forskolin  (Selleck Chemicals)


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    Name:
    Colforsin
    Description:
    Colforsin Forskolin Coleonol is a ubiquitous activator of eukaryotic adenylyl cyclase AC in a wide variety of cell types commonly used to raise levels of cAMP in the study and research of cell physiology Forskolin also activates PXR and FXR activity Forskolin stimulates autophagy
    Catalog Number:
    s2449-10mg
    Price:
    110.0
    Size:
    10mg
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    Structured Review

    Selleck Chemicals forskolin
    Colforsin
    Colforsin Forskolin Coleonol is a ubiquitous activator of eukaryotic adenylyl cyclase AC in a wide variety of cell types commonly used to raise levels of cAMP in the study and research of cell physiology Forskolin also activates PXR and FXR activity Forskolin stimulates autophagy
    https://www.bioz.com/result/forskolin/product/Selleck Chemicals
    Average 94 stars, based on 1 article reviews
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    forskolin - by Bioz Stars, 2021-03
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    Images

    1) Product Images from "Glucagon-like peptide-1 modulates RAW264.7 macrophage polarization by interfering with the JNK/STAT3 signaling pathway"

    Article Title: Glucagon-like peptide-1 modulates RAW264.7 macrophage polarization by interfering with the JNK/STAT3 signaling pathway

    Journal: Experimental and Therapeutic Medicine

    doi: 10.3892/etm.2019.7347

    GLP-1/GLP-1 receptor signaling inhibits M1 activation and induces M2 activation via the cyclic adenosine monophosphate/PKA signaling pathway in RAW264.7 cells. (A) RAW264.7 cells were preincubated with the adenylyl cyclase activator Forskolin (10 µM) or the PKA inhibitor H89 (10 µM) for 30 min, followed by treatment without or with GLP-1 (10 nmol/l) for 24 h. *P
    Figure Legend Snippet: GLP-1/GLP-1 receptor signaling inhibits M1 activation and induces M2 activation via the cyclic adenosine monophosphate/PKA signaling pathway in RAW264.7 cells. (A) RAW264.7 cells were preincubated with the adenylyl cyclase activator Forskolin (10 µM) or the PKA inhibitor H89 (10 µM) for 30 min, followed by treatment without or with GLP-1 (10 nmol/l) for 24 h. *P

    Techniques Used: Activation Assay

    GLP-1 inhibits JNK activation via the cyclic adenosine monophosphate/PKA signaling pathway in RAW264.7 cells. RAW264.7 cells were preincubated with the adenylyl cyclase activator Forskolin (10 µM) or the PKA inhibitor H89 (10 µM) for 30 min, followed by treatment without or with GLP-1 (10 nmol/l) for 24 h. Cell extracts were prepared and analyzed by western blotting using anti-p-JNK, anti-JNK and anti-GAPDH antibodies. The results are representative of three independent experiments. P
    Figure Legend Snippet: GLP-1 inhibits JNK activation via the cyclic adenosine monophosphate/PKA signaling pathway in RAW264.7 cells. RAW264.7 cells were preincubated with the adenylyl cyclase activator Forskolin (10 µM) or the PKA inhibitor H89 (10 µM) for 30 min, followed by treatment without or with GLP-1 (10 nmol/l) for 24 h. Cell extracts were prepared and analyzed by western blotting using anti-p-JNK, anti-JNK and anti-GAPDH antibodies. The results are representative of three independent experiments. P

    Techniques Used: Activation Assay, Western Blot

    GLP-1 increases intracellular cAMP levels in RAW264.7 macrophages. RAW 264.7 cells were incubated with various concentrations of GLP-1 (0.5–30 nmol/l) for 8 h. The Forskolin (10 µM) group was set as the positive control. Cell extracts were prepared, followed by determination of the cAMP levels in RAW264.7 cells by enzyme immunoassay. The results are representative of three independent experiments. P
    Figure Legend Snippet: GLP-1 increases intracellular cAMP levels in RAW264.7 macrophages. RAW 264.7 cells were incubated with various concentrations of GLP-1 (0.5–30 nmol/l) for 8 h. The Forskolin (10 µM) group was set as the positive control. Cell extracts were prepared, followed by determination of the cAMP levels in RAW264.7 cells by enzyme immunoassay. The results are representative of three independent experiments. P

    Techniques Used: Incubation, Positive Control, Enzyme-linked Immunosorbent Assay

    2) Product Images from "Inhibition of TPL2 by interferon-α suppresses bladder cancer through activation of PDE4D"

    Article Title: Inhibition of TPL2 by interferon-α suppresses bladder cancer through activation of PDE4D

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    doi: 10.1186/s13046-018-0971-4

    Suppression of COX-2 expression by IFN-α via inhibition of the TPL2 and cAMP/CREB. ( a ) T24 cells were treated with IFN-α (1 × 10 4 U/mL) for specific time points; or treated using various concentrations of IFN-α for 24 h. The cell lysates were immunoblotted with COX-2 antibody. β-Tubulin staining is shown as a loading control. ( b ) T24 cells were treated with IFN-α (1 × 10 4 U/mL) for specific time points. The TPL2, p-TPL2, IKKα/β, p- IKKα/β, IκBα and p-IκBα were analyzed by performing western blotting. ( c ) T24 cells were treated with IFN-α (1 × 10 4 U/ml), TPL2i (2 μM), and PD98059 (40 μM) for 12 h. The COX-2, TPL2, p-TPL2, ERK, p- ERK, IKKα/β, p-IKKα/β, IκBα and p-IκBα were analyzed by performing western blotting. β-Tubulin staining is shown as a loading control. ( d ) The intracellular cAMP level was detected after T24 cells were treated with IFN-α (1 × 10 4 U/mL), TPL2i (2 μM), and PD98059 (40 μM) for 4 h. ( e ) T24 cells were treated with IFN-α (1 × 10 4 U/mL), TPL2i (2 μM), PD98059 (40 μM), or forskolin (50 μM) for 24 h. The expression levels of COX-2, CREB, and p-CREB were analyzed by western blotting. The β-tubulin was used as the loading control. ( f ) T24 cells were treated with IFN-α (1 × 10 4 U/mL), PD98059 (40 μM), and EGF (25 ng/mL) for 12 h. The COX-2 expression was analyzed by performing western blotting. ( g ) Cell viability was detected after T24 cells were treated using forskolin (50 μM), TPL2i (2 μM), and PD98059 (40 μM) for 72 h. Data represent the results of three independent experiments. Error bars indicate mean ± SD. *, P
    Figure Legend Snippet: Suppression of COX-2 expression by IFN-α via inhibition of the TPL2 and cAMP/CREB. ( a ) T24 cells were treated with IFN-α (1 × 10 4 U/mL) for specific time points; or treated using various concentrations of IFN-α for 24 h. The cell lysates were immunoblotted with COX-2 antibody. β-Tubulin staining is shown as a loading control. ( b ) T24 cells were treated with IFN-α (1 × 10 4 U/mL) for specific time points. The TPL2, p-TPL2, IKKα/β, p- IKKα/β, IκBα and p-IκBα were analyzed by performing western blotting. ( c ) T24 cells were treated with IFN-α (1 × 10 4 U/ml), TPL2i (2 μM), and PD98059 (40 μM) for 12 h. The COX-2, TPL2, p-TPL2, ERK, p- ERK, IKKα/β, p-IKKα/β, IκBα and p-IκBα were analyzed by performing western blotting. β-Tubulin staining is shown as a loading control. ( d ) The intracellular cAMP level was detected after T24 cells were treated with IFN-α (1 × 10 4 U/mL), TPL2i (2 μM), and PD98059 (40 μM) for 4 h. ( e ) T24 cells were treated with IFN-α (1 × 10 4 U/mL), TPL2i (2 μM), PD98059 (40 μM), or forskolin (50 μM) for 24 h. The expression levels of COX-2, CREB, and p-CREB were analyzed by western blotting. The β-tubulin was used as the loading control. ( f ) T24 cells were treated with IFN-α (1 × 10 4 U/mL), PD98059 (40 μM), and EGF (25 ng/mL) for 12 h. The COX-2 expression was analyzed by performing western blotting. ( g ) Cell viability was detected after T24 cells were treated using forskolin (50 μM), TPL2i (2 μM), and PD98059 (40 μM) for 72 h. Data represent the results of three independent experiments. Error bars indicate mean ± SD. *, P

    Techniques Used: Expressing, Inhibition, Staining, Western Blot

    3) Product Images from "Conversion of Fibroblast into Functional Leydig-like Cell Using Defined Small Molecules"

    Article Title: Conversion of Fibroblast into Functional Leydig-like Cell Using Defined Small Molecules

    Journal: Stem Cell Reports

    doi: 10.1016/j.stemcr.2020.07.002

    Conversion of MEFs into Leydig-like Cells by the Combination of Forskolin, 20α-Hydroxycholesterol, LH, and SB431542 (4C) (A) Schematic of the experimental procedure. (B) Cells morphology was transformed from a fibroblast morphology to a Leydig cell morphology during the induction process. Representative images from days 0, 7, and 14 are shown. Scale bars, 100 μm. (C–E) qRT-PCR analysis of key Leydig transcription factor genes (C, Nr5a1 ; D, Gata4 , and E, Dmrt1 ) after induction with 4C. (F and G) qRT-PCR analysis of fibroblast genes (F, Postn ; G, Col5a2 ) after induction with 4C. (H) Cell proliferation curves during the induction process. Three wells were harvested for cell counting. The data were obtained from three independent experiments and are presented as the mean ± SD values. ∗ p
    Figure Legend Snippet: Conversion of MEFs into Leydig-like Cells by the Combination of Forskolin, 20α-Hydroxycholesterol, LH, and SB431542 (4C) (A) Schematic of the experimental procedure. (B) Cells morphology was transformed from a fibroblast morphology to a Leydig cell morphology during the induction process. Representative images from days 0, 7, and 14 are shown. Scale bars, 100 μm. (C–E) qRT-PCR analysis of key Leydig transcription factor genes (C, Nr5a1 ; D, Gata4 , and E, Dmrt1 ) after induction with 4C. (F and G) qRT-PCR analysis of fibroblast genes (F, Postn ; G, Col5a2 ) after induction with 4C. (H) Cell proliferation curves during the induction process. Three wells were harvested for cell counting. The data were obtained from three independent experiments and are presented as the mean ± SD values. ∗ p

    Techniques Used: Transformation Assay, Quantitative RT-PCR, Cell Counting

    Characteristics of CiLCs (A) Morphology of MEFs, MEFs-4C, PLCs, and ALCs. MEFs-4C: MEFs were treated with the combination of four small molecules (forskolin [FSK], 20α-hydroxycholesterol, LH, and SB431542) for 21 days. Scale bars, 100 μm. (B) qRT-PCR analysis of key Leydig cell-related genes in MEFs, MEFs-4C, PLCs, and ALCs. (C) Steroidogenic pathway in PLCs and ALCs. (D–F) Production of androsterone (D), testosterone (E), and dihydrotestosterone (F) by MEFs-4C, PLCs, and ALCs. (G) Immunostaining of the Leydig cell markers StAR and HSD3B in MEFs-4C and PLCs. (H) Representative western blotting for the protein expression in MEFs-4C and PLCs. The data were obtained from at least three independent experiments and were presented as mean ± SD values. ns, not significant. See also Figure S4 .
    Figure Legend Snippet: Characteristics of CiLCs (A) Morphology of MEFs, MEFs-4C, PLCs, and ALCs. MEFs-4C: MEFs were treated with the combination of four small molecules (forskolin [FSK], 20α-hydroxycholesterol, LH, and SB431542) for 21 days. Scale bars, 100 μm. (B) qRT-PCR analysis of key Leydig cell-related genes in MEFs, MEFs-4C, PLCs, and ALCs. (C) Steroidogenic pathway in PLCs and ALCs. (D–F) Production of androsterone (D), testosterone (E), and dihydrotestosterone (F) by MEFs-4C, PLCs, and ALCs. (G) Immunostaining of the Leydig cell markers StAR and HSD3B in MEFs-4C and PLCs. (H) Representative western blotting for the protein expression in MEFs-4C and PLCs. The data were obtained from at least three independent experiments and were presented as mean ± SD values. ns, not significant. See also Figure S4 .

    Techniques Used: Quantitative RT-PCR, Immunostaining, Western Blot, Expressing

    4) Product Images from "Essential Oils of Alpinia nantoensis Retard Forskolin-Induced Melanogenesis via ERK1/2-Mediated Proteasomal Degradation of MITF"

    Article Title: Essential Oils of Alpinia nantoensis Retard Forskolin-Induced Melanogenesis via ERK1/2-Mediated Proteasomal Degradation of MITF

    Journal: Plants

    doi: 10.3390/plants9121672

    Inhibitory effect of leaf (LEO) and rhizome (REO) essential oils on melanin content and tyrosinase enzyme activity in forskolin (FRK)-induced B16-F10 melanoma cells. ( A ) Cells were treated with indicated concentrations of LEO, REO, arbutin (AB) and kojic acid (KA) and stimulated with FRK for 48 h. Melanin content was assessed by with an absorbance at 405 nm. Cellular melanin content was calculated by comparison with a melanin standard curve. ( B ) The relative activity of mushroom tyrosinase on L-DOPA in the presence of increasing concentrations of LEO, REO, KA and ascorbic acid (AA), compared to the control (100%). ( C ) Effect of cellular tyrosinase activity was determined using whole cell lysates. After treatment with indicated concentration of LEO, REO, AB and KA for 48 h. Cell lysates were used as enzyme source and L-DOPA as substrate. The effects on L-DOPA oxidation velocity was measured at 492 nm. ( D – F ) Relative mRNA expression levels of tyrosinase, TRP-1 and dopachrome tautomerase (DCT) were determined by Q-PCR analysis. ( G – I ) Protein expression levels of tyrosinase, TRP-1 and DCT were determined by Western blot analysis and the histogram showed relative protein expression, which were normalized with loading control GAPDH. Data represent the mean ± SD of three experiments. Statistical significance was set at # p
    Figure Legend Snippet: Inhibitory effect of leaf (LEO) and rhizome (REO) essential oils on melanin content and tyrosinase enzyme activity in forskolin (FRK)-induced B16-F10 melanoma cells. ( A ) Cells were treated with indicated concentrations of LEO, REO, arbutin (AB) and kojic acid (KA) and stimulated with FRK for 48 h. Melanin content was assessed by with an absorbance at 405 nm. Cellular melanin content was calculated by comparison with a melanin standard curve. ( B ) The relative activity of mushroom tyrosinase on L-DOPA in the presence of increasing concentrations of LEO, REO, KA and ascorbic acid (AA), compared to the control (100%). ( C ) Effect of cellular tyrosinase activity was determined using whole cell lysates. After treatment with indicated concentration of LEO, REO, AB and KA for 48 h. Cell lysates were used as enzyme source and L-DOPA as substrate. The effects on L-DOPA oxidation velocity was measured at 492 nm. ( D – F ) Relative mRNA expression levels of tyrosinase, TRP-1 and dopachrome tautomerase (DCT) were determined by Q-PCR analysis. ( G – I ) Protein expression levels of tyrosinase, TRP-1 and DCT were determined by Western blot analysis and the histogram showed relative protein expression, which were normalized with loading control GAPDH. Data represent the mean ± SD of three experiments. Statistical significance was set at # p

    Techniques Used: Activity Assay, Concentration Assay, Expressing, Polymerase Chain Reaction, Western Blot

    5) Product Images from "Canonical TGFβ signaling induces collective invasion in colorectal carcinogenesis through a Snail1- and Zeb1-independent partial EMT"

    Article Title: Canonical TGFβ signaling induces collective invasion in colorectal carcinogenesis through a Snail1- and Zeb1-independent partial EMT

