblot showing TRPV4 in WT and PI3Kγ KO MLFs treated ±TGF-β. (H) Quantification of TRPV4 band density relative to GAPDH in (G). All graphs show means ± SEM from N = 3 independent experiments (*P≤0.05, ***P≤0.005, using ANOVA followed by Student-Newman-Keuls multiple comparisons test).

Article Snippet: Lentiviral Constructs Lentiviral constructs for HA-tagged WT PI3Kγ (full length), HA-N-terminal PI3Kγ (N-term, contains only AA 1-142), HA-N-terminal deleted PI3Kγ (N-del, missing the N-terminal (ΔAA 1-142) and the ATP binding site (ΔAA 948-981)), and GFP-tagged full length TRPV4 were produced by Cyagen Biosciences.

Techniques: Fluorescence, Western Blot

Journal: Science signaling

Article Title: Translocation of TRPV4-PI3Kγ complexes to the plasma membrane drives myofibroblast transdifferentiation

doi: 10.1126/scisignal.aau1533

Figure Lengend Snippet: Our data suggest that TRPV4 binds to the non-catalytic, N-terminal domain of PI3Kγ, forming a TRPV4-PI3Kγ complex. Upon TGF-β stimulation, the N-terminus of PI3Kγ acts as a scaffold, recruiting TRPV4-PI3Kγ complexes from the cytoplasm to the plasma membrane. This translocation leads to an increase in TRPV4-mediated Ca2+ influx, which promotes myofibroblast transdifferentiation.

Article Snippet: Lentiviral Constructs Lentiviral constructs for HA-tagged WT PI3Kγ (full length), HA-N-terminal PI3Kγ (N-term, contains only AA 1-142), HA-N-terminal deleted PI3Kγ (N-del, missing the N-terminal (ΔAA 1-142) and the ATP binding site (ΔAA 948-981)), and GFP-tagged full length TRPV4 were produced by Cyagen Biosciences.

Techniques: Translocation Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Exosomal transfer of miR-15b-3p enhances tumorigenesis and malignant transformation through the DYNLT1/Caspase-3/Caspase-9 signaling pathway in gastric cancer

doi: 10.1186/s13046-019-1511-6

Figure Lengend Snippet: DYNLT1 is a direct downstream miR-15b-3p target in GC cells. a miR-15b-3p target gene prediction using four bioinformatics tools (miRDB, RNA22, TarBase and TargetScan). b DYNLT1 mRNA levels in GC tissues and paired adjacent non-GC tissues ( n = 108) analyzed using qRT-PCR. c GC tissue DYNLT1 expression was found to be significantly decreased, based on the TCGA database. d-f Western blotting and IHC analysis of DYNLT1 protein levels in GC tissues and adjacent non-GC tissues. Scale bar, 200 μm. g miR-15b-3p and DYNLT1 expression level association analysis using the 108 GC tissues. h-i Immunoblotting assays and qRT-PCR on DYNLT1 expression of miR-15b-3p inhibitor/inhibitor-NC/mimics/NC transfected BGC-823 and SGC-7901 cells. j DYNLT1 binding site of the wild-type (WT) and mutated type with miR-15b-3p. k Direct recognition of DYNLT1 3′-UTR by miR-15b-3p. Co-transfection of BGC-823 and SGC-7901 cells with WT or Mutant DYNLT1 3′-UTR and miR-15b-3p mimics, inhibitor or their corresponding normal control (NC or inhibitor-NC). The relative luciferase activity of BGC-823 and SGC-7901 cells were determined. Mean ± SEM of the results are presented

Article Snippet: In 24-well plates, the luciferase construct containing wild-type (WT) or mutated binding site of DYNLT1 (constructed by Genechem Inc., China) were transfected into target cells.