    Journal: bioRxiv

    doi: 10.1101/2020.12.11.420851

    Oncogenically transformed intestinal organoids display no cell-intrinsic invasiveness in vitro . a , Strategy for generating oncogenically transformed small intestinal and colonic organoids from genetically engineered mice carrying a Villin-CreER T2 transgene, two floxed Apc alleles, as well as heterozygous Kras LSL-G12D/+ and Trp53 LSL-R172H/+ loci. Floxed stop cassettes (LSL) prevent expression of the mutant Kras and Trp53 alleles. For recombination, organoids were treated with 4-hydroxy-tamoxifen (4-OHT), yielding TKA organoids. b , Whole mounts and hematoxylin/eosin (H E) stained sections of floxed and TKA organoids (line 815) cultured in 7 mg/ml Matrigel and visualized by phase contrast (PhC) or bright field microscopy. Scale bars: 200 μm. c , Representative images of immunofluorescence staining of pan-laminin, E-cadherin, atypical protein kinase C (aPKC), and β-catenin in sections of floxed and TKA organoids (line 815) cultured in 7 mg/ml Matrigel. Nuclei were stained by DAPI; n > 3. Scale bars: 50 μm. d, Top: Microscopy of floxed and TKA organoids (line 815) in 7 mg/ml Matrigel at the beginning (0 h) and the end (8 h) of forskolin and DMSO treatment. Scale bar: 200 μm. Bottom: Quantification of forskolin-induced organoid swelling. TKA organoids were exposed to DMSO or forskolin for the indicated time periods. Normalized changes in organoid diameter were calculated by first computing at each time point and for each organoid under consideration the increase in diameter relative to the corresponding value at t=0 h, followed by normalization of forskolin-induced relative changes in diameter to those of DMSO-treated control samples. At least five organoids treated with DMSO or forskolin were analyzed per biological replicate and organoid line. e , Left: set-up for cultivating organoids in type I collagen at an air-liquid-interface and representative H E stainings of organoid displaying different histological features (line 815). Scale bar: 200 μm. Right: Quantification of organoids following histological classification (morib.: moribund; dyspl.: dysplastic). Quantitative experiments in ( d and e ) were performed with three floxed/TKA organoid lines (815: n=3; 931: n=3; 947: n=3). Dots represent results of independent biological replicates and dot color identifies the organoid lines.
    Figure Legend Snippet: Oncogenically transformed intestinal organoids display no cell-intrinsic invasiveness in vitro . a , Strategy for generating oncogenically transformed small intestinal and colonic organoids from genetically engineered mice carrying a Villin-CreER T2 transgene, two floxed Apc alleles, as well as heterozygous Kras LSL-G12D/+ and Trp53 LSL-R172H/+ loci. Floxed stop cassettes (LSL) prevent expression of the mutant Kras and Trp53 alleles. For recombination, organoids were treated with 4-hydroxy-tamoxifen (4-OHT), yielding TKA organoids. b , Whole mounts and hematoxylin/eosin (H E) stained sections of floxed and TKA organoids (line 815) cultured in 7 mg/ml Matrigel and visualized by phase contrast (PhC) or bright field microscopy. Scale bars: 200 μm. c , Representative images of immunofluorescence staining of pan-laminin, E-cadherin, atypical protein kinase C (aPKC), and β-catenin in sections of floxed and TKA organoids (line 815) cultured in 7 mg/ml Matrigel. Nuclei were stained by DAPI; n > 3. Scale bars: 50 μm. d, Top: Microscopy of floxed and TKA organoids (line 815) in 7 mg/ml Matrigel at the beginning (0 h) and the end (8 h) of forskolin and DMSO treatment. Scale bar: 200 μm. Bottom: Quantification of forskolin-induced organoid swelling. TKA organoids were exposed to DMSO or forskolin for the indicated time periods. Normalized changes in organoid diameter were calculated by first computing at each time point and for each organoid under consideration the increase in diameter relative to the corresponding value at t=0 h, followed by normalization of forskolin-induced relative changes in diameter to those of DMSO-treated control samples. At least five organoids treated with DMSO or forskolin were analyzed per biological replicate and organoid line. e , Left: set-up for cultivating organoids in type I collagen at an air-liquid-interface and representative H E stainings of organoid displaying different histological features (line 815). Scale bar: 200 μm. Right: Quantification of organoids following histological classification (morib.: moribund; dyspl.: dysplastic). Quantitative experiments in ( d and e ) were performed with three floxed/TKA organoid lines (815: n=3; 931: n=3; 947: n=3). Dots represent results of independent biological replicates and dot color identifies the organoid lines.

    Techniques Used: Transformation Assay, In Vitro, Mouse Assay, Expressing, Mutagenesis, Staining, Cell Culture, Microscopy, Immunofluorescence

    6) Product Images from "High-Throughput Phenotypic Screening of Kinase Inhibitors to Identify Drug Targets for Polycystic Kidney Disease"

    Article Title: High-Throughput Phenotypic Screening of Kinase Inhibitors to Identify Drug Targets for Polycystic Kidney Disease

    Journal: Slas Discovery

    doi: 10.1177/2472555217716056

    mIMCD3 sh Pkd1 cyst growth can be enhanced with forskolin, and this increase can be prevented with positive control compounds rapamycin, sorafenib, roscovitine, and NVP-BEZ-235. ( A ) High-resolution images obtained from an unexposed (0.2% DMSO, top panel) and a forskolin-treated (bottom panel) cyst. ( B ) Using Ominer analysis tools, cyst size could be expressed as a function of forskolin concentration. Data points represent means ± SD of eight replicate wells (technical replicates). ( C ) Representative images of control compounds after coexposure with forskolin, including unstimulated (0.2% DMSO) control. ( D ) Quantification of average cyst size from C. Whiskers represent min to max. Rapamycin, roscovitine, sorafenib, and NVP-BEZ-235 tested in quadruplicate (technical replicates), unstimulated ( n = 16 replicate wells) and stimulated ( n = 8 replicate wells). These experiments have been performed independently more than three times ( Suppl. Figs. S1B,C and S5A,C also show the performance of control molecules in biological replicates).
    Figure Legend Snippet: mIMCD3 sh Pkd1 cyst growth can be enhanced with forskolin, and this increase can be prevented with positive control compounds rapamycin, sorafenib, roscovitine, and NVP-BEZ-235. ( A ) High-resolution images obtained from an unexposed (0.2% DMSO, top panel) and a forskolin-treated (bottom panel) cyst. ( B ) Using Ominer analysis tools, cyst size could be expressed as a function of forskolin concentration. Data points represent means ± SD of eight replicate wells (technical replicates). ( C ) Representative images of control compounds after coexposure with forskolin, including unstimulated (0.2% DMSO) control. ( D ) Quantification of average cyst size from C. Whiskers represent min to max. Rapamycin, roscovitine, sorafenib, and NVP-BEZ-235 tested in quadruplicate (technical replicates), unstimulated ( n = 16 replicate wells) and stimulated ( n = 8 replicate wells). These experiments have been performed independently more than three times ( Suppl. Figs. S1B,C and S5A,C also show the performance of control molecules in biological replicates).

    Techniques Used: Positive Control, Concentration Assay

    Phenotypic analysis discriminates potentially undesirable compound effects. ( A ) Validation of compounds from each target identified in Figure 3 . Mean percent inhibition (technical replicates representing triplicate wells) of cyst growth is depicted by a color scale from yellow (no inhibition of forskolin-induced cyst growth) to blue (complete inhibition of forskolin-induced cyst growth). Standard deviations are not included in this plot; for reference purposes, several dose curves are included in Supplemental Figure S6 . A mean Z′ factor of +0.36 between stimulated and unstimulated control conditions was calculated over six plates. ( B,C ) Multiparametric (PCA) analysis (PCA plot summarizes 84% of variation in the entire dataset) identifies different compound clusters, as shown by the contour plots. Forskolin-stimulated controls (large cysts) are represented as empty circles; unstimulated controls (small cysts) are represented as black dots. Data points represent single wells. ( C ) PCA plot summarizing 84% of variation in the entire dataset. Trajectories of different compound types are indicated by arrows (mTOR inhibitors, blue; CDK inhibitors, green; DNA-PK inhibitors, red; aurora kinase inhibitors, orange). Data points represent single wells; point size correlates with molecule concentration (legend omitted for presentation purposes). ( D ) Representative images from conditions shown as in panels A–C. Novel phenotypes identified in panels B and C are illustrated by images of 100 nM dinaciclib and 1 µM PIK-75.
    Figure Legend Snippet: Phenotypic analysis discriminates potentially undesirable compound effects. ( A ) Validation of compounds from each target identified in Figure 3 . Mean percent inhibition (technical replicates representing triplicate wells) of cyst growth is depicted by a color scale from yellow (no inhibition of forskolin-induced cyst growth) to blue (complete inhibition of forskolin-induced cyst growth). Standard deviations are not included in this plot; for reference purposes, several dose curves are included in Supplemental Figure S6 . A mean Z′ factor of +0.36 between stimulated and unstimulated control conditions was calculated over six plates. ( B,C ) Multiparametric (PCA) analysis (PCA plot summarizes 84% of variation in the entire dataset) identifies different compound clusters, as shown by the contour plots. Forskolin-stimulated controls (large cysts) are represented as empty circles; unstimulated controls (small cysts) are represented as black dots. Data points represent single wells. ( C ) PCA plot summarizing 84% of variation in the entire dataset. Trajectories of different compound types are indicated by arrows (mTOR inhibitors, blue; CDK inhibitors, green; DNA-PK inhibitors, red; aurora kinase inhibitors, orange). Data points represent single wells; point size correlates with molecule concentration (legend omitted for presentation purposes). ( D ) Representative images from conditions shown as in panels A–C. Novel phenotypes identified in panels B and C are illustrated by images of 100 nM dinaciclib and 1 µM PIK-75.

    Techniques Used: Inhibition, Concentration Assay

    mTOR inhibitors, but not PI3K inhibitors, prevent forskolin-induced cyst swelling. ( A ) PCA plot comprising 84% of variation in the entire dataset. mTOR inhibitors (red circles) cluster together with unstimulated control (black dots), whereas PI3K inhibitors (blue circles) cluster with the stimulated condition (empty circles). Data points represent single wells; inhibitor point size correlates with molecule concentration (legend omitted for presentation purposes). ( B ) Validation of mTOR inhibitors at concentrations of 6–100 nM. Cyst growth inhibition displayed by a color gradient ranging from yellow (no inhibition) to blue (inhibition). The color represents the mean value of triplicate wells (technical replicates; mean Z′ factor over eight plates of 0.28 between stimulated and unstimulated control conditions; also for panels C and D). ( C ) Validation of PI3K inhibitors; color scale as in panel B. ( D ) Validation of dual PI3K/mTOR inhibitors; color scale as in panel B. ( E ) Combination of mTOR inhibitor torin 1 with PI3K inhibitor buparlisib (NVP-BKM120) to assess synergy in mIMRFNPKD 5E4 cells. Values represent mean ± SD of quadruplicate wells (technical replicates; Z′ factor of +0.64 between stimulated and unstimulated control conditions).
    Figure Legend Snippet: mTOR inhibitors, but not PI3K inhibitors, prevent forskolin-induced cyst swelling. ( A ) PCA plot comprising 84% of variation in the entire dataset. mTOR inhibitors (red circles) cluster together with unstimulated control (black dots), whereas PI3K inhibitors (blue circles) cluster with the stimulated condition (empty circles). Data points represent single wells; inhibitor point size correlates with molecule concentration (legend omitted for presentation purposes). ( B ) Validation of mTOR inhibitors at concentrations of 6–100 nM. Cyst growth inhibition displayed by a color gradient ranging from yellow (no inhibition) to blue (inhibition). The color represents the mean value of triplicate wells (technical replicates; mean Z′ factor over eight plates of 0.28 between stimulated and unstimulated control conditions; also for panels C and D). ( C ) Validation of PI3K inhibitors; color scale as in panel B. ( D ) Validation of dual PI3K/mTOR inhibitors; color scale as in panel B. ( E ) Combination of mTOR inhibitor torin 1 with PI3K inhibitor buparlisib (NVP-BKM120) to assess synergy in mIMRFNPKD 5E4 cells. Values represent mean ± SD of quadruplicate wells (technical replicates; Z′ factor of +0.64 between stimulated and unstimulated control conditions).

    Techniques Used: Concentration Assay, Inhibition

    SelleckChem kinase inhibitor library screen. Kinase inhibitors were screened in quadruplicate (technical replicates) at 1 and 0.1 µM, in the presence of 2.5 µM forskolin, to stimulate cyst growth (procedure shown in Fig. 1 ). Data were z score normalized to the plate median and subsequently to the unstimulated control median (unstimulated control, black striped line). Cyst growth induction by forskolin is presented by a red striped line. Compound effects are represented by means of quadruplicate wells of two tested concentrations. Top 15 hit molecules are presented in the right cutout. The replicate-adjusted Z′ factor for this primary screen reached −0.93 between stimulated and unstimulated control conditions.
    Figure Legend Snippet: SelleckChem kinase inhibitor library screen. Kinase inhibitors were screened in quadruplicate (technical replicates) at 1 and 0.1 µM, in the presence of 2.5 µM forskolin, to stimulate cyst growth (procedure shown in Fig. 1 ). Data were z score normalized to the plate median and subsequently to the unstimulated control median (unstimulated control, black striped line). Cyst growth induction by forskolin is presented by a red striped line. Compound effects are represented by means of quadruplicate wells of two tested concentrations. Top 15 hit molecules are presented in the right cutout. The replicate-adjusted Z′ factor for this primary screen reached −0.93 between stimulated and unstimulated control conditions.

    Techniques Used:

    7) Product Images from "mTORC1-Mediated Inhibition of 4EBP1 is Essential for Hedgehog Signaling-Driven Translation and Medulloblastoma"

    Article Title: mTORC1-Mediated Inhibition of 4EBP1 is Essential for Hedgehog Signaling-Driven Translation and Medulloblastoma

    Journal: Developmental cell

    doi: 10.1016/j.devcel.2017.10.011

    HH signaling increases SMO protein levels via a noncanonical pathway (A) Schematic showing a simplified HH signaling pathway with reagents used to modulate the signaling. (B) SAG increased GLI1 and SMO protein levels in GNPs in culture within 24 h. The bars show the fold changes in the SMO levels relative to those in untreated cells (SAG − ) (mean ± SD, n=3). (C, D) Western blots and quantifications of GLI1 and SMO protein levels in NIH 3T3 cells at the indicated times after SHH treatment. The bars show the fold changes in the protein levels relative to those in untreated cells (mean ± SD, n=3). (E) SMO and GLI1 protein levels in NIH 3T3 cells stimulated with SHH or FBS in the presence or absence of SANT-1. (F) SAG-induced GLI1 and SMO protein levels in NIH 3T3 cells were suppressed in cells expressing shRNA against Ift88 ( shIft88 ) or Smo ( shSmo ). (G) GLI1 and SMO protein levels in NIH 3T3 cells expressing shSufu at the indicated times after SHH treatment. Note that SMO, but not GLI1, was induced by SHH. (H) Forskolin blocked SAG from increasing GLI1 but not SMO protein levels. (I) SAG increased Gli1 but not Smo mRNA levels in NIH 3T3 cells. The bars show the fold changes in the mRNA levels relative to those in untreated cells (mean ± SD, n=3). * P
    Figure Legend Snippet: HH signaling increases SMO protein levels via a noncanonical pathway (A) Schematic showing a simplified HH signaling pathway with reagents used to modulate the signaling. (B) SAG increased GLI1 and SMO protein levels in GNPs in culture within 24 h. The bars show the fold changes in the SMO levels relative to those in untreated cells (SAG − ) (mean ± SD, n=3). (C, D) Western blots and quantifications of GLI1 and SMO protein levels in NIH 3T3 cells at the indicated times after SHH treatment. The bars show the fold changes in the protein levels relative to those in untreated cells (mean ± SD, n=3). (E) SMO and GLI1 protein levels in NIH 3T3 cells stimulated with SHH or FBS in the presence or absence of SANT-1. (F) SAG-induced GLI1 and SMO protein levels in NIH 3T3 cells were suppressed in cells expressing shRNA against Ift88 ( shIft88 ) or Smo ( shSmo ). (G) GLI1 and SMO protein levels in NIH 3T3 cells expressing shSufu at the indicated times after SHH treatment. Note that SMO, but not GLI1, was induced by SHH. (H) Forskolin blocked SAG from increasing GLI1 but not SMO protein levels. (I) SAG increased Gli1 but not Smo mRNA levels in NIH 3T3 cells. The bars show the fold changes in the mRNA levels relative to those in untreated cells (mean ± SD, n=3). * P

    Techniques Used: Western Blot, Expressing, shRNA

    8) Product Images from "Forskolin exerts anticancer roles in non-Hodgkin’s lymphomas via regulating Axin/β-catenin signaling pathway"

    Article Title: Forskolin exerts anticancer roles in non-Hodgkin’s lymphomas via regulating Axin/β-catenin signaling pathway

    Journal: Cancer Management and Research

    doi: 10.2147/CMAR.S180754

    Forskolin reduced the nuclear accumulation of β-catenin and the expression of c-myc and cyclin D1. Notes: ( A, B ) The protein expression of cytoplasmic and nuclear β-catenin in Toledo and NK-92 cells in the presence of 0, 10, 20, or 40 μM forskolin was detected by Western blotting. ( C, D ) Western blotting analysis of the total protein expression of c-myc and cyclin D1 influenced by 0, 10, 20, or 40 μM of forskolin in Toledo and NK-92 cells (n=3, * P
    Figure Legend Snippet: Forskolin reduced the nuclear accumulation of β-catenin and the expression of c-myc and cyclin D1. Notes: ( A, B ) The protein expression of cytoplasmic and nuclear β-catenin in Toledo and NK-92 cells in the presence of 0, 10, 20, or 40 μM forskolin was detected by Western blotting. ( C, D ) Western blotting analysis of the total protein expression of c-myc and cyclin D1 influenced by 0, 10, 20, or 40 μM of forskolin in Toledo and NK-92 cells (n=3, * P