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Transfection, Binding Assay, Cotransfection, Mutagenesis, Control, Luciferase, Activity Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Exosomal transfer of miR-15b-3p enhances tumorigenesis and malignant transformation through the DYNLT1/Caspase-3/Caspase-9 signaling pathway in gastric cancer

doi: 10.1186/s13046-019-1511-6

Figure Lengend Snippet: Exosomal transfer of miR-15b-3p enhances malignant transformation in vitro. Proliferation of SGC-7901 and GES-1 cells co-cultured with PBS alone or exosomes containing miR-15b-3p mimics/NC/inhibitor/inhibitor-NC were assessed using colony formation ( a ), CCK-8 ( b ) and EdU ( c ) assays. Scale bar, 100 μm. d Migration and invasion assays of SGC-7901 and GES-1 cell treated with PBS, Exo-NC, Exo-15b-3p-mimics, Exo-inhibitor-NC or Exo-15b-3p-inhibitor. Cells that had migrated and invaded were counted. Representative images are shown. e The SGC-7901 and GES-1 cell apoptosis, in the presence of PBS or exosomes (wrapped with 15b-3p-mimics, inhibitor or their corresponding normal control) were detected using flow cytometry. f miR-15b-3p mimics/NC/inhibitor/inhibitor-NC-enriched exosomes or only PBS was incubated with GES-1 and SGC-7901 cells for 24 h, followed by TUNEL assay. g Western blotting assay of DYNLT1, BAX, BCL-2, Cleaved Caspase-9 and Cleaved Caspase-3 in SGC-7901 and GES-1 with the treatments indicated. The internal control used was β-Actin. Mean ± SEM of three independent experiments are presented

Article Snippet: In 24-well plates, the luciferase construct containing wild-type (WT) or mutated binding site of DYNLT1 (constructed by Genechem Inc., China) were transfected into target cells.

Techniques: Transformation Assay, In Vitro, Cell Culture, CCK-8 Assay, Migration, Control, Flow Cytometry, Incubation, TUNEL Assay, Western Blot

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Exosomal transfer of miR-15b-3p enhances tumorigenesis and malignant transformation through the DYNLT1/Caspase-3/Caspase-9 signaling pathway in gastric cancer

doi: 10.1186/s13046-019-1511-6

Figure Lengend Snippet: Exo-miR-15b-3p regulates tumor growth in vivo. a miR-15b-3p expression levels in BGC-823 cells (stably transfected with Lv-miR-15b-3p/Lv-NC or Lv-inhibitor/Lv-inNC) or exosomes isolated from BGC-823 cells were detected using qRT-PCR. SGC-7901 cells were treated with PBS or exosomes loaded with Lv-miR-15b-3p/Lv-NC or Lv-inhibitor/Lv-inNC and were subsequently injected into the nude mice ( n = 5). The xenografts ( b) and tumor growth curve ( c) show that Exo-Lv-miR-15b-3p promotes, while Exo-Lv-inhibitor suppresses xenograft tumor growth in nude mice. d Representative images of tumor growth of the mice treated with exosomes derived from stably transfected-BGC-823 cells or PBS, were determined using luciferase-based bioluminescence imaging. e Representative images of TUNEL staining of the xenograft tumors for the ectopic expression or silencing of Exo-miR-15b-3p and their corresponding control or PBS groups. Scale bar, 100 μm. f Quantification of TUNEL-positive cells. g qRT-PCR analysis of miR-15b-3p and DYNLT1 expression in xenograft tumors with the treatment indicated. h Immunohistochemical analysis of DYNLT1 expression in the xenografts. Scale bar, 50 μm. i Western blotting analysis of DYNLT1, BAX, BCL-2, Cleaved Caspase-9 and Cleaved Caspase-3 in xenograft tumor tissues among the different groups. The internal control used was β-Actin. Mean ± SEM of the results are presented

Article Snippet: In 24-well plates, the luciferase construct containing wild-type (WT) or mutated binding site of DYNLT1 (constructed by Genechem Inc., China) were transfected into target cells.

Techniques: In Vivo, Expressing, Stable Transfection, Transfection, Isolation, Quantitative RT-PCR, Injection, Derivative Assay, Luciferase, Imaging, TUNEL Assay, Staining, Control, Immunohistochemical staining, Western Blot

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Exosomal transfer of miR-15b-3p enhances tumorigenesis and malignant transformation through the DYNLT1/Caspase-3/Caspase-9 signaling pathway in gastric cancer

doi: 10.1186/s13046-019-1511-6

Figure Lengend Snippet: Schematic illustrating the function and mechanism of GC cells-derived exo-miR-15b-3p in recipient cells. The exo-miR-15b-3p released by GC cells promotes the proliferation, migration, invasion and inhibits apoptosis of recipient cells via the DYNLT1/Caspase-3/Caspase-9 signaling pathway, thereby promoting tumorigenesis and malignant transformation. Serum exo-miR-15b-3p may function as a potential GC diagnostic biomarker

Article Snippet: In 24-well plates, the luciferase construct containing wild-type (WT) or mutated binding site of DYNLT1 (constructed by Genechem Inc., China) were transfected into target cells.