    Techniques Used: Expressing, Western Blot

    Forskolin reduced the expression of β-catenin through upregulation of the expression of Axin. Notes: ( A, B ) The expressions of Axin, Frz, APC, GSK-3β, and CK1 in forskolin (40 μM)-treated or control Toledo and NK-92 cells were examined by Western blotting analysis (n=3, * P
    Figure Legend Snippet: Forskolin reduced the expression of β-catenin through upregulation of the expression of Axin. Notes: ( A, B ) The expressions of Axin, Frz, APC, GSK-3β, and CK1 in forskolin (40 μM)-treated or control Toledo and NK-92 cells were examined by Western blotting analysis (n=3, * P

    Techniques Used: Expressing, Western Blot

    Forskolin promoted the protein expression of cleaved caspase-3/9 and repressed cell growth in NHL cells. Notes: ( A, B ) Toledo, NK-92, RL, and Farage cells were treated with 30 μM of forskolin for 48 hours, then the cells were harvested for Western blotting to assess the expression of total or cleaved caspase-3/9. ( C ) MTT assay was conducted to test the cell growth after Toledo cells were administered 0, 20, 40, 80, or 160 μM of forskolin for 1, 2, 3, 4, 5, or 6 days. ( D ) Toledo cells were administered 0, 20, 40, 80, or 160 μM of forskolin for 48 hours, then MTT assay was used to evaluate the IC 50 of forskolin. ( E ) Toledo cells were incubated with 0, 20, 40, 80, or 160 μM of forskolin for 48 hours, then Western blotting analysis was carried out to detect the expression of total or cleaved caspase-3/9. ( F ) MTT assay was conducted to test the cell growth after NK-92 cells were administered 0, 20, 40, 80, or 160 μM of forskolin for 1, 2, 3, 4, 5, or 6 days. ( G ) NK-92 cells were administered 0, 20, 40, 80, or 160 μM of fors kolin for 48 hours, then MTT assay was used to evaluate the IC 50 of forskolin. ( H ) NK-92 cells were incubated with 0, 20, 40, 80, or 160 μM of forskolin for 48 hours, then Western blotting analysis was carried out to detect the expression of total or cleaved caspase-3/9 (n=3, * P
    Figure Legend Snippet: Forskolin promoted the protein expression of cleaved caspase-3/9 and repressed cell growth in NHL cells. Notes: ( A, B ) Toledo, NK-92, RL, and Farage cells were treated with 30 μM of forskolin for 48 hours, then the cells were harvested for Western blotting to assess the expression of total or cleaved caspase-3/9. ( C ) MTT assay was conducted to test the cell growth after Toledo cells were administered 0, 20, 40, 80, or 160 μM of forskolin for 1, 2, 3, 4, 5, or 6 days. ( D ) Toledo cells were administered 0, 20, 40, 80, or 160 μM of forskolin for 48 hours, then MTT assay was used to evaluate the IC 50 of forskolin. ( E ) Toledo cells were incubated with 0, 20, 40, 80, or 160 μM of forskolin for 48 hours, then Western blotting analysis was carried out to detect the expression of total or cleaved caspase-3/9. ( F ) MTT assay was conducted to test the cell growth after NK-92 cells were administered 0, 20, 40, 80, or 160 μM of forskolin for 1, 2, 3, 4, 5, or 6 days. ( G ) NK-92 cells were administered 0, 20, 40, 80, or 160 μM of fors kolin for 48 hours, then MTT assay was used to evaluate the IC 50 of forskolin. ( H ) NK-92 cells were incubated with 0, 20, 40, 80, or 160 μM of forskolin for 48 hours, then Western blotting analysis was carried out to detect the expression of total or cleaved caspase-3/9 (n=3, * P

    Techniques Used: Expressing, Western Blot, MTT Assay, Incubation

    Forskolin/SP600125 reduced tumor growth through downregulating β-catenin in vivo. Notes: ( A ) Mouse tumor-bearing assay was used to evaluate the tumorigenesis of NK-92 cells with different treatments. ( B ) Immunohistochemical analysis of the expression of ki-67 in the tumor tissues from different groups in NK-92 cells (scale bar =100 μm) (n=6, * P
    Figure Legend Snippet: Forskolin/SP600125 reduced tumor growth through downregulating β-catenin in vivo. Notes: ( A ) Mouse tumor-bearing assay was used to evaluate the tumorigenesis of NK-92 cells with different treatments. ( B ) Immunohistochemical analysis of the expression of ki-67 in the tumor tissues from different groups in NK-92 cells (scale bar =100 μm) (n=6, * P

    Techniques Used: In Vivo, Immunohistochemistry, Expressing

    SP600125 enhanced cell apoptosis induced by forskolin in NHL cells. Notes: ( A, B ) The expression of the total or cleaved caspase-3/9 was decided by Western blotting after Toledo or NK-92 cells were treated with SP600125 (20 μM) or not (n=3, ** P
    Figure Legend Snippet: SP600125 enhanced cell apoptosis induced by forskolin in NHL cells. Notes: ( A, B ) The expression of the total or cleaved caspase-3/9 was decided by Western blotting after Toledo or NK-92 cells were treated with SP600125 (20 μM) or not (n=3, ** P

    Techniques Used: Expressing, Western Blot

    Forskolin reduced β-catenin expression while it increased its ubiquitination. Notes: Toledo and NK-92 cells were incubated with 0, 20, 40, 80, or 160 μM of forskolin for 48 hours, then ( A, B ) Western blotting was recruited to determine the expression of YAP1, β-catenin, AKT, p-AKT, STAT, and p-STAT, and ( C ) IP assay was used to examine the expression of ubiquitin, which binds to β-catenin protein. (n=3, * P
    Figure Legend Snippet: Forskolin reduced β-catenin expression while it increased its ubiquitination. Notes: Toledo and NK-92 cells were incubated with 0, 20, 40, 80, or 160 μM of forskolin for 48 hours, then ( A, B ) Western blotting was recruited to determine the expression of YAP1, β-catenin, AKT, p-AKT, STAT, and p-STAT, and ( C ) IP assay was used to examine the expression of ubiquitin, which binds to β-catenin protein. (n=3, * P

    Techniques Used: Expressing, Incubation, Western Blot

    Forskolin increased the stability of Axin protein and reduced its ubiquitylation. Notes: ( A ) Toledo and NK-92 cells were treated with 0, 10, 20, or 40 μM of forskolin for 48 hours, then cells were collected for Western blotting analysis to determine the expression of Axin. ( B ) Toledo and NK-92 cells were treated with 0, 10, 20, or 40 μM of forskolin for 48 hours, then the culture medium was replaced with CHX (100 mg/mL) and incubated for 0, 1, 2, 4, 8, and 24 hours, next cells were harvested for Western blotting analysis with antibody against Axin. ( C, D ) Bar graph of the expression of Axin related to Figure 5B . ( E ) IP assay was used to analyze the expression of Ub combining with Axin protein in the presence or absence of forskolin (40 μM) (n=3, * P
    Figure Legend Snippet: Forskolin increased the stability of Axin protein and reduced its ubiquitylation. Notes: ( A ) Toledo and NK-92 cells were treated with 0, 10, 20, or 40 μM of forskolin for 48 hours, then cells were collected for Western blotting analysis to determine the expression of Axin. ( B ) Toledo and NK-92 cells were treated with 0, 10, 20, or 40 μM of forskolin for 48 hours, then the culture medium was replaced with CHX (100 mg/mL) and incubated for 0, 1, 2, 4, 8, and 24 hours, next cells were harvested for Western blotting analysis with antibody against Axin. ( C, D ) Bar graph of the expression of Axin related to Figure 5B . ( E ) IP assay was used to analyze the expression of Ub combining with Axin protein in the presence or absence of forskolin (40 μM) (n=3, * P

    Techniques Used: Western Blot, Expressing, Incubation

    9) Product Images from "Direct conversion of human fibroblasts into dopaminergic neuron-like cells using small molecules and protein factors"

    Article Title: Direct conversion of human fibroblasts into dopaminergic neuron-like cells using small molecules and protein factors

    Journal: Military Medical Research

    doi: 10.1186/s40779-020-00284-2

    Chemical induction of human lung fetal IMR-90 fibroblasts into neuron-like cells. a A schematic diagram describing the chemical induction procedure. V, VPA; R, Repsox; K, kenpaullone; F, forskolin; Y, Y-27632; P, purmorphamine; L, L-ascorbic acid. b Representative microscopy images depicting the morphological changes after chemical induction. Scale bars=100 μm. c Characterization of the cultured IMR-90 fibroblasts by immunostaining assay for the fibroblast marker VIMENTIN, neural markers Class III β-tubulin 1 (TUJ1) and microtubule-associated protein 2 (MAP2), neural progenitor/stem cell markers PAX6, SOX2, and NESTIN, glial cell marker GFAP, and neural crest cell marker p75. Scale bars=25 μm. d Expression of TUJ1 after 7 days of chemical inductions. Scale bar = 25 μm. e Dynamic changes of VIMENTIN and TUJ1 in fibroblasts at early 12 h and 24 h of chemical induction. Scale bars=25 μm. f Dynamic changes of VIMENTIN and TUJ1 at early 24 h and 72 h in the control medium with small molecules and protein factors. Scale bars=25 μm
    Figure Legend Snippet: Chemical induction of human lung fetal IMR-90 fibroblasts into neuron-like cells. a A schematic diagram describing the chemical induction procedure. V, VPA; R, Repsox; K, kenpaullone; F, forskolin; Y, Y-27632; P, purmorphamine; L, L-ascorbic acid. b Representative microscopy images depicting the morphological changes after chemical induction. Scale bars=100 μm. c Characterization of the cultured IMR-90 fibroblasts by immunostaining assay for the fibroblast marker VIMENTIN, neural markers Class III β-tubulin 1 (TUJ1) and microtubule-associated protein 2 (MAP2), neural progenitor/stem cell markers PAX6, SOX2, and NESTIN, glial cell marker GFAP, and neural crest cell marker p75. Scale bars=25 μm. d Expression of TUJ1 after 7 days of chemical inductions. Scale bar = 25 μm. e Dynamic changes of VIMENTIN and TUJ1 in fibroblasts at early 12 h and 24 h of chemical induction. Scale bars=25 μm. f Dynamic changes of VIMENTIN and TUJ1 at early 24 h and 72 h in the control medium with small molecules and protein factors. Scale bars=25 μm

    Techniques Used: Microscopy, Cell Culture, Immunostaining, Marker, Expressing

    10) Product Images from "cAMP regulates 11β-hydroxysteroid dehydrogenase-2 and Sp1 expression in MLO-Y4/MC3T3-E1 cells"

    Article Title: cAMP regulates 11β-hydroxysteroid dehydrogenase-2 and Sp1 expression in MLO-Y4/MC3T3-E1 cells

    Journal: Experimental and Therapeutic Medicine

    doi: 10.3892/etm.2020.8942

    Effects of forskolin and SQ22536 on MLO-Y4 and MC3T3-E1 cell viability. The NC group was treated with 0 µmol forskolin or SQ22536. (A) Effect of forskolin on MLO-Y4 and MC3T3-E1 cell viability. Compared with the NC group, the 100 µmol forskolin group exhibited the most significant increase in MLO-Y4 cell viability (P
    Figure Legend Snippet: Effects of forskolin and SQ22536 on MLO-Y4 and MC3T3-E1 cell viability. The NC group was treated with 0 µmol forskolin or SQ22536. (A) Effect of forskolin on MLO-Y4 and MC3T3-E1 cell viability. Compared with the NC group, the 100 µmol forskolin group exhibited the most significant increase in MLO-Y4 cell viability (P

    Techniques Used:

    11) Product Images from "Orkambi® and amplifier co‐therapy improves function from a rare CFTR mutation in gene‐edited cells and patient tissue"

    Article Title: Orkambi® and amplifier co‐therapy improves function from a rare CFTR mutation in gene‐edited cells and patient tissue

    Journal: EMBO Molecular Medicine

    doi: 10.15252/emmm.201607137

    Small molecule correctors of ΔF508‐CFTR partially rescue processing and conformational defect of ΔI1234_R1239‐CFTR in the HEK‐293 expression system Immunoblots of steady‐state expression of ΔI1234_R1239‐CFTR following treatments with ΔF508‐CFTR modulators. Class 1 and Class 2 correctors are labeled in red and blue, respectively. ΔF508‐CFTR and WT‐CFTR are included as controls. Quantitation of maturation by most efficacious modulators; VX‐809 was used as a representative Class 1 corrector. Maturation was benchmarked to ΔF508‐CFTR and WT‐CFTR (mean ± SEM, n = 3 biological replicates, and statistical significance tested using two‐way ANOVA with Tukey's multiple comparisons test). For ΔF508‐CFTR: DMSO versus VX‐809, ** P = 0.0046. For ΔI1234_R1239‐CFTR: DMSO versus VX‐809, ** P = 0.0032; DMSO versus C4, *** P = 0.0009; DMSO versus VX‐809 + C4, *** P = 0.0005. For ΔF508‐CFTR (DMSO) versus ΔI1234_R1239‐CFTR (DMSO), ** P = 0.0022. Quantitation of rate of activation (chloride efflux assay) of ΔI1234_R1239‐CFTR following chronic treatment with ΔF508‐CFTR correctors and acute activation (forskolin/IBMX). Activation rate was benchmarked to ΔF508‐CFTR and WT‐CFTR (mean ± SEM, n = 4 biological replicates, and statistical significance tested using two‐way ANOVA with Tukey's multiple comparisons test). For ΔF508‐CFTR: DMSO versus VX‐809, *** P = 0.0003. For ΔI1234_R1239‐CFTR: DMSO versus VX‐809, ** P = 0.0018; DMSO versus C4, * P = 0.0408; DMSO versus VX‐809 + C4, * P = 0.0199. For ΔF508‐CFTR (VX‐809) versus ΔI1234_R1239‐CFTR (VX‐809), * P = 0.0222. Immunoblots of proteolytic digestion of full‐length WT‐CFTR, ΔF508‐CFTR and ΔI1234_R1239‐CFTR. Band B, black arrowhead; band C, white arrowhead. Square brackets denote NBD1 and NBD2 fragments of interest, and those generated from ΔI1234_R1239‐CFTR were quantitatively analyzed in panel (G). Quantitation of proteolytic susceptibility of full‐length WT‐CFTR, ΔF508‐CFTR and ΔI1234_R1239‐CFTR (mean ± SEM, n = 3 biological replicates, and statistical significance tested using unpaired t ‐tests). For 1.6 μg/ml trypsin: ΔF508‐CFTR versus ΔI1234_R1239‐CFTR, * P = 0.0206. Quantitation of proteolytic susceptibility of ΔI1234_R1239‐CFTR in the absence (DMSO) or presence of small molecule correctors of ΔF508‐CFTR (i.e., VX‐809, C4, or VX‐809 + C4; mean ± SEM, n = 3 biological replicates, and statistical significance tested using unpaired t ‐tests). For ΔI1234_R1239‐CFTR (3.1 μg/ml trypsin): DMSO versus VX‐809, * P = 0.0151; DMSO versus C4, * P = 0.0458; DMSO versus VX‐809 + C4, * P = 0.0422. For ΔI1234_R1239‐CFTR (6.3 μg/ml trypsin): DMSO versus VX‐809, * P = 0.0412; DMSO versus C4, * P = 0.0233; DMSO versus VX‐809 + C4, * P = 0.0308. Quantitation of proteolytic digestion of NBD1 and NBD2 fragments of ΔI1234_R1239‐CFTR in the presence of small molecule correctors (VX‐809, C4, or VX‐809 + C4; mean ± SEM, n = 3 biological replicates, and statistical significance tested using two‐way ANOVA with Tukey's multiple comparisons test). For NBD1: DMSO versus C4, * P = 0.0141; DMSO versus VX‐809 + C4, * P = 0.0200; C4 versus VX‐809 + C4, * P = 0.0353.
    Figure Legend Snippet: Small molecule correctors of ΔF508‐CFTR partially rescue processing and conformational defect of ΔI1234_R1239‐CFTR in the HEK‐293 expression system Immunoblots of steady‐state expression of ΔI1234_R1239‐CFTR following treatments with ΔF508‐CFTR modulators. Class 1 and Class 2 correctors are labeled in red and blue, respectively. ΔF508‐CFTR and WT‐CFTR are included as controls. Quantitation of maturation by most efficacious modulators; VX‐809 was used as a representative Class 1 corrector. Maturation was benchmarked to ΔF508‐CFTR and WT‐CFTR (mean ± SEM, n = 3 biological replicates, and statistical significance tested using two‐way ANOVA with Tukey's multiple comparisons test). For ΔF508‐CFTR: DMSO versus VX‐809, ** P = 0.0046. For ΔI1234_R1239‐CFTR: DMSO versus VX‐809, ** P = 0.0032; DMSO versus C4, *** P = 0.0009; DMSO versus VX‐809 + C4, *** P = 0.0005. For ΔF508‐CFTR (DMSO) versus ΔI1234_R1239‐CFTR (DMSO), ** P = 0.0022. Quantitation of rate of activation (chloride efflux assay) of ΔI1234_R1239‐CFTR following chronic treatment with ΔF508‐CFTR correctors and acute activation (forskolin/IBMX). Activation rate was benchmarked to ΔF508‐CFTR and WT‐CFTR (mean ± SEM, n = 4 biological replicates, and statistical significance tested using two‐way ANOVA with Tukey's multiple comparisons test). For ΔF508‐CFTR: DMSO versus VX‐809, *** P = 0.0003. For ΔI1234_R1239‐CFTR: DMSO versus VX‐809, ** P = 0.0018; DMSO versus C4, * P = 0.0408; DMSO versus VX‐809 + C4, * P = 0.0199. For ΔF508‐CFTR (VX‐809) versus ΔI1234_R1239‐CFTR (VX‐809), * P = 0.0222. Immunoblots of proteolytic digestion of full‐length WT‐CFTR, ΔF508‐CFTR and ΔI1234_R1239‐CFTR. Band B, black arrowhead; band C, white arrowhead. Square brackets denote NBD1 and NBD2 fragments of interest, and those generated from ΔI1234_R1239‐CFTR were quantitatively analyzed in panel (G). Quantitation of proteolytic susceptibility of full‐length WT‐CFTR, ΔF508‐CFTR and ΔI1234_R1239‐CFTR (mean ± SEM, n = 3 biological replicates, and statistical significance tested using unpaired t ‐tests). For 1.6 μg/ml trypsin: ΔF508‐CFTR versus ΔI1234_R1239‐CFTR, * P = 0.0206. Quantitation of proteolytic susceptibility of ΔI1234_R1239‐CFTR in the absence (DMSO) or presence of small molecule correctors of ΔF508‐CFTR (i.e., VX‐809, C4, or VX‐809 + C4; mean ± SEM, n = 3 biological replicates, and statistical significance tested using unpaired t ‐tests). For ΔI1234_R1239‐CFTR (3.1 μg/ml trypsin): DMSO versus VX‐809, * P = 0.0151; DMSO versus C4, * P = 0.0458; DMSO versus VX‐809 + C4, * P = 0.0422. For ΔI1234_R1239‐CFTR (6.3 μg/ml trypsin): DMSO versus VX‐809, * P = 0.0412; DMSO versus C4, * P = 0.0233; DMSO versus VX‐809 + C4, * P = 0.0308. Quantitation of proteolytic digestion of NBD1 and NBD2 fragments of ΔI1234_R1239‐CFTR in the presence of small molecule correctors (VX‐809, C4, or VX‐809 + C4; mean ± SEM, n = 3 biological replicates, and statistical significance tested using two‐way ANOVA with Tukey's multiple comparisons test). For NBD1: DMSO versus C4, * P = 0.0141; DMSO versus VX‐809 + C4, * P = 0.0200; C4 versus VX‐809 + C4, * P = 0.0353.