Techniques: Derivative Assay, Migration, Transformation Assay, Diagnostic Assay, Biomarker Discovery

Journal: Molecular cancer therapeutics

Article Title: ALK inhibitor response in melanomas expressing EML4-ALK fusions and alternate ALK isoforms

doi: 10.1158/1535-7163.MCT-17-0472

Figure Lengend Snippet: Driver gene mutations in ALK expressing melanomas

Article Snippet: Lentiviral constructs and expression cDNA inserts for ALK fusions, wt ALK , and ALK ATI were synthesized (GenScript) and cloned into the pLVX-EF1α-IRES-ZsGreen1 lentivirus expression vector (Clontech).

Techniques: Expressing

Journal: Scientific Reports

Article Title: pH Induced Conformational Transitions in the Transforming Growth Factor β-Induced Protein (TGFβIp) Associated Corneal Dystrophy Mutants

doi: 10.1038/srep23836

Figure Lengend Snippet: ( a ) Schematic representation of the domain arrangement and boundaries of the full-length TGFβIp and 4 th _FAS1 domains of the wild-type, non-amyloidogenic (R555W) and amyloidogenic (H572R) mutants used in the study. ( b ) SDS-PAGE gel showing the purified fractions of the 4 th _FAS1 domains of the WT, R555W and H572R mutants. ( c–e ) Far UV CD spectra of the 4 th _FAS1 domains of WT ( d ), R555W ( e ) and H572R ( f ) incubated for 16 hours at acidic pH conditions (pH 3, 4.5, 5.5 and 7). The R555W mutant displayed clear changes in the CD spectra at 222 nm and 207 nm with decrease in pH ( f ). The CD intensity at 222 nm decreased with decrease in pH confirming the unfolding of the secondary structures. The CD spectra for the WT ( c ) and H572R ( e ) mutant remained almost unchanged. Plotting the intensities at 222 nm at varying pH ( g ) showed that the non-amyloidogenic phenotype, R555W, was more sensitive to pH and the amyloidogenic phenotype, H572R, remained more stable to pH changes at room temperature.

Article Snippet: The cDNA constructs of the 4 th _FAS1 domains of the WT TGFβIp, the mutants R555W and H572R were bought from Genscript (Piscataway, NJ) in pUC57 vectors.

Techniques: SDS Page, Purification, Circular Dichroism, Incubation, Mutagenesis

Journal: Scientific Reports

Article Title: pH Induced Conformational Transitions in the Transforming Growth Factor β-Induced Protein (TGFβIp) Associated Corneal Dystrophy Mutants

doi: 10.1038/srep23836

Figure Lengend Snippet: Effects of mutation on the charge and hydrophobicity.

Article Snippet: The cDNA constructs of the 4 th _FAS1 domains of the WT TGFβIp, the mutants R555W and H572R were bought from Genscript (Piscataway, NJ) in pUC57 vectors.

Techniques: Mutagenesis

Journal: Scientific Reports

Article Title: pH Induced Conformational Transitions in the Transforming Growth Factor β-Induced Protein (TGFβIp) Associated Corneal Dystrophy Mutants

doi: 10.1038/srep23836

Figure Lengend Snippet: ( a , b ) Far UV CD spectra of the 4 th _FAS1 domain of WT at pH 7.0 and pH 8.0 before heating (black), after heating to 70 °C (red) and cooling back to 20 °C (blue). ( c,d ) Far UV CD spectra of the 4 th _FAS1 domains of R555W at pH 7 ( c ) and pH 8 ( d ) before heating (black) and after heating to 70 °C (red) and cooling back to 20 °C (blue). ( e , f ) Far UV CD spectra of the 4 th _FAS1 domains of H572R a pH 7.0 ( e ) and pH 8.0 ( f ) before heating (black) and after heating to 70 °C (red) and cooling back to 20 °C (blue). The WT and the mutants did not display any significant changes in structure at pH 7.0 and pH 8.0.

Article Snippet: The cDNA constructs of the 4 th _FAS1 domains of the WT TGFβIp, the mutants R555W and H572R were bought from Genscript (Piscataway, NJ) in pUC57 vectors.

Techniques: Circular Dichroism

Journal: Scientific Reports

Article Title: pH Induced Conformational Transitions in the Transforming Growth Factor β-Induced Protein (TGFβIp) Associated Corneal Dystrophy Mutants

doi: 10.1038/srep23836

Figure Lengend Snippet: ( a – c ) Far UV CD spectra of the 4 th _FAS1 domain of WT at pH 3.0 ( a ), pH 4.5 ( b ) and pH 5.5 ( c ) before heating (black) and after heating to 70 °C (red) and cooling back to 20 °C (blue). ( d – f ) Far UV CD spectra of the 4 th _FAS1 domain of R555W at pH 3 ( d ), pH 4.5 ( e ) and pH 5.5 ( f ) before heating (black) and after heating to 70 °C (red) and cooling back to 20 °C (blue). ( g – i ) Far UV CD spectra of the 4 th _FAS1 domain of H572R at pH 3.0 ( g ), pH 4.5 ( h ) and pH 5.5 ( i ) before heating (black) and after heating to 70 °C (red) and cooling back to 20 °C (blue). While the WT did not show any changes in structure, both the mutants displayed a very clear transition to β-sheet under acidic conditions.

Article Snippet: The cDNA constructs of the 4 th _FAS1 domains of the WT TGFβIp, the mutants R555W and H572R were bought from Genscript (Piscataway, NJ) in pUC57 vectors.

Techniques: Circular Dichroism

Journal: Scientific Reports

Article Title: pH Induced Conformational Transitions in the Transforming Growth Factor β-Induced Protein (TGFβIp) Associated Corneal Dystrophy Mutants

doi: 10.1038/srep23836

Figure Lengend Snippet: ( a – e ) Variable temperature CD curves at 222 nm of WT (black), R555W (red) and H572R (blue) proteins heated from 20 °C to 70 °C at various pH (3.0 [ a ], 4.5 [ b ], 5.5 [ c ], 7.0 [ d ] and 8.0 [ e ]) and the CD intensities at 222 nm were plotted as a function of temperature. The baseline subtracted curves of the WT (black), R555W (red) and H572R (blue) proteins show that while there was no transition observed in the WT in all the conditions as observed from the unchanged straight line in black, little or no changes were seen in pH 7 and pH 8 for the mutants. However, clear transitions to β-sheet were observed at acidic pH (pH 3, pH 4.5 and pH 5.5) for both the mutants. In all cases, we observe transition (Tt) is higher for R555W compared to H572R. A clear shift in their thermal denaturation curves between the mutants at acidic pH (pH 3.0, 4.5 and 5.5) is observed. A difference in T t of 5–12 °C is observed at various pH conditions.

Article Snippet: The cDNA constructs of the 4 th _FAS1 domains of the WT TGFβIp, the mutants R555W and H572R were bought from Genscript (Piscataway, NJ) in pUC57 vectors.

Techniques:

Journal: Scientific Reports

Article Title: pH Induced Conformational Transitions in the Transforming Growth Factor β-Induced Protein (TGFβIp) Associated Corneal Dystrophy Mutants