    Techniques Used: Expressing, Western Blot, Labeling, Quantitation Assay, Activation Assay, Generated

    Nasal epithelial cultures derived from siblings homozygous for the c.3700 A > G mutation exhibit low CFTR channel function relative to non‐CF family members and modest rescue by VX‐809 and VX‐770 Pedigree of the relationship of family members toward the index patients and their genotype status. Relative generation levels are marked as I and II; the proband is marked with an arrow; heterozygote carriers are represented by a half‐filled square (father), circle (mother), or diamond (three siblings), while homozygous WT (two siblings) or c.3700 A > G individuals (CF‐1 and CF‐2) are unfilled or filled, respectively. A representative tracing shows Ussing chamber measurements (symmetrical chloride concentrations) of CFTR function in nasal cell cultures from the non‐CF father. Summary of CFTR‐dependent activation (black, forskolin alone for non‐CF individuals, forskolin plus VX‐770 for CF patients) and inhibition (gray, CFTRinh172) responses for several family members (mean ± SEM). Representative tracings show Ussing chamber measurements of transepithelial potential difference traces of CFTR function in nasal cell cultures from affected patients (CF‐1 and CF‐2) in the absence or presence of the small molecule corrector VX‐809. Bar graph showing mean (± SEM) forskolin and VX‐770 (1 μM) activated Ieq for nasal cultures from CF‐1 and CF‐2 after pre‐treatment with VX‐809 (48 h, 3 μM); replicate measurements of n = 6 different cultures (CF‐1) and n = 5 (CF‐2). The VX‐809 and VX‐770 rescued function for CF‐1 remains significantly less than that measured in similarly treated nasal cultures derived from CF patients homozygous for ΔF508 (nasal cultures from six different patients donating to CFIT (CF Canada and SickKids Foundation sponsored program, box and whisker plots; middle lines indicate the median, box ranges indicate the lower and upper quartiles, and whiskers indicate the lowest and highest values)). Statistical significance of comparisons assessed using unpaired t ‐tests. Representative tracing shows Ussing chamber measurements of calcium‐activated chloride channel (CaCC) activity in nasal cell cultures from CF‐2 in response to the P2Y2 receptor agonist ATP.
    Figure Legend Snippet: Nasal epithelial cultures derived from siblings homozygous for the c.3700 A > G mutation exhibit low CFTR channel function relative to non‐CF family members and modest rescue by VX‐809 and VX‐770 Pedigree of the relationship of family members toward the index patients and their genotype status. Relative generation levels are marked as I and II; the proband is marked with an arrow; heterozygote carriers are represented by a half‐filled square (father), circle (mother), or diamond (three siblings), while homozygous WT (two siblings) or c.3700 A > G individuals (CF‐1 and CF‐2) are unfilled or filled, respectively. A representative tracing shows Ussing chamber measurements (symmetrical chloride concentrations) of CFTR function in nasal cell cultures from the non‐CF father. Summary of CFTR‐dependent activation (black, forskolin alone for non‐CF individuals, forskolin plus VX‐770 for CF patients) and inhibition (gray, CFTRinh172) responses for several family members (mean ± SEM). Representative tracings show Ussing chamber measurements of transepithelial potential difference traces of CFTR function in nasal cell cultures from affected patients (CF‐1 and CF‐2) in the absence or presence of the small molecule corrector VX‐809. Bar graph showing mean (± SEM) forskolin and VX‐770 (1 μM) activated Ieq for nasal cultures from CF‐1 and CF‐2 after pre‐treatment with VX‐809 (48 h, 3 μM); replicate measurements of n = 6 different cultures (CF‐1) and n = 5 (CF‐2). The VX‐809 and VX‐770 rescued function for CF‐1 remains significantly less than that measured in similarly treated nasal cultures derived from CF patients homozygous for ΔF508 (nasal cultures from six different patients donating to CFIT (CF Canada and SickKids Foundation sponsored program, box and whisker plots; middle lines indicate the median, box ranges indicate the lower and upper quartiles, and whiskers indicate the lowest and highest values)). Statistical significance of comparisons assessed using unpaired t ‐tests. Representative tracing shows Ussing chamber measurements of calcium‐activated chloride channel (CaCC) activity in nasal cell cultures from CF‐2 in response to the P2Y2 receptor agonist ATP.

    Techniques Used: Derivative Assay, Mutagenesis, Activation Assay, Inhibition, Whisker Assay, Activity Assay

    VX‐809 and VX‐770 partially rescue functional expression of ΔI1234_R1239‐CFTR in a HEK‐293 expression system Representative traces (membrane depolarization assay) of ΔI1234_R1239‐CFTR (ΔIR) and ΔF508‐CFTR (ΔF) function following chronic treatment with VX‐809 and acute activation (forskolin/VX‐770). Quantitation of relative activated responses of ΔI1234_R1239‐CFTR and ΔF508‐CFTR (mean ± SEM, n = 3 biological replicates). Activation of WT‐CFTR by forskolin (without added compound, n = 3, shown for comparison). Statistical significance tested using two‐way ANOVA with Tukey's multiple comparisons test.
    Figure Legend Snippet: VX‐809 and VX‐770 partially rescue functional expression of ΔI1234_R1239‐CFTR in a HEK‐293 expression system Representative traces (membrane depolarization assay) of ΔI1234_R1239‐CFTR (ΔIR) and ΔF508‐CFTR (ΔF) function following chronic treatment with VX‐809 and acute activation (forskolin/VX‐770). Quantitation of relative activated responses of ΔI1234_R1239‐CFTR and ΔF508‐CFTR (mean ± SEM, n = 3 biological replicates). Activation of WT‐CFTR by forskolin (without added compound, n = 3, shown for comparison). Statistical significance tested using two‐way ANOVA with Tukey's multiple comparisons test.

    Techniques Used: Functional Assay, Expressing, Activation Assay, Quantitation Assay

    HBE cells edited by CRISPR/Cas9 to express ΔI1234_R1239‐CFTR recapitulate defects observed in patient‐derived nasal epithelial cultures and provide a model for functional repair using novel small molecule amplifiers Left panel: Immunoblot of steady‐state expression of WT‐CFTR in parental HBE cells and ΔI1234_R1239‐CFTR (ΔIR) in gene‐edited cells. CNX is used as a loading control. Right panel: Immunoblot of steady‐state expression of ΔI1234_R1239‐CFTR (ΔIR) in edited cells and patient‐derived nasal epithelial tissue (CF‐1). CNX is used as a loading control. Band B, black arrowhead; band C, white arrowhead. Representative traces (membrane depolarization assay) of WT‐CFTR, ΔI1234_R1239‐CFTR (ΔIR), and CFTR knockout (KO) HBE cell lines; ΔI1234_R1239‐CFTR function is measured following chronic treatment with VX‐809. Black arrow, acute activation (forskolin + VX‐770); gray arrow, acute inhibition (CFTRinh‐172). Inset shows comparison of mean ± SEM change in membrane potential with activation in edited cells expressing WT‐CFTR ( n = 4 platings, 4 technical replicates) or the mutant (ΔIR, n = 9 platings, 4 technical replicates). Statistical significance of comparisons assessed by unpaired t ‐test. ΔI1234_R1239‐CFTR mRNA levels in HBE‐ΔIR after 24‐h treatment with vehicle (DMSO), PTI‐CH (1 μM), or DMSO or PTI‐CH in the presence of α‐amanitin (50 μg/ml) to inhibit transcription (mean ± SEM, n = 4). Statistical significance tested using two‐way ANOVA with Tukey's multiple comparisons test. Immunoblot of steady‐state CFTR expression levels in HBE‐KO (DMSO) and HBE‐ΔIR cells following 24‐h treatment with vehicle (DMSO), VX‐809 (3 μM), PTI‐CH (1 μM), or VX‐809 + PTI‐CH. CNX was used as a loading control. Band B, black arrowhead; band C, white arrowhead. Changes in band B or band C abundance after VX‐809 plus PTI‐CH pre‐treatment were greater than those measured with VX‐809 alone (mean ± SEM n = 3 biological replicates, significance assessed using paired t ‐test). Representative traces (membrane depolarization assay) of ΔI1234_R1239‐CFTR function in HBE‐ΔIR cells following 24‐h treatment with vehicle (DMSO), VX‐809, or VX‐809 + PTI‐CH. Black arrow, acute activation (forskolin + VX‐770); gray arrow, acute inhibition (CFTRinh‐172). Quantitation (mean ± SEM) of peak responses to acute activation of ΔI1234_R1239‐CFTR in HBE‐ΔIR cells pretreated with DMSO ( n = 8), VX‐809 alone ( n = 8) or VX‐809 plus PTI‐CH ( n = 4). Statistical significance of comparisons assessed using unpaired t ‐test.
    Figure Legend Snippet: HBE cells edited by CRISPR/Cas9 to express ΔI1234_R1239‐CFTR recapitulate defects observed in patient‐derived nasal epithelial cultures and provide a model for functional repair using novel small molecule amplifiers Left panel: Immunoblot of steady‐state expression of WT‐CFTR in parental HBE cells and ΔI1234_R1239‐CFTR (ΔIR) in gene‐edited cells. CNX is used as a loading control. Right panel: Immunoblot of steady‐state expression of ΔI1234_R1239‐CFTR (ΔIR) in edited cells and patient‐derived nasal epithelial tissue (CF‐1). CNX is used as a loading control. Band B, black arrowhead; band C, white arrowhead. Representative traces (membrane depolarization assay) of WT‐CFTR, ΔI1234_R1239‐CFTR (ΔIR), and CFTR knockout (KO) HBE cell lines; ΔI1234_R1239‐CFTR function is measured following chronic treatment with VX‐809. Black arrow, acute activation (forskolin + VX‐770); gray arrow, acute inhibition (CFTRinh‐172). Inset shows comparison of mean ± SEM change in membrane potential with activation in edited cells expressing WT‐CFTR ( n = 4 platings, 4 technical replicates) or the mutant (ΔIR, n = 9 platings, 4 technical replicates). Statistical significance of comparisons assessed by unpaired t ‐test. ΔI1234_R1239‐CFTR mRNA levels in HBE‐ΔIR after 24‐h treatment with vehicle (DMSO), PTI‐CH (1 μM), or DMSO or PTI‐CH in the presence of α‐amanitin (50 μg/ml) to inhibit transcription (mean ± SEM, n = 4). Statistical significance tested using two‐way ANOVA with Tukey's multiple comparisons test. Immunoblot of steady‐state CFTR expression levels in HBE‐KO (DMSO) and HBE‐ΔIR cells following 24‐h treatment with vehicle (DMSO), VX‐809 (3 μM), PTI‐CH (1 μM), or VX‐809 + PTI‐CH. CNX was used as a loading control. Band B, black arrowhead; band C, white arrowhead. Changes in band B or band C abundance after VX‐809 plus PTI‐CH pre‐treatment were greater than those measured with VX‐809 alone (mean ± SEM n = 3 biological replicates, significance assessed using paired t ‐test). Representative traces (membrane depolarization assay) of ΔI1234_R1239‐CFTR function in HBE‐ΔIR cells following 24‐h treatment with vehicle (DMSO), VX‐809, or VX‐809 + PTI‐CH. Black arrow, acute activation (forskolin + VX‐770); gray arrow, acute inhibition (CFTRinh‐172). Quantitation (mean ± SEM) of peak responses to acute activation of ΔI1234_R1239‐CFTR in HBE‐ΔIR cells pretreated with DMSO ( n = 8), VX‐809 alone ( n = 8) or VX‐809 plus PTI‐CH ( n = 4). Statistical significance of comparisons assessed using unpaired t ‐test.

    Techniques Used: CRISPR, Derivative Assay, Functional Assay, Expressing, Knock-Out, Activation Assay, Inhibition, Mutagenesis, Quantitation Assay

    Potential deleterious effect of VX‐770 at high concentrations observed for ΔI1234_R1239‐CFTR in a heterologous expression system Immunoblot (A) and quantitation (B) of ΔI1234_R1239‐CFTR expression following pharmacological correction (VX‐809) in the absence (DMSO) and presence of chronic (24 h) VX‐770 treatment (0.1, 1, and 10 μM; mean ± SEM, n = 3 biological replicates). Band B, black arrowhead; band C, white arrowhead. Statistical significance tested using two‐way ANOVA with Tukey's multiple comparisons test. Representative traces (membrane depolarization assay) (C) and quantitation (D) of ΔI1234_R1239‐CFTR function following pharmacological correction (VX‐809) in the absence (DMSO) and presence of chronic (24 h) VX‐770 treatment (10 μM) and acute activation (VX‐770/forskolin; mean ± SEM, n = 8 biological replicates). Statistical significance tested using two‐way ANOVA with Tukey's multiple comparisons test.
    Figure Legend Snippet: Potential deleterious effect of VX‐770 at high concentrations observed for ΔI1234_R1239‐CFTR in a heterologous expression system Immunoblot (A) and quantitation (B) of ΔI1234_R1239‐CFTR expression following pharmacological correction (VX‐809) in the absence (DMSO) and presence of chronic (24 h) VX‐770 treatment (0.1, 1, and 10 μM; mean ± SEM, n = 3 biological replicates). Band B, black arrowhead; band C, white arrowhead. Statistical significance tested using two‐way ANOVA with Tukey's multiple comparisons test. Representative traces (membrane depolarization assay) (C) and quantitation (D) of ΔI1234_R1239‐CFTR function following pharmacological correction (VX‐809) in the absence (DMSO) and presence of chronic (24 h) VX‐770 treatment (10 μM) and acute activation (VX‐770/forskolin; mean ± SEM, n = 8 biological replicates). Statistical significance tested using two‐way ANOVA with Tukey's multiple comparisons test.