doi: 10.1038/srep23836

Figure Lengend Snippet: ( a – b ) Transmission Electron Microscopy. TEM images of the β-oligomers of the 4 th _FAS1 domains of R555W ( a ) and H572R ( b ) mutants were acquired with a JEOL JEM-1010 transmission electron microscope using Digital Micrograph™ 1.81.78 for GMS 1.8.0. The β-oligomers of the amyloidogenic phenotype were larger measuring between 10–40 nm, mean size ~19.1 nm ± 4.9 nm ( a ) compared to the non-amyloidogenic β-oligomers that measured 4–8 nm, with a mean size ~5.1 nm ± 1.79 nm ( b ). Inset figures – particle size distribution of the β-oligomers. While the amyloidogenic β-oligomers were larger and displayed rugged edges and varying diameters, the non-amyloidogenic β-oligomers were smaller in size with smoother edges and were more homogenous. ( c ) ThT assay. Emission fluorescence intensities at 485 nm recorded after incubating the WT TGFβIp and the β-oligomers of R555W and H572R with ThT dye. An amyloid forming peptide (611-633aa - pN622K) of TGFβIp was used as the positive control. The β-oligomers of the amyloidogenic mutant H572R showed significant fluorescence intensity (**P < 0.01) almost 3 times more fluorescence compared to the non-amyloidogenic R555W. ( d ) DLS. %intensity plots plotted against the apparent hydrodynamic radii (R H ). The R H values calculated from the distribution curves for R555W (~39.58 nm| pH 3.0 , ~51.9 nm| pH 4.5 and ~68.2 nm| pH 5.5 ) and H572R (~89.95 nm| pH 3.0 , ~69 nm| pH 4.5 and ~155 nm| pH 5.5 ) show that H572R b-oligomers are larger in size and slightly more heterogeneous. ( e , f ) Mass Spectrometric analyses - Identification of the aggregation hotspots in the mutants and β-oligomers. ( d ) Peptide map generated following the insilico trypsin digestion of TGFβIp displaying a series of peptides. ( e ) The β-oligomers formed from the mutant 4 th _FAS1 domains were digested with trypsin and the resulting fragments were analyzed by LC–MS/MS. The regions inaccessible for trypsin digestion have been underlined in red. It is clearly seen that the non-amyloidogenic R555W shows more regions inaccessible for trypsin digestion.

Article Snippet: The cDNA constructs of the 4 th _FAS1 domains of the WT TGFβIp, the mutants R555W and H572R were bought from Genscript (Piscataway, NJ) in pUC57 vectors.

Techniques: Transmission Assay, Electron Microscopy, Microscopy, ThT Assay, Fluorescence, Positive Control, Mutagenesis, Generated, Liquid Chromatography with Mass Spectroscopy

Journal: Scientific Reports

Article Title: pH Induced Conformational Transitions in the Transforming Growth Factor β-Induced Protein (TGFβIp) Associated Corneal Dystrophy Mutants

doi: 10.1038/srep23836

Figure Lengend Snippet: ( a – d ) Thermal stability. Variable temperature CD values at 222 nm of the 4 th _FAS1 domains of R555W ( a ) and H572R ( c ) in buffer at pH 5.5 when heated from 20 °C to 70 °C. Far UV CD spectra of the 4 th _FAS1 domains of R555W ( b ) and H572R ( d ) after heating (red) to 70 °C and cooling (blue) back to 20 °C. ( e , f ) Stability at physiological pH. Far UV CD spectra of the 4 th _FAS1 domains (black) of R555W ( e ) and H572R ( f ) at pH 5.5 under native conditions (black), after heating to 70 °C (red) and after reconstituting in buffer at pH 7.0 (blue) after incubation at room temperature for 4 weeks.

Article Snippet: The cDNA constructs of the 4 th _FAS1 domains of the WT TGFβIp, the mutants R555W and H572R were bought from Genscript (Piscataway, NJ) in pUC57 vectors.

Techniques: Circular Dichroism, Incubation

Journal: Scientific Reports

Article Title: pH Induced Conformational Transitions in the Transforming Growth Factor β-Induced Protein (TGFβIp) Associated Corneal Dystrophy Mutants

doi: 10.1038/srep23836

Figure Lengend Snippet: ( a ) The growth and proliferation of the pHCSF cells following the treatment with the β-oligomers and the mutant 4 th _FAS1 domains monitored for 48 hours in an xCELLigence system. ( b ) The cell survival after treatment for 24 hours plotted as area under the curve (AUC). While the WT and R555W show low cytotoxicity, almost similar to the media control, the amyloidogenic H572R mutant was cytotoxic compared to the control (**P < 0.01). Interestingly, the β-oligomers of both the amyloidogenic and non-amyloidogenic mutants showed high cytotoxicity (**P < 0.01) with the approximately 7 times more for the non-amyloidogenic mutant and 12 times more for the amyloidogenic mutant. To obtain detailed information on the cytotoxicity of the soluble and insoluble fractions, β-oligomers of the two mutants were prepared at various acidic pH conditions (pH 3.0, pH 4.5, pH 5.5) and their cytotoxicity were examined on pHCSFs from 3 different donors (n = 3) using xCELLigence ( c ) and MTT assays ( d ). ( c ) xCELLigence assay. The WT shows low cytotoxicity, almost similar to the control. The soluble β-oligomers of both the mutants showed high cytotoxicity (**P < 0.01) compared to the controls. The insoluble β-oligomers were relatively less cytotoxic compared to the soluble β-oligomers. ( d ) MTT assay. The cytotoxicity of the β-oligomers was also tested using an MTT assay. Similar to xCELLigence, we could see that soluble β-oligomers derived from both the mutants displayed potent cytotoxic effect (**P < 0.01) compared to the controls and insoluble aggregates. Though the insoluble oligomers were relatively less cytotoxic than the soluble oligomers, they displayed cytotoxicity compared to the controls.