    Techniques Used: Expressing, Quantitation Assay, Activation Assay

    Amplifying ΔI1234_R1239‐CFTR expression enhances functional rescue effect of VX‐809 + VX‐770 in primary nasal cultures Ussing chamber responses (symmetrical chloride concentrations) exhibited by nasal epithelial cultures obtained from CF‐1 after 24‐h pre‐treatment with vehicle (DMSO), VX‐809, or VX‐809 + PTI‐CH. Responses to VX‐770 (1 μM, gray arrow), forskolin (10 μM, thick black arrow), and CFTRinh‐172 (5 μM, thin black arrow) were tested. ΔI1234_R1239‐CFTR protein expression in nasal cultures from CF‐1 after 24‐h pre‐treatment with vehicle (DMSO), VX‐809 (3 μM), or VX‐809 + PTI‐CH (3 μM and 1 μM, respectively). Band B, black arrowhead; band C, white arrowhead; calnexin was used as a loading control. Summary of forskolin‐ and VX‐770‐dependent responses in Ussing chamber studies of nasal cultures from subject (CF‐1). Bars represent mean and SEM of six biological replicates (different nasal cell seedings). The combination of VX‐809 (3 μM) + PTI‐CH (1 μM; black bar) significantly increased forskolin and VX‐770 activated Ieq relative to VX‐809 alone (hatched bars corresponding to primary nasal culture data shown previously in Fig 6 E). Statistical significance of comparisons assessed using paired t ‐tests.
    Figure Legend Snippet: Amplifying ΔI1234_R1239‐CFTR expression enhances functional rescue effect of VX‐809 + VX‐770 in primary nasal cultures Ussing chamber responses (symmetrical chloride concentrations) exhibited by nasal epithelial cultures obtained from CF‐1 after 24‐h pre‐treatment with vehicle (DMSO), VX‐809, or VX‐809 + PTI‐CH. Responses to VX‐770 (1 μM, gray arrow), forskolin (10 μM, thick black arrow), and CFTRinh‐172 (5 μM, thin black arrow) were tested. ΔI1234_R1239‐CFTR protein expression in nasal cultures from CF‐1 after 24‐h pre‐treatment with vehicle (DMSO), VX‐809 (3 μM), or VX‐809 + PTI‐CH (3 μM and 1 μM, respectively). Band B, black arrowhead; band C, white arrowhead; calnexin was used as a loading control. Summary of forskolin‐ and VX‐770‐dependent responses in Ussing chamber studies of nasal cultures from subject (CF‐1). Bars represent mean and SEM of six biological replicates (different nasal cell seedings). The combination of VX‐809 (3 μM) + PTI‐CH (1 μM; black bar) significantly increased forskolin and VX‐770 activated Ieq relative to VX‐809 alone (hatched bars corresponding to primary nasal culture data shown previously in Fig 6 E). Statistical significance of comparisons assessed using paired t ‐tests.

    Techniques Used: Expressing, Functional Assay

    Summary of forskolin‐ and VX‐770‐dependent responses in Ussing chamber studies of nasal cultures from subject CF‐2 Bars represent mean and SEM of five biological replicates (different nasal cell seedings). Statistical significance assessed using paired t ‐tests. The combination of VX‐809 (3 μM) + PTI‐CH (1 μM; black bar) did not enhance forskolin and VX‐770 activated Ieq relative to VX‐809 alone (hatched bar corresponding to primary nasal culture data shown in Fig 6 E).
    Figure Legend Snippet: Summary of forskolin‐ and VX‐770‐dependent responses in Ussing chamber studies of nasal cultures from subject CF‐2 Bars represent mean and SEM of five biological replicates (different nasal cell seedings). Statistical significance assessed using paired t ‐tests. The combination of VX‐809 (3 μM) + PTI‐CH (1 μM; black bar) did not enhance forskolin and VX‐770 activated Ieq relative to VX‐809 alone (hatched bar corresponding to primary nasal culture data shown in Fig 6 E).

    Techniques Used:

    12) Product Images from "Fasting and Systemic Insulin Signaling Regulate Phosphorylation of Brain Proteins That Modulate Cell Morphology and Link to Neurological Disorders *"

    Article Title: Fasting and Systemic Insulin Signaling Regulate Phosphorylation of Brain Proteins That Modulate Cell Morphology and Link to Neurological Disorders *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M115.668103

    Forskolin induces the PAS antibody-reactive protein phosphorylation in primary neurons. A , primary cortical neurons were isolated from neonatal mice and subjected to stimulation with forskolin. The PAS antibody-reactive phosphorylation was detected in cell lysates with FLOT-1 as a loading control. B , primary cerebellar granule cells were isolated from neonatal mice and subjected to stimulation with forskolin in the presence or absence of H-89. The PAS antibody-reactive phosphorylation was detected in cell lysates with GAPDH as a loading control. C–E , the indicated proteins (srGAP3, MARK3, and MARK4) were immunoprecipitated from lysates of primary cortical neurons that were stimulated with or without forskolin, and their phosphorylation was detected using the PAS antibody. IB , immunoblot.
    Figure Legend Snippet: Forskolin induces the PAS antibody-reactive protein phosphorylation in primary neurons. A , primary cortical neurons were isolated from neonatal mice and subjected to stimulation with forskolin. The PAS antibody-reactive phosphorylation was detected in cell lysates with FLOT-1 as a loading control. B , primary cerebellar granule cells were isolated from neonatal mice and subjected to stimulation with forskolin in the presence or absence of H-89. The PAS antibody-reactive phosphorylation was detected in cell lysates with GAPDH as a loading control. C–E , the indicated proteins (srGAP3, MARK3, and MARK4) were immunoprecipitated from lysates of primary cortical neurons that were stimulated with or without forskolin, and their phosphorylation was detected using the PAS antibody. IB , immunoblot.

    Techniques Used: Isolation, Mouse Assay, Immunoprecipitation

    Phosphorylation of the srGAP3 induced by fasting in mouse brain and forkolin in primary neurons. A , the srGAP3 proteins were immunoprecipitated ( IP ) from brain lysates and subjected to phosphomapping analysis via mass spectrometry. A.U. , arbitrary unit. B , the srGAP3 proteins were immunoprecipitated from the brains of mice that were either fasted overnight (16 h) or intraperitoneally injected with insulin for 20 min after an overnight fast. Phosphorylated Ser 858 was detected and quantified by measuring the area detected by the relevant extracted ion chromatograms on the immunoprecipitated srGAP3 proteins. The quantified intensities under fasting conditions were set to one, and the intensities under insulin injection conditions were normalized against the respective values under fasting conditions. The values shown are the mean of two experiments. C , The HA-tagged wild-type and mutant srGAP3 proteins were expressed in HEK293 cells. Phosphorylation of the HA-tagged srGAP3 proteins was detected using the PAS antibody and pS858-srGAP3 phospho-specific antibody. Total HA-tagged srGAP3 was detected using the anti-HA antibody with GAPDH as a loading control. The surrounding sequence for Ser 1029 and Ser 1030 is as follows: RRS ss SSTEMM (Ser 1029 and Ser 1030 shown in lowercase bold letters). D , HEK293 cells expressing the HA-tagged wild-type and mutant srGAP3 proteins were stimulated with or without forskolin. Phosphorylation of the HA-tagged srGAP3 proteins was detected using the PAS antibody and pS858-srGAP3 phospho-specific antibody. Phosphorylated VASP was detected using the pS157-VASP phospho-specific antibody with GAPDH as a loading control. E , the srGAP3 proteins were immunoprecipitated using the srGAP3 antibody from brain lysates of mice subjected to overnight fasting (16 h) or to intraperitoneal insulin injection (20 min) after an overnight fast. The Ser 858 phosphorylation of srGAP3 was detected in the immunoprecipitates using the pS858-srGAP3 phospho-specific antibody. F and G , primary cortical neurons ( F ) and cerebellar granule cells ( G ) were isolated from neonatal mice and subjected to stimulation with forskolin ( FSK ) in the presence or absence of H-89. The Ser 858 phosphorylation of srGAP3 was detected in cell lysates using the pS858-srGAP3 phospho-specific antibody. Total and phosphorylated CREB were detected in cell lysates with GAPDH as a loading control. The arrowhead in F indicates the signals for Ser 858 phosphorylation of srGAP3. H , primary cerebellar granule cells were isolated from neonatal mice and subjected to stimulation with forskolin, insulin, or both. Total and phosphorylated PKB, srGAP3, and CREB were detected in cell lysates. IP , immunoprecipitate; IB , immunoblot.
    Figure Legend Snippet: Phosphorylation of the srGAP3 induced by fasting in mouse brain and forkolin in primary neurons. A , the srGAP3 proteins were immunoprecipitated ( IP ) from brain lysates and subjected to phosphomapping analysis via mass spectrometry. A.U. , arbitrary unit. B , the srGAP3 proteins were immunoprecipitated from the brains of mice that were either fasted overnight (16 h) or intraperitoneally injected with insulin for 20 min after an overnight fast. Phosphorylated Ser 858 was detected and quantified by measuring the area detected by the relevant extracted ion chromatograms on the immunoprecipitated srGAP3 proteins. The quantified intensities under fasting conditions were set to one, and the intensities under insulin injection conditions were normalized against the respective values under fasting conditions. The values shown are the mean of two experiments. C , The HA-tagged wild-type and mutant srGAP3 proteins were expressed in HEK293 cells. Phosphorylation of the HA-tagged srGAP3 proteins was detected using the PAS antibody and pS858-srGAP3 phospho-specific antibody. Total HA-tagged srGAP3 was detected using the anti-HA antibody with GAPDH as a loading control. The surrounding sequence for Ser 1029 and Ser 1030 is as follows: RRS ss SSTEMM (Ser 1029 and Ser 1030 shown in lowercase bold letters). D , HEK293 cells expressing the HA-tagged wild-type and mutant srGAP3 proteins were stimulated with or without forskolin. Phosphorylation of the HA-tagged srGAP3 proteins was detected using the PAS antibody and pS858-srGAP3 phospho-specific antibody. Phosphorylated VASP was detected using the pS157-VASP phospho-specific antibody with GAPDH as a loading control. E , the srGAP3 proteins were immunoprecipitated using the srGAP3 antibody from brain lysates of mice subjected to overnight fasting (16 h) or to intraperitoneal insulin injection (20 min) after an overnight fast. The Ser 858 phosphorylation of srGAP3 was detected in the immunoprecipitates using the pS858-srGAP3 phospho-specific antibody. F and G , primary cortical neurons ( F ) and cerebellar granule cells ( G ) were isolated from neonatal mice and subjected to stimulation with forskolin ( FSK ) in the presence or absence of H-89. The Ser 858 phosphorylation of srGAP3 was detected in cell lysates using the pS858-srGAP3 phospho-specific antibody. Total and phosphorylated CREB were detected in cell lysates with GAPDH as a loading control. The arrowhead in F indicates the signals for Ser 858 phosphorylation of srGAP3. H , primary cerebellar granule cells were isolated from neonatal mice and subjected to stimulation with forskolin, insulin, or both. Total and phosphorylated PKB, srGAP3, and CREB were detected in cell lysates. IP , immunoprecipitate; IB , immunoblot.

    Techniques Used: Immunoprecipitation, Mass Spectrometry, Mouse Assay, Injection, Mutagenesis, Sequencing, Expressing, Isolation

    13) Product Images from "Long‐term expanding human airway organoids for disease modeling"

    Article Title: Long‐term expanding human airway organoids for disease modeling

    Journal: The EMBO Journal

    doi: 10.15252/embj.2018100300

    Airway organoids to study cystic fibrosis Box‐and‐whisker plot showing concentration‐dependent forskolin‐induced swelling of AOs in the absence and presence of CFTR inhibitors CFTRinh‐172 and GlyH101. Upon CFTR inhibition, swelling is noticeably decreased but not absent. Shown are pooled data from three different AO lines used in each of three independent experiments. Whiskers indicate smallest and largest values, boxes indicate 25 th to 75 th percentile, and horizontal solid line indicates median. AUC, area under the curve. Box‐and‐whisker plot showing concentration‐dependent E act ‐induced swelling of AOs, but not rectal organoids (black outlines). Forskolin causes swelling in both organoid types (gray outlines). Shown are pooled data from three different AO and two different rectal organoid lines used in three to four independent experiments. Whiskers indicate smallest and largest values, boxes indicate 25 th to 75 th percentile, and horizontal solid line indicates median. Swelling was linear for 2 h for AOs, but only 1 h for rectal organoids. See Appendix Fig S3A for respective time course plots. Representative histological sections of periodic acid–Schiff (PAS)‐stained organoids from a CF patient with CFTR F508del/F508del mutation. Note the thick layer of PAS‐positive polysaccharides apically lining the airway epithelium. Rectal organoids were generated from rectal biopsies; AOs were generated from broncho‐alveolar lavages (BALs). Scale bars equal 50 μm. See Appendix Fig S3B for PAS‐stained wild‐type and CFTR R334W/R334W organoid sections. Box‐and‐whisker plot showing swelling assays of several CF patient AO lines carrying the indicated CFTR mutations (G542X is a premature stop associated with severe disease and no functional CFTR protein; F508del is the most common CFTR mutation in subjects with CF and severely reduces apical trafficking and function, leading to severe disease (high sweat chloride, high pancreas insufficiency, high pseudomonas infection rate); R334W is a milder CFTR mutation associated as indicated by lower pseudomonas infection rates and pancreas sufficiency with reduced ion channel conductivity, normal apical expression, and some residual function). Forskolin‐induced swelling rarely exceeds vehicle controls in CF AOs, but increases in the presence of the CFTR modulating drugs VX‐770 and VX‐809. E act ‐induced swelling exceeds forskolin‐induced swelling to a similar extent as pre‐treatment with VX‐770 and VX‐809 in four out of five CF AO lines. In contrast, BAL‐derived WT AOs swell following forskolin stimulation independent of the presence of VX‐770 and VX‐809. Of note, E act ‐induced swelling is not observed in this AO line. Shown are pooled data of four to five independent experiments (CF AOs) or two independent experiments (WT AOs). Whiskers indicate smallest and largest values, boxes indicate 25 th to 75 th percentile, and horizontal solid line indicates median. See Appendix Fig S3C for selected time course plots. Whole‐mounted immunofluorescence analysis of BAL AO‐derived ALI cultures. After 3 weeks of differentiation, ALI cultures show ciliated airway epithelium (green, β‐tubulin). Counterstained are the actin cytoskeleton (red) and nuclei (blue). Transepithelial electrical measurements of BAL AO‐derived ALI cultures. Shown are equivalent current ( I eq ) traces of a CFTR wt/wt (gray) and CFTR F508del/F508del donor, treated with vehicle (blue) or VX‐809 and VX‐770 (red). Inhibition of the epithelial sodium channel (ENaC) with benzamil was observed in all traces (declining I eq ). Forskolin increased the current in CFTR wt/wt and CFTR F508del/F50del donor treated with VX‐809 and VX‐770, but not in the vehicle treated. A decline was observed with CFTRinh‐172 in all examined conditions. The bar graphs quantify the peak forskolin activated‐ and CFTRinh‐172 inhibited‐currents. Shown are pooled data of four independent measurements for each donor. Results are shown as mean ± s.d.
    Figure Legend Snippet: Airway organoids to study cystic fibrosis Box‐and‐whisker plot showing concentration‐dependent forskolin‐induced swelling of AOs in the absence and presence of CFTR inhibitors CFTRinh‐172 and GlyH101. Upon CFTR inhibition, swelling is noticeably decreased but not absent. Shown are pooled data from three different AO lines used in each of three independent experiments. Whiskers indicate smallest and largest values, boxes indicate 25 th to 75 th percentile, and horizontal solid line indicates median. AUC, area under the curve. Box‐and‐whisker plot showing concentration‐dependent E act ‐induced swelling of AOs, but not rectal organoids (black outlines). Forskolin causes swelling in both organoid types (gray outlines). Shown are pooled data from three different AO and two different rectal organoid lines used in three to four independent experiments. Whiskers indicate smallest and largest values, boxes indicate 25 th to 75 th percentile, and horizontal solid line indicates median. Swelling was linear for 2 h for AOs, but only 1 h for rectal organoids. See Appendix Fig S3A for respective time course plots. Representative histological sections of periodic acid–Schiff (PAS)‐stained organoids from a CF patient with CFTR F508del/F508del mutation. Note the thick layer of PAS‐positive polysaccharides apically lining the airway epithelium. Rectal organoids were generated from rectal biopsies; AOs were generated from broncho‐alveolar lavages (BALs). Scale bars equal 50 μm. See Appendix Fig S3B for PAS‐stained wild‐type and CFTR R334W/R334W organoid sections. Box‐and‐whisker plot showing swelling assays of several CF patient AO lines carrying the indicated CFTR mutations (G542X is a premature stop associated with severe disease and no functional CFTR protein; F508del is the most common CFTR mutation in subjects with CF and severely reduces apical trafficking and function, leading to severe disease (high sweat chloride, high pancreas insufficiency, high pseudomonas infection rate); R334W is a milder CFTR mutation associated as indicated by lower pseudomonas infection rates and pancreas sufficiency with reduced ion channel conductivity, normal apical expression, and some residual function). Forskolin‐induced swelling rarely exceeds vehicle controls in CF AOs, but increases in the presence of the CFTR modulating drugs VX‐770 and VX‐809. E act ‐induced swelling exceeds forskolin‐induced swelling to a similar extent as pre‐treatment with VX‐770 and VX‐809 in four out of five CF AO lines. In contrast, BAL‐derived WT AOs swell following forskolin stimulation independent of the presence of VX‐770 and VX‐809. Of note, E act ‐induced swelling is not observed in this AO line. Shown are pooled data of four to five independent experiments (CF AOs) or two independent experiments (WT AOs). Whiskers indicate smallest and largest values, boxes indicate 25 th to 75 th percentile, and horizontal solid line indicates median. See Appendix Fig S3C for selected time course plots. Whole‐mounted immunofluorescence analysis of BAL AO‐derived ALI cultures. After 3 weeks of differentiation, ALI cultures show ciliated airway epithelium (green, β‐tubulin). Counterstained are the actin cytoskeleton (red) and nuclei (blue). Transepithelial electrical measurements of BAL AO‐derived ALI cultures. Shown are equivalent current ( I eq ) traces of a CFTR wt/wt (gray) and CFTR F508del/F508del donor, treated with vehicle (blue) or VX‐809 and VX‐770 (red). Inhibition of the epithelial sodium channel (ENaC) with benzamil was observed in all traces (declining I eq ). Forskolin increased the current in CFTR wt/wt and CFTR F508del/F50del donor treated with VX‐809 and VX‐770, but not in the vehicle treated. A decline was observed with CFTRinh‐172 in all examined conditions. The bar graphs quantify the peak forskolin activated‐ and CFTRinh‐172 inhibited‐currents. Shown are pooled data of four independent measurements for each donor. Results are shown as mean ± s.d.

    Techniques Used: Whisker Assay, Concentration Assay, Inhibition, Activated Clotting Time Assay, Staining, Mutagenesis, Generated, Functional Assay, Infection, Expressing, Derivative Assay, Immunofluorescence

    14) Product Images from "Evaluation of both exonic and intronic variants for effects on RNA splicing allows for accurate assessment of the effectiveness of precision therapies"

    Article Title: Evaluation of both exonic and intronic variants for effects on RNA splicing allows for accurate assessment of the effectiveness of precision therapies

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1009100

    CFTR intronic splice site variants with residual function are responsive to CFTR modulators. (A) Sashimi plots showing skipping of exon 16 caused by 2657+5G > A variant . RNA-seq was performed on the total RNA extracted from nasal cells of the individual harboring c.2657+5G > A/W1282X genotype. RNA from healthy individual served as a control. Per-base expression is plotted on y-axis of Sashimi plot. ‘E’ refers to exon locations on x-axis. +5 on the Sashimi plot indicates variant is located in intron 16 (not to scale). Numbers on the Sashimi plot indicate counts of reads spanning exon junctions. ɸ on the illustration indicates relative location of the variant c.2657+5 G > A. (B) Residual full-length CFTR transcript allows for modulator response in a c.2657+5G > A/W1282X individual . Representative short circuit current (I sc ) tracings on Human nasal epithelial (HNE) cells harvested from an individual harboring c.2657+5G > A/W1282X. CFTR channel function and CFTR modulator response were evaluated in well-differentiated airway liquid interface (ALI) culture. (C) Primary cells from an individual harboring c.3717+40A > G/F508del respond to modulator therapy. Representative short circuit current (I sc ) tracings on Human nasal epithelial (HNE) cells harvested from an individual harboring c.3717+40A > G/F508del. CFTR channel function and CFTR modulator response were evaluated in well- differentiated airway liquid interface (ALI) culture. (D) Intronic variants that allow for residual normal splicing respond to modulator therapies. Stacked bar graphs indicate effect of modulator treatment on CFBE stable cells expressing different CFTR intronic variants. Change in current (ΔI sc ) was defined as the current inhibited by Inh-172 after sustained I sc responses were achieved upon stimulation with forskolin alone or sequentially with ivacaftor. Data shown as mean±SEM (minimum of three independent measurements per condition). p value was determined by one-way ANOVA. ****( p ≤0.0001), n.s. (not significant, p > 0.05) when compared to DMSO treated vehicle control. Data underlying graphs in this figure reported in S5 Data .
    Figure Legend Snippet: CFTR intronic splice site variants with residual function are responsive to CFTR modulators. (A) Sashimi plots showing skipping of exon 16 caused by 2657+5G > A variant . RNA-seq was performed on the total RNA extracted from nasal cells of the individual harboring c.2657+5G > A/W1282X genotype. RNA from healthy individual served as a control. Per-base expression is plotted on y-axis of Sashimi plot. ‘E’ refers to exon locations on x-axis. +5 on the Sashimi plot indicates variant is located in intron 16 (not to scale). Numbers on the Sashimi plot indicate counts of reads spanning exon junctions. ɸ on the illustration indicates relative location of the variant c.2657+5 G > A. (B) Residual full-length CFTR transcript allows for modulator response in a c.2657+5G > A/W1282X individual . Representative short circuit current (I sc ) tracings on Human nasal epithelial (HNE) cells harvested from an individual harboring c.2657+5G > A/W1282X. CFTR channel function and CFTR modulator response were evaluated in well-differentiated airway liquid interface (ALI) culture. (C) Primary cells from an individual harboring c.3717+40A > G/F508del respond to modulator therapy. Representative short circuit current (I sc ) tracings on Human nasal epithelial (HNE) cells harvested from an individual harboring c.3717+40A > G/F508del. CFTR channel function and CFTR modulator response were evaluated in well- differentiated airway liquid interface (ALI) culture. (D) Intronic variants that allow for residual normal splicing respond to modulator therapies. Stacked bar graphs indicate effect of modulator treatment on CFBE stable cells expressing different CFTR intronic variants. Change in current (ΔI sc ) was defined as the current inhibited by Inh-172 after sustained I sc responses were achieved upon stimulation with forskolin alone or sequentially with ivacaftor. Data shown as mean±SEM (minimum of three independent measurements per condition). p value was determined by one-way ANOVA. ****( p ≤0.0001), n.s. (not significant, p > 0.05) when compared to DMSO treated vehicle control. Data underlying graphs in this figure reported in S5 Data .

    Techniques Used: Variant Assay, RNA Sequencing Assay, Expressing

    15) Product Images from "Cartilage-specific deletion of Alk5 gene results in a progressive osteoarthritis-like phenotype in mice"

    Article Title: Cartilage-specific deletion of Alk5 gene results in a progressive osteoarthritis-like phenotype in mice

    Journal: Osteoarthritis and cartilage

    doi: 10.1016/j.joca.2017.07.010

    TGF-β1/ALK5 signaling induces Prg4 expression via the PKA-CREB signaling pathway in chondrocytes A, B, G and H. Femoral head cartilage isolated from Prg4 GFPCreERT2/+ mice was pretreated with selective inhibitor for 30 min, followed by an additional 24 h incubation with TGF-β1, inhibitor, or a combination of both. qRT-PCR and western blotting were performed to detect the expression of Prg4 mRNA (A and G, two-way ANOVA followed by Tukey’s test, n = 3 femoral head cartilage per group) and the GFP protein expression (B and H, quantitative data were shown in the lower panel, two-way ANOVA followed by Tukey’s test, n = 3 femoral head cartilage per group), respectively. C and D, Primary chondrocytes isolated from Cre-negative mice were pretreated with 10 μM H89 (C) or 1 μM SB-505124 (D) for 30 min, followed by an additional 30 min incubation of 10 ng/ml TGF-β1,10 μM H89/1 μM SB-505124, or a combination of both. Western blotting was performed to detect the pCREB and pSmad3 protein expressions. Quantitative data were shown in the right panel (two-way ANOVA followed by Tukey’s test, n = 4 mice per group). E, Western blotting analysis of pCREB in Cre-negative or Alk5 -deficient primary chondrocytes treated with TGF-β1 for 30 min. Quantitative data were shown in the right panel (two-way ANOVA followed by Tukey’s test, n = 4 mice per group). F, IHC analysis of pCREB protein expression in articular cartilage of 2-month-old Alk5 cKO mice and Cre-negative control mice. Quantitative data were shown in the right panel (The percentage of positive cells in Cre-negative mice was defined as 1, student’s unpaired t -test, n = 4 mice per group). I, qRT-PCR analysis of Prg4 mRNA expression in Cre-negative or Alk5 -deficient femoral head cartilage treated with increasing concentrations of forskolin (two-way ANOVA followed by Tukey’s test, n = 3 femoral head cartilage per group). J. Schematic illustration of the mechanisms of ALK5 signaling in the maintenance of articular cartilage. Data were expressed as the mean ± 95% confidence intervals. In F, symbols represent individual mice. In A, B, C, D, E, G and H, symbols represent three technical replicates.
    Figure Legend Snippet: TGF-β1/ALK5 signaling induces Prg4 expression via the PKA-CREB signaling pathway in chondrocytes A, B, G and H. Femoral head cartilage isolated from Prg4 GFPCreERT2/+ mice was pretreated with selective inhibitor for 30 min, followed by an additional 24 h incubation with TGF-β1, inhibitor, or a combination of both. qRT-PCR and western blotting were performed to detect the expression of Prg4 mRNA (A and G, two-way ANOVA followed by Tukey’s test, n = 3 femoral head cartilage per group) and the GFP protein expression (B and H, quantitative data were shown in the lower panel, two-way ANOVA followed by Tukey’s test, n = 3 femoral head cartilage per group), respectively. C and D, Primary chondrocytes isolated from Cre-negative mice were pretreated with 10 μM H89 (C) or 1 μM SB-505124 (D) for 30 min, followed by an additional 30 min incubation of 10 ng/ml TGF-β1,10 μM H89/1 μM SB-505124, or a combination of both. Western blotting was performed to detect the pCREB and pSmad3 protein expressions. Quantitative data were shown in the right panel (two-way ANOVA followed by Tukey’s test, n = 4 mice per group). E, Western blotting analysis of pCREB in Cre-negative or Alk5 -deficient primary chondrocytes treated with TGF-β1 for 30 min. Quantitative data were shown in the right panel (two-way ANOVA followed by Tukey’s test, n = 4 mice per group). F, IHC analysis of pCREB protein expression in articular cartilage of 2-month-old Alk5 cKO mice and Cre-negative control mice. Quantitative data were shown in the right panel (The percentage of positive cells in Cre-negative mice was defined as 1, student’s unpaired t -test, n = 4 mice per group). I, qRT-PCR analysis of Prg4 mRNA expression in Cre-negative or Alk5 -deficient femoral head cartilage treated with increasing concentrations of forskolin (two-way ANOVA followed by Tukey’s test, n = 3 femoral head cartilage per group). J. Schematic illustration of the mechanisms of ALK5 signaling in the maintenance of articular cartilage. Data were expressed as the mean ± 95% confidence intervals. In F, symbols represent individual mice. In A, B, C, D, E, G and H, symbols represent three technical replicates.

    Techniques Used: Expressing, Isolation, Mouse Assay, Incubation, Quantitative RT-PCR, Western Blot, Immunohistochemistry, Negative Control

    16) Product Images from "Metabolic stress regulates ERK activity by controlling KSR‐RAF heterodimerization"

    Article Title: Metabolic stress regulates ERK activity by controlling KSR‐RAF heterodimerization

    Journal: EMBO Reports

    doi: 10.15252/embr.201744524

    Metabolic stressors promote the dissociation of the 14‐3‐3 proteins from the N‐terminus of CRAF and induce a RAS‐independent activation of CRAF in NRAS‐mutant cells MelJuso cells were treated with 2DG (11 mM), rotenone (Rot; 5 μM), metformin (Met; 10 mM), and forskolin (Fo; 10 μM) for 2 h or with 2DG (5.5 mM), rotenone (Rot; 5 μM), metformin (Met; 5 mM), and forskolin (Fo; 10 μM) for 14 h. Endogenous NRAS was immunoprecipitated (IP), and the immunocomplexes were Western‐blotted for endogenous NRAS and CRAF. Endogenous NRAS, CRAF, phospho‐MEK1/2 (pMEK1/2), and total MEK2 levels in the cell lysates are also shown. MelJuso cells were transfected with two different siRNAs against NRAS (si#1, si#2) and their combination (siPool) or with a non‐targeted siRNA sequence (NTsiRNA). A double siRNA transfection (48 and 72 h) was performed to have an optimal knockdown. Cells were treated with 2DG (11 mM) for 4 h. Cell lysates were Western‐blotted for NRAS, phospho‐MEK2 (pMEK2), total MEK2, and α‐tubulin. Myc‐epitope‐tagged CRAF WT or CRAF R89L were transfected into MelJuso cells. After 24 h, cells were treated with 2DG (11 mM) for 4 h. Myc‐tagged CRAF WT and myc‐tagged CRAF R89L were immunoprecipitated and subjected to kinase assay in the presence of recombinant kinase‐dead MEK1 (K97M) (500 ng) as a substrate and ATP (20 μM). CRAF R89L activity (%) is relative to the untreated CRAF R89L sample. Bars show mean % ± SEM ( n = 3). Difference between untreated and 2DG‐treated samples was examined with unpaired t ‐test (** P = 0.0076). MelJuso cells were treated with 2DG (11 mM), rotenone (Rot; 5 μM), metformin (Met; 10 mM), and forskolin (Fo; 10 μM) for 2, 4, and 8 h. Cell extracts were Western‐blotted for phospho‐CRAF (pCRAF) S259. CRAF levels in the lysate are shown as a loading control. MelJuso cells were treated with 2DG (11 mM) or rotenone (Rot; 5 μM) for 4 h. Endogenous 14‐3‐3 proteins were immunoprecipitated (IP), and the immunocomplexes were Western‐blotted for endogenous 14‐3‐3 and CRAF. Endogenous 14‐3‐3 and CRAF levels in the cell lysates are also shown. FLAG‐epitope‐tagged CRAF WT and FLAG‐epitope‐tagged CRAF S259A were transfected into MelJuso cells. Cells were treated with 2DG (11 mM) for 4 h. FLAG‐tagged CRAF was immunoprecipitated (IP), and the immunocomplexes were Western‐blotted for endogenous 14‐3‐3 and FLAG‐CRAF. Endogenous 14‐3‐3 and FLAG‐CRAF levels in the cell lysates are also shown. Source data are available online for this figure.
    Figure Legend Snippet: Metabolic stressors promote the dissociation of the 14‐3‐3 proteins from the N‐terminus of CRAF and induce a RAS‐independent activation of CRAF in NRAS‐mutant cells MelJuso cells were treated with 2DG (11 mM), rotenone (Rot; 5 μM), metformin (Met; 10 mM), and forskolin (Fo; 10 μM) for 2 h or with 2DG (5.5 mM), rotenone (Rot; 5 μM), metformin (Met; 5 mM), and forskolin (Fo; 10 μM) for 14 h. Endogenous NRAS was immunoprecipitated (IP), and the immunocomplexes were Western‐blotted for endogenous NRAS and CRAF. Endogenous NRAS, CRAF, phospho‐MEK1/2 (pMEK1/2), and total MEK2 levels in the cell lysates are also shown. MelJuso cells were transfected with two different siRNAs against NRAS (si#1, si#2) and their combination (siPool) or with a non‐targeted siRNA sequence (NTsiRNA). A double siRNA transfection (48 and 72 h) was performed to have an optimal knockdown. Cells were treated with 2DG (11 mM) for 4 h. Cell lysates were Western‐blotted for NRAS, phospho‐MEK2 (pMEK2), total MEK2, and α‐tubulin. Myc‐epitope‐tagged CRAF WT or CRAF R89L were transfected into MelJuso cells. After 24 h, cells were treated with 2DG (11 mM) for 4 h. Myc‐tagged CRAF WT and myc‐tagged CRAF R89L were immunoprecipitated and subjected to kinase assay in the presence of recombinant kinase‐dead MEK1 (K97M) (500 ng) as a substrate and ATP (20 μM). CRAF R89L activity (%) is relative to the untreated CRAF R89L sample. Bars show mean % ± SEM ( n = 3). Difference between untreated and 2DG‐treated samples was examined with unpaired t ‐test (** P = 0.0076). MelJuso cells were treated with 2DG (11 mM), rotenone (Rot; 5 μM), metformin (Met; 10 mM), and forskolin (Fo; 10 μM) for 2, 4, and 8 h. Cell extracts were Western‐blotted for phospho‐CRAF (pCRAF) S259. CRAF levels in the lysate are shown as a loading control. MelJuso cells were treated with 2DG (11 mM) or rotenone (Rot; 5 μM) for 4 h. Endogenous 14‐3‐3 proteins were immunoprecipitated (IP), and the immunocomplexes were Western‐blotted for endogenous 14‐3‐3 and CRAF. Endogenous 14‐3‐3 and CRAF levels in the cell lysates are also shown. FLAG‐epitope‐tagged CRAF WT and FLAG‐epitope‐tagged CRAF S259A were transfected into MelJuso cells. Cells were treated with 2DG (11 mM) for 4 h. FLAG‐tagged CRAF was immunoprecipitated (IP), and the immunocomplexes were Western‐blotted for endogenous 14‐3‐3 and FLAG‐CRAF. Endogenous 14‐3‐3 and FLAG‐CRAF levels in the cell lysates are also shown. Source data are available online for this figure.