Article Snippet: The cDNA constructs of the 4 th _FAS1 domains of the WT TGFβIp, the mutants R555W and H572R were bought from Genscript (Piscataway, NJ) in pUC57 vectors.

Techniques: Mutagenesis, Control, MTT Assay, Derivative Assay

Journal: Biochemical pharmacology

Article Title: H 2 S-induced S -sulfhydration of lactate dehydrogenase A (LDHA) stimulates cellular bioenergetics in HCT116 colon cancer cells

doi: 10.1016/j.bcp.2017.03.025

Figure Lengend Snippet: (A) Protein sequences of WT LDHA and C163A LDHA. Protein sequences of WT LDHA and C163A LDHA. A His tag was added to the end of each protein to aid in the protein isolation and purification steps (letters shown in red). The Cys residue substituted to Ala in C163A LDHA is shown bold and highlighted. (B) WT LDHA or C163A LDHA (1 μg) were pre-incubated with 10–300 μM NaHS for 30 min at 37°C. LDHA enzymatic analysis was carried out as described in the Materials and Methods. Data represent mean ± SEM and expressed as a percentage of the corresponding untreated WT LDHA group. n = 4–6, for each group; *P < 0.05 vs. WT LDHA CTL (based on one-way ANOVA corrected with Tukey’s post-hoc test).

Article Snippet: 2.6 Production of WT LDHA and C163A LDHA GenScript (Piscataway, NJ) was contracted to synthesize and produce full length protein constructs of normal human LDHA (WT) and C163A LDHA, which were verified by DNA sequencing.

Techniques: Isolation, Purification, Residue, Incubation

Journal: Biochemical pharmacology

Article Title: H 2 S-induced S -sulfhydration of lactate dehydrogenase A (LDHA) stimulates cellular bioenergetics in HCT116 colon cancer cells

doi: 10.1016/j.bcp.2017.03.025

Figure Lengend Snippet: MS/MS/LC was used to identify and quantity the post-translational modification of NaHS-induced S-sulfhydration (SSH) of LDHA. (A) Figure shows the area under the peak of SSH-Cys163 and the total digested protein without the SSH modification in both WT and C163A LDHA. (Total wild-type LDHA protein used in the assay was 1 μg; total C163A LDHA was increased to 2.5 μg in order to increase the detection of residual sulfhydrated cysteines.) Only minimal amount of sulfhydrated cysteines was detected in the C163A LDHA. (B) Figure represents the ratio of the area under the peak of SSH-Cys163 vs. the area under the peak of the digested peptide without S-sulfhydration modification in WT LDHA. (C) Extracted Ion Chromatogram (XIC) of WT LDHA (top) and C163A LDHA (bottom) treated with 10 μM NaHS. NaHS-treated WT LDHA produced a significant SSH-Cys163 peak at retention time (RT) 20.16 min, which was absent in NaHS-treated C163A LDHA.

Article Snippet: 2.6 Production of WT LDHA and C163A LDHA GenScript (Piscataway, NJ) was contracted to synthesize and produce full length protein constructs of normal human LDHA (WT) and C163A LDHA, which were verified by DNA sequencing.

Techniques: Tandem Mass Spectroscopy, Modification, Produced

Journal: Biochemical pharmacology

Article Title: H 2 S-induced S -sulfhydration of lactate dehydrogenase A (LDHA) stimulates cellular bioenergetics in HCT116 colon cancer cells

doi: 10.1016/j.bcp.2017.03.025

Figure Lengend Snippet: 3.2 H 2 S activates human LDHA via S -sulfhydrating Cys163

Article Snippet: 2.6 Production of WT LDHA and C163A LDHA GenScript (Piscataway, NJ) was contracted to synthesize and produce full length protein constructs of normal human LDHA (WT) and C163A LDHA, which were verified by DNA sequencing.

Techniques: Sequencing, Modification, Residue