    Techniques Used: Activation Assay, Mutagenesis, Immunoprecipitation, Western Blot, Transfection, Sequencing, Kinase Assay, Recombinant, Activity Assay, FLAG-tag

    17) Product Images from "Capitalizing on the heterogeneous effects of CFTR nonsense and frameshift variants to inform therapeutic strategy for cystic fibrosis"

    Article Title: Capitalizing on the heterogeneous effects of CFTR nonsense and frameshift variants to inform therapeutic strategy for cystic fibrosis

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1007723

    Corrector treatment increases CFTR activation response of nonsense variants in exon 22 that result in mature truncated CFTR. (A) Schematic of CFTR-Expression Minigene with abridged introns 21 and abridged intron 22 (EMG-i21-i22) constructed in pcDNA5FRT plasmid. CFTR exons are shown in boxes and two abridged introns in dashed lines. The location of each studied variant is shown relative to the CFTR exons and regions predicted to elicit NMD. (B) RT-qPCR showing relative steady state levels of CFTR transcript in HEK293 stable cells expressing wild-type EMG or EMGs with truncations at residue position, as indicated on the labels. Values were normalized to B2M . Mean ± SEM ( n = 3) measured in triplicates. P value was determined by one way ANOVA. **** indicates significant difference ( P ≤0.0001) when compared with CFTR mRNA abundance in cells expressing WT-EMG. (C) Steady state levels of CFTR protein from HEK293 cells transiently transfected with wild-type EMG or EMGs with different nonsense variants. 40 μg of total cell lysates were electrophoresed and IB was probed with anti-CFTR antibody-MM13-4 (EMD Millipore). (D) Representative IB showing sensitivity of CFTR to PNGaseF and Endo H. Mature complex glycosylated band is sensitive to PNGase only, whereas immature core glycosylated band is sensitive to both PNGase and EndoH. (E). Schematic illustration of the nonsense variants in the protein context showing their classification into two groups based on mRNA stability and protein maturity. Each nonsense variant truncates CFTR at intracellular loop 6 (ICL6) just before NBD2. (F) A representative Ussing chamber tracing of CFBE cells stably expressing S1196X-EMG. Short-circuit (I sc ) measurements were recorded in Ussing chambers after treatment of cells with 0.03% DMSO (vehicle) or 3 μM corrector compounds (lumacaftor/tezacaftor or both) for 48 h. ( G and H) Stacked bar graphs indicate effect of modulator treatment on CFBE (G) and MDCK (H) stable cells expressing different CFTR 3’ nonsense variants. Change in I sc (ΔIsc) was defined as the current inhibited by Inh-172 after sustained Isc responses were achieved upon stimulation with forskolin alone or sequentially with ivacaftor. Mean ± SEM ( n = 3–8). WT-CFTR function represents forskolin stimulated I sc without modulator treatment in cells expressing EMG i21-i22. P value was determined by one way ANOVA. **** indicates significant difference ( P ≤0.0001), n.s. (not significant, P > 0.05); when compared with forskolin stimulated CFTR function in DMSO (vehicle) treated cells expressing respective variant.
    Figure Legend Snippet: Corrector treatment increases CFTR activation response of nonsense variants in exon 22 that result in mature truncated CFTR. (A) Schematic of CFTR-Expression Minigene with abridged introns 21 and abridged intron 22 (EMG-i21-i22) constructed in pcDNA5FRT plasmid. CFTR exons are shown in boxes and two abridged introns in dashed lines. The location of each studied variant is shown relative to the CFTR exons and regions predicted to elicit NMD. (B) RT-qPCR showing relative steady state levels of CFTR transcript in HEK293 stable cells expressing wild-type EMG or EMGs with truncations at residue position, as indicated on the labels. Values were normalized to B2M . Mean ± SEM ( n = 3) measured in triplicates. P value was determined by one way ANOVA. **** indicates significant difference ( P ≤0.0001) when compared with CFTR mRNA abundance in cells expressing WT-EMG. (C) Steady state levels of CFTR protein from HEK293 cells transiently transfected with wild-type EMG or EMGs with different nonsense variants. 40 μg of total cell lysates were electrophoresed and IB was probed with anti-CFTR antibody-MM13-4 (EMD Millipore). (D) Representative IB showing sensitivity of CFTR to PNGaseF and Endo H. Mature complex glycosylated band is sensitive to PNGase only, whereas immature core glycosylated band is sensitive to both PNGase and EndoH. (E). Schematic illustration of the nonsense variants in the protein context showing their classification into two groups based on mRNA stability and protein maturity. Each nonsense variant truncates CFTR at intracellular loop 6 (ICL6) just before NBD2. (F) A representative Ussing chamber tracing of CFBE cells stably expressing S1196X-EMG. Short-circuit (I sc ) measurements were recorded in Ussing chambers after treatment of cells with 0.03% DMSO (vehicle) or 3 μM corrector compounds (lumacaftor/tezacaftor or both) for 48 h. ( G and H) Stacked bar graphs indicate effect of modulator treatment on CFBE (G) and MDCK (H) stable cells expressing different CFTR 3’ nonsense variants. Change in I sc (ΔIsc) was defined as the current inhibited by Inh-172 after sustained Isc responses were achieved upon stimulation with forskolin alone or sequentially with ivacaftor. Mean ± SEM ( n = 3–8). WT-CFTR function represents forskolin stimulated I sc without modulator treatment in cells expressing EMG i21-i22. P value was determined by one way ANOVA. **** indicates significant difference ( P ≤0.0001), n.s. (not significant, P > 0.05); when compared with forskolin stimulated CFTR function in DMSO (vehicle) treated cells expressing respective variant.

    Techniques Used: Activation Assay, Expressing, Construct, Plasmid Preparation, Variant Assay, Quantitative RT-PCR, Transfection, Stable Transfection

    CFTR nonsense variant with splicing defect has residual function that benefits from modulator treatments. (A) A schematic illustration of CFTR-Expression Minigene with introns 14–18 (introns 14 and 16 are full-length, and 15, 17, 18 are abridged). Arrow indicates location of E831X (top). CFTR mRNA splicing patterns of the total RNA extracted from HEK293 cells transiently transfected with E831X-EMG. (B) Steady state amounts of different isoforms of CFTR produced from E831X-EMG-i14-i18 expressed transiently in HEK293 cells. Lysates from cells expressing WT-EMG i14-i18, intronless WT CFTR or F508del served as positive controls, and empty vector as negative control. Immunoblot (IB) was probed with anti-CFTR antibody, 596 (CFFT). Horizontal arrows indicate to isoforms corresponding to (i) a normal codon (E831) substituted with a stop codon, (ii) deletion of complete exon 16, and (iii) deletion of a single amino acid E831. Beta-Actin was used as loading control. (C) Short-circuit (I sc ) tracing of CFTR function observed in CFBE-stable cells expressing E831X mounted on Ussing chamber. Cells were treated for 48 h with correctors (lumacaftor/tezacaftor or both, 3 μM each) and acutely with potentiator (ivacaftor, 10 μM). Change in I sc (ΔI sc ) was defined as the current inhibited by Inh-172 after sustained Isc responses achieved upon stimulation with forskolin alone or sequentially with ivacaftor. Data are presented as mean±SEM (n = 3). P value was determined by one way ANOVA. **** indicates significant difference ( P ≤0.0001) when compared with forskolin stimulated CFTR function in DMSO (vehicle) treated cells. WT-CFTR function represents forskolin stimulated Isc without modulator treatment in cells expressing EMG i14-i18. (D ) A tracing of CFTR function observed in primary nasal epithelial cells of an individual harboring E831X/F508del. CF-Human nasal epithelial (HNE) cells were treated for 24 h with lumacaftor and tezacaftor, 3 μM each, and acutely with Ivacaftor (10 μM). Stacked bar graph is a comparison of improvement in CFTR function of E831X/F508del vs F508del/Indel. Alternate Indel alleles were either 2184insA, 2183delAA > G, or 3659del C. P value was determined by one way ANOVA. ** indicates significant difference ( P ≤0.01) when compared with forskolin stimulated CFTR function in DMSO (vehicle) treated E831X/F508del HNEs. * ( P ≤0.05) when compared with CFTR function in modulator treated F508del/Indel HNEs.
    Figure Legend Snippet: CFTR nonsense variant with splicing defect has residual function that benefits from modulator treatments. (A) A schematic illustration of CFTR-Expression Minigene with introns 14–18 (introns 14 and 16 are full-length, and 15, 17, 18 are abridged). Arrow indicates location of E831X (top). CFTR mRNA splicing patterns of the total RNA extracted from HEK293 cells transiently transfected with E831X-EMG. (B) Steady state amounts of different isoforms of CFTR produced from E831X-EMG-i14-i18 expressed transiently in HEK293 cells. Lysates from cells expressing WT-EMG i14-i18, intronless WT CFTR or F508del served as positive controls, and empty vector as negative control. Immunoblot (IB) was probed with anti-CFTR antibody, 596 (CFFT). Horizontal arrows indicate to isoforms corresponding to (i) a normal codon (E831) substituted with a stop codon, (ii) deletion of complete exon 16, and (iii) deletion of a single amino acid E831. Beta-Actin was used as loading control. (C) Short-circuit (I sc ) tracing of CFTR function observed in CFBE-stable cells expressing E831X mounted on Ussing chamber. Cells were treated for 48 h with correctors (lumacaftor/tezacaftor or both, 3 μM each) and acutely with potentiator (ivacaftor, 10 μM). Change in I sc (ΔI sc ) was defined as the current inhibited by Inh-172 after sustained Isc responses achieved upon stimulation with forskolin alone or sequentially with ivacaftor. Data are presented as mean±SEM (n = 3). P value was determined by one way ANOVA. **** indicates significant difference ( P ≤0.0001) when compared with forskolin stimulated CFTR function in DMSO (vehicle) treated cells. WT-CFTR function represents forskolin stimulated Isc without modulator treatment in cells expressing EMG i14-i18. (D ) A tracing of CFTR function observed in primary nasal epithelial cells of an individual harboring E831X/F508del. CF-Human nasal epithelial (HNE) cells were treated for 24 h with lumacaftor and tezacaftor, 3 μM each, and acutely with Ivacaftor (10 μM). Stacked bar graph is a comparison of improvement in CFTR function of E831X/F508del vs F508del/Indel. Alternate Indel alleles were either 2184insA, 2183delAA > G, or 3659del C. P value was determined by one way ANOVA. ** indicates significant difference ( P ≤0.01) when compared with forskolin stimulated CFTR function in DMSO (vehicle) treated E831X/F508del HNEs. * ( P ≤0.05) when compared with CFTR function in modulator treated F508del/Indel HNEs.

    Techniques Used: Variant Assay, Expressing, Transfection, Produced, Plasmid Preparation, Negative Control

    Nonsense and frameshift mutations at 3’ region that synthesize complex glycosylated truncated protein can respond to CFTR modulators. (A) Schematic of CFTR-Expression Minigene with full-length introns 25 and 26 (EMG-i25-i26) constructed in pcDNA5FRT plasmid. CFTR expression is driven by a CMV promoter. The location of each studied variant is shown relative to CFTR exons and regions predicted to elicit NMD. (B) Real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) showing relative steady state levels of CFTR transcript in HEK293 stable cells expressing wild-type EMG or EMGs with nonsense or frameshift variants, as indicated. Values were normalized to B2M . Mean ± SEM ( n = 3) measured in triplicates. P value was determined by one way ANOVA. **** indicates significant difference ( P ≤0.0001) when compared with CFTR mRNA abundance in cells expressing WT-EMG. (C) Immunoblot (IB) of the steady state amounts of immature core-glycosylated ( band B ) and the mature complex-glycosylated mature CFTR protein ( band C ). Lysates were collected from HEK293 cells expressing WT-EMG or EMGs with different PTC-generating variants. Lysates from cells expressing either intronless WT CFTR or F508del served as controls, or empty vector as negative control. 40 μg of total cell lysates were electrophoresed and IB was probed with anti-CFTR antibody (596 # Cystic Fibrosis Foundation Therapeutics). (D) Schematic illustration showing three groups of 3’- nonsense variants based on mRNA stability and protein maturity. (E) Immunoblot of HEK293 stable cells expressing Q1390X or E1418X. The cells were incubated for 48 h with DMSO (.03%) or corrector compounds (lumacaftor and tezacaftor either alone or in combination—3 μM each). CFTR was visualized with anti-CFTR antibody, 596 (CFFT). (F) A representative Ussing chamber tracing of EMG E1418X-expressing CFBE stable cells grown on snap-wells. Short-circuit current (I sc ) measurements were recorded in Ussing chambers after treatment of cells with 0.03% DMSO (vehicle) or 3 μM corrector compounds (lumacaftor/tezacaftor or both) for 48 h. Cells were mounted on Ussing chambers to measure CFTR mediated chloride channel activity as a proxy of CFTR function. After stabilization of the basal current, forskolin (10 μM) was added to the basolateral chamber followed by potentiator, ivacaftor (10 μM), and CFTR Inhibitor 172 (10 μM) added to the apical chambers. Inh-172 was added earlier in DMSO no ivacaftor (dashed blue line) treated cells. (G and H) Stacked bar graphs indicate effect of modulator treatment on CFBE (G) and MDCK (H) stable cells expressing different CFTR 3’ nonsense variants. Change in I sc (ΔIsc) was defined as the current inhibited by Inh-172 after sustained Isc responses were achieved upon stimulation with forskolin alone or sequentially with ivacaftor. Mean ± SEM ( n = 3–8). WT-CFTR function represents forskolin stimulated Isc without modulator treatment in cells expressing EMG i25-i26. P value was determined by one way ANOVA. **** P ≤0.0001, ** P ≤0.01, * P ≤0.05, and n.s. (not significant, P > 0.05); when compared with forskolin stimulated CFTR function in DMSO (vehicle) treated cells expressing respective variant.
    Figure Legend Snippet: Nonsense and frameshift mutations at 3’ region that synthesize complex glycosylated truncated protein can respond to CFTR modulators. (A) Schematic of CFTR-Expression Minigene with full-length introns 25 and 26 (EMG-i25-i26) constructed in pcDNA5FRT plasmid. CFTR expression is driven by a CMV promoter. The location of each studied variant is shown relative to CFTR exons and regions predicted to elicit NMD. (B) Real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) showing relative steady state levels of CFTR transcript in HEK293 stable cells expressing wild-type EMG or EMGs with nonsense or frameshift variants, as indicated. Values were normalized to B2M . Mean ± SEM ( n = 3) measured in triplicates. P value was determined by one way ANOVA. **** indicates significant difference ( P ≤0.0001) when compared with CFTR mRNA abundance in cells expressing WT-EMG. (C) Immunoblot (IB) of the steady state amounts of immature core-glycosylated ( band B ) and the mature complex-glycosylated mature CFTR protein ( band C ). Lysates were collected from HEK293 cells expressing WT-EMG or EMGs with different PTC-generating variants. Lysates from cells expressing either intronless WT CFTR or F508del served as controls, or empty vector as negative control. 40 μg of total cell lysates were electrophoresed and IB was probed with anti-CFTR antibody (596 # Cystic Fibrosis Foundation Therapeutics). (D) Schematic illustration showing three groups of 3’- nonsense variants based on mRNA stability and protein maturity. (E) Immunoblot of HEK293 stable cells expressing Q1390X or E1418X. The cells were incubated for 48 h with DMSO (.03%) or corrector compounds (lumacaftor and tezacaftor either alone or in combination—3 μM each). CFTR was visualized with anti-CFTR antibody, 596 (CFFT). (F) A representative Ussing chamber tracing of EMG E1418X-expressing CFBE stable cells grown on snap-wells. Short-circuit current (I sc ) measurements were recorded in Ussing chambers after treatment of cells with 0.03% DMSO (vehicle) or 3 μM corrector compounds (lumacaftor/tezacaftor or both) for 48 h. Cells were mounted on Ussing chambers to measure CFTR mediated chloride channel activity as a proxy of CFTR function. After stabilization of the basal current, forskolin (10 μM) was added to the basolateral chamber followed by potentiator, ivacaftor (10 μM), and CFTR Inhibitor 172 (10 μM) added to the apical chambers. Inh-172 was added earlier in DMSO no ivacaftor (dashed blue line) treated cells. (G and H) Stacked bar graphs indicate effect of modulator treatment on CFBE (G) and MDCK (H) stable cells expressing different CFTR 3’ nonsense variants. Change in I sc (ΔIsc) was defined as the current inhibited by Inh-172 after sustained Isc responses were achieved upon stimulation with forskolin alone or sequentially with ivacaftor. Mean ± SEM ( n = 3–8). WT-CFTR function represents forskolin stimulated Isc without modulator treatment in cells expressing EMG i25-i26. P value was determined by one way ANOVA. **** P ≤0.0001, ** P ≤0.01, * P ≤0.05, and n.s. (not significant, P > 0.05); when compared with forskolin stimulated CFTR function in DMSO (vehicle) treated cells expressing respective variant.

    Techniques Used: Expressing, Construct, Plasmid Preparation, Variant Assay, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Negative Control, Incubation, Activity Assay

    NMD inhibition has a synergistic effect on corrector-potentiator combination response in stable cells expressing nonsense variants in exon 22 that produce mature truncated CFTR. (A) Relative expression of the alternate CFTR allele in the primary nasal cells of CF individuals carrying exon 22 nonsense variant. Pyrosequencing assay was designed such that exon 22 with upstream and downstream flanking exons was amplified from the corresponding cDNA preparations. Sequencing primer yielded relative abundances of alternate alleles at the respective loci where nucleotide change occurred. (B ) CFTR mRNA decay in HEK293 cells stably expressing wild type EMG or EMG harboring nonsense variants R1158X or S1196X. Actinomycin D (3 μg/ml) was added at time 0 to induce transcriptional shut-down. Cells were collected at the indicated time points. Levels of the CFTR mRNAs were assessed by RT-qPCR, normalized to B2M mRNA and displayed as a percentage of the levels at t = 0. Mean ± SEM ( n = 3) (C) Efficiency of siRNA mediated knock down of UPF1 detected on IB of whole cell lysates collected from HEK293 cells stably expressing either R1158X or S1196X. GAPDH siRNA and non-target (NT) siRNA were used as positive and negative controls respectively. Beta-Actin was used as loading control. (D) Effect of direct NMD inhibition on the level of CFTR transcript by siRNA mediated knock down of UPF1 in HEK293 cells stably expressing either R1158X or S1196X. Levels of the CFTR mRNAs were assessed by RT-qPCR and normalized to B2M mRNA. Mean ± SEM, n = 3 independent biological triplicates, P value was determined by two way ANOVA. ** ( P ≤0.01) and *** ( P ≤0.001) indicate significant difference when compared with CFTR mRNA abundance in untreated cells (E) Short-circuit (I sc ) tracings of CFBE-S1196X stable cells recorded in Ussing chambers after direct inhibition of NMD by UPF1 . Cells were transfected with Upf1 siRNA at 50% confluency for 4 days before being mounted on Ussing chambers. GAPDH and non-targeted (NT) siRNA transfections were used as controls. Cells were incubated with lumacaftor (3 μM) or DMSO (0.03%) during last 48h of siRNA transfections. (F) Stacked bar graphs indicate effect of UPF1 siRNA in combination with CFTR modulators. Change in I sc (ΔI sc ) was defined as the current inhibited by Inh-172 after sustained Isc responses were achieved upon stimulation with forskolin alone or sequentially with ivacaftor. Mean ± SEM ( n = 3). P value was determined by one way ANOVA. ** ( P ≤0.01) indicate significant difference when compared with forskolin stimulated CFTR function in NT siRNA transfected cells and **** ( P ≤0.0001) indicate significant difference when compared with ivacaftor activated CFTR function in NT siRNA transfected cells incubated with or without lumacaftor. WT-CFTR function represents forskolin stimulated ΔI sc without modulator treatment in cells expressing EMG i21-i22.
    Figure Legend Snippet: NMD inhibition has a synergistic effect on corrector-potentiator combination response in stable cells expressing nonsense variants in exon 22 that produce mature truncated CFTR. (A) Relative expression of the alternate CFTR allele in the primary nasal cells of CF individuals carrying exon 22 nonsense variant. Pyrosequencing assay was designed such that exon 22 with upstream and downstream flanking exons was amplified from the corresponding cDNA preparations. Sequencing primer yielded relative abundances of alternate alleles at the respective loci where nucleotide change occurred. (B ) CFTR mRNA decay in HEK293 cells stably expressing wild type EMG or EMG harboring nonsense variants R1158X or S1196X. Actinomycin D (3 μg/ml) was added at time 0 to induce transcriptional shut-down. Cells were collected at the indicated time points. Levels of the CFTR mRNAs were assessed by RT-qPCR, normalized to B2M mRNA and displayed as a percentage of the levels at t = 0. Mean ± SEM ( n = 3) (C) Efficiency of siRNA mediated knock down of UPF1 detected on IB of whole cell lysates collected from HEK293 cells stably expressing either R1158X or S1196X. GAPDH siRNA and non-target (NT) siRNA were used as positive and negative controls respectively. Beta-Actin was used as loading control. (D) Effect of direct NMD inhibition on the level of CFTR transcript by siRNA mediated knock down of UPF1 in HEK293 cells stably expressing either R1158X or S1196X. Levels of the CFTR mRNAs were assessed by RT-qPCR and normalized to B2M mRNA. Mean ± SEM, n = 3 independent biological triplicates, P value was determined by two way ANOVA. ** ( P ≤0.01) and *** ( P ≤0.001) indicate significant difference when compared with CFTR mRNA abundance in untreated cells (E) Short-circuit (I sc ) tracings of CFBE-S1196X stable cells recorded in Ussing chambers after direct inhibition of NMD by UPF1 . Cells were transfected with Upf1 siRNA at 50% confluency for 4 days before being mounted on Ussing chambers. GAPDH and non-targeted (NT) siRNA transfections were used as controls. Cells were incubated with lumacaftor (3 μM) or DMSO (0.03%) during last 48h of siRNA transfections. (F) Stacked bar graphs indicate effect of UPF1 siRNA in combination with CFTR modulators. Change in I sc (ΔI sc ) was defined as the current inhibited by Inh-172 after sustained Isc responses were achieved upon stimulation with forskolin alone or sequentially with ivacaftor. Mean ± SEM ( n = 3). P value was determined by one way ANOVA. ** ( P ≤0.01) indicate significant difference when compared with forskolin stimulated CFTR function in NT siRNA transfected cells and **** ( P ≤0.0001) indicate significant difference when compared with ivacaftor activated CFTR function in NT siRNA transfected cells incubated with or without lumacaftor. WT-CFTR function represents forskolin stimulated ΔI sc without modulator treatment in cells expressing EMG i21-i22.

    Techniques Used: Inhibition, Expressing, Variant Assay, Pyrosequencing Assay, Amplification, Sequencing, Stable Transfection, Quantitative RT-PCR, Transfection, Incubation

    5’ nonsense variants that do not undergo NMD are the potential targets of read-through agents. (A) Left panel—Sanger sequencing, and right panel—fragment analysis. Total RNA was extracted from the conditionally reprogrammed nasal epithelial cells of CF individual with F508del/L88X genotype. RT-PCRs were performed using CFTR-specific primers to amplify L88X and F508del regions. Area under the peak was used to determine expression of L88X transcript compared to F508del. (B) RNA-seq of the primary human nasal epithelial cells of healthy and L88X/F508del individuals. Density profile of all expressed genes (top), and relative transcript counts of L88X compared to F508del (bottom). (C) Schematic of CFTR-Expression Minigene with abridged introns 1, 2, 3, 4 and 5 constructed in pcDNA5FRT plasmid. CFTR exons are shown in boxes and abridged introns in dashed lines. The location of each studied variant is shown relative to the CFTR exons and regions predicted to elicit NMD. (D) Graph shows relative steady state levels of CFTR transcript in HEK293 stable cells expressing wild-type EMG or EMGs with truncations at residue position, as indicated on the labels. Values were normalized to B2M. Mean ± SEM ( n = 3) measured in triplicates. P value was determined by one way ANOVA. No significant difference (n.s.) ( P > 0.01) when compared with CFTR mRNA abundance in cells expressing WT-EMG. (E) Immunoblot of the naturally occurring 5’-truncations on the steady state amounts of CFTR protein expressed in HEK293 cells. CFTR was visualized with anti-CFTR antibody-596 (CFFT), and anti-Na + K + ATPase served as control. (F) CFTR function measured in CFBE stable expressing L88X. Cells were incubated for 24 h with readthrough compound (G418, 5 μM and 25 μM)/ corrector (lumacaftor, 3 μM) or both. Short-circuit (I sc ) tracing of CFTR function observed in CFBE-stable cells expressing L88X mounted on Ussing chamber. Change in Isc (ΔI sc ) was defined as the current inhibited by Inh-172 after sustained Isc responses achieved upon stimulation with forskolin alone or sequentially with ivacaftor. Data are presented as mean ± SEM (n = 3). P value was determined by one way ANOVA. **** ( P ≤0.0001), and *** ( P ≤0.001) indicate significant difference when compared with forskolin stimulated CFTR function in DMSO (vehicle) treated cells. WT-CFTR function represents forskolin stimulated Isc without modulator treatment in cells expressing EMG i1-i5.
    Figure Legend Snippet: 5’ nonsense variants that do not undergo NMD are the potential targets of read-through agents. (A) Left panel—Sanger sequencing, and right panel—fragment analysis. Total RNA was extracted from the conditionally reprogrammed nasal epithelial cells of CF individual with F508del/L88X genotype. RT-PCRs were performed using CFTR-specific primers to amplify L88X and F508del regions. Area under the peak was used to determine expression of L88X transcript compared to F508del. (B) RNA-seq of the primary human nasal epithelial cells of healthy and L88X/F508del individuals. Density profile of all expressed genes (top), and relative transcript counts of L88X compared to F508del (bottom). (C) Schematic of CFTR-Expression Minigene with abridged introns 1, 2, 3, 4 and 5 constructed in pcDNA5FRT plasmid. CFTR exons are shown in boxes and abridged introns in dashed lines. The location of each studied variant is shown relative to the CFTR exons and regions predicted to elicit NMD. (D) Graph shows relative steady state levels of CFTR transcript in HEK293 stable cells expressing wild-type EMG or EMGs with truncations at residue position, as indicated on the labels. Values were normalized to B2M. Mean ± SEM ( n = 3) measured in triplicates. P value was determined by one way ANOVA. No significant difference (n.s.) ( P > 0.01) when compared with CFTR mRNA abundance in cells expressing WT-EMG. (E) Immunoblot of the naturally occurring 5’-truncations on the steady state amounts of CFTR protein expressed in HEK293 cells. CFTR was visualized with anti-CFTR antibody-596 (CFFT), and anti-Na + K + ATPase served as control. (F) CFTR function measured in CFBE stable expressing L88X. Cells were incubated for 24 h with readthrough compound (G418, 5 μM and 25 μM)/ corrector (lumacaftor, 3 μM) or both. Short-circuit (I sc ) tracing of CFTR function observed in CFBE-stable cells expressing L88X mounted on Ussing chamber. Change in Isc (ΔI sc ) was defined as the current inhibited by Inh-172 after sustained Isc responses achieved upon stimulation with forskolin alone or sequentially with ivacaftor. Data are presented as mean ± SEM (n = 3). P value was determined by one way ANOVA. **** ( P ≤0.0001), and *** ( P ≤0.001) indicate significant difference when compared with forskolin stimulated CFTR function in DMSO (vehicle) treated cells. WT-CFTR function represents forskolin stimulated Isc without modulator treatment in cells expressing EMG i1-i5.

    Techniques Used: Sequencing, Expressing, RNA Sequencing Assay, Construct, Plasmid Preparation, Variant Assay, Incubation

    18) Product Images from "mPGES-1/PGE2 promotes the growth of T-ALL cells in vitro and in vivo by regulating the expression of MTDH via the EP3/cAMP/PKA/CREB pathway"

    Article Title: mPGES-1/PGE2 promotes the growth of T-ALL cells in vitro and in vivo by regulating the expression of MTDH via the EP3/cAMP/PKA/CREB pathway

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-020-2380-9

    mPGES-1/PGE2 regulated MTDH expression via the cAMP/PKA-CREB pathway. The adenylate cyclase (AC) agonist forskolin increased the expression of p-CREB, CREB and MTDH, while the protein kinase A (PKA) inhibitor H89 induced the opposite effects on these proteins ( a , b ). The relative luciferase activity (firefly/Renilla) in group 1 (cells cotransfected with pCMV-GFP-Puro-01-CREB1, pGL3-MTDH and pRL-TK) was almost 3.43 times that in group 2 (cells cotransfected with pCMV-GFP-Puro-NC, pGL3-MTDH and pRL-TK) ( c ). ** p
    Figure Legend Snippet: mPGES-1/PGE2 regulated MTDH expression via the cAMP/PKA-CREB pathway. The adenylate cyclase (AC) agonist forskolin increased the expression of p-CREB, CREB and MTDH, while the protein kinase A (PKA) inhibitor H89 induced the opposite effects on these proteins ( a , b ). The relative luciferase activity (firefly/Renilla) in group 1 (cells cotransfected with pCMV-GFP-Puro-01-CREB1, pGL3-MTDH and pRL-TK) was almost 3.43 times that in group 2 (cells cotransfected with pCMV-GFP-Puro-NC, pGL3-MTDH and pRL-TK) ( c ). ** p

    Techniques Used: Expressing, Luciferase, Activity Assay

    19) Product Images from "Controlling the Switch from Neurogenesis to Pluripotency during Marmoset Monkey Somatic Cell Reprogramming with Self-Replicating mRNAs and Small Molecules"

    Article Title: Controlling the Switch from Neurogenesis to Pluripotency during Marmoset Monkey Somatic Cell Reprogramming with Self-Replicating mRNAs and Small Molecules

    Journal: Cells

    doi: 10.3390/cells9112422

    Generation of marmoset induced pluripotent stem cells (iPSCs) with the Tomato-modified self-replicating mRNA based on the Venezuelan equine encephalitis virus (VEE-OKS-iM-iTomato). ( A ) Structure of the reprogramming VEE-OKS-iM-iTomato mRNA. ( B ) Scheme of the reprogramming process. ( C ) Image of marmoset fetal fibroblasts (cjFFs) transfected with VEE-OKS-iM-iTomato at day 3 post-transfection. Transfected cells are recognizable by their red fluorescence. ( D ) An intermediate primary colony at day 27 post-transfection with red VEE-OKS-iM-iTomato fluorescence. ( E ) An intermediate primary colony at day 27 post-transfection with characteristic compact morphology and clearly defined borders (indicated with arrowheads). The cells beyond the upper and left borders of the colony are non-reprogrammed cjFFs. ( F ) An intermediate primary colony stained for alkaline phosphatase (AP). ( G ) Marmoset iPSC colony after second round of reprogramming of the intermediate primary cells with IWR1, CHIR99021, CGP77675, human recombinant leukemia inhibitory factor (hrLIF), and Forskolin. ( H ) Morphology of marmoset iPSC colonies growing on Geltrex at P27. (All scale bars = 200 μm).
    Figure Legend Snippet: Generation of marmoset induced pluripotent stem cells (iPSCs) with the Tomato-modified self-replicating mRNA based on the Venezuelan equine encephalitis virus (VEE-OKS-iM-iTomato). ( A ) Structure of the reprogramming VEE-OKS-iM-iTomato mRNA. ( B ) Scheme of the reprogramming process. ( C ) Image of marmoset fetal fibroblasts (cjFFs) transfected with VEE-OKS-iM-iTomato at day 3 post-transfection. Transfected cells are recognizable by their red fluorescence. ( D ) An intermediate primary colony at day 27 post-transfection with red VEE-OKS-iM-iTomato fluorescence. ( E ) An intermediate primary colony at day 27 post-transfection with characteristic compact morphology and clearly defined borders (indicated with arrowheads). The cells beyond the upper and left borders of the colony are non-reprogrammed cjFFs. ( F ) An intermediate primary colony stained for alkaline phosphatase (AP). ( G ) Marmoset iPSC colony after second round of reprogramming of the intermediate primary cells with IWR1, CHIR99021, CGP77675, human recombinant leukemia inhibitory factor (hrLIF), and Forskolin. ( H ) Morphology of marmoset iPSC colonies growing on Geltrex at P27. (All scale bars = 200 μm).

    Techniques Used: Modification, Transfection, Fluorescence, Staining, Recombinant

    Role of small molecule inhibitors in maintaining pluripotency gene expression of marmoset iPSCs in long-term culture. ( A ) Marmoset iPSCs cultured either with full culture medium, with omission of individual factors (-IWR1, -CHIR99021, -CGP77675, -hrLIF, -Forskolin), or without any inhibitors and hrLIF. (All scale bars = 50 μm). ( B ) Pluripotency gene expression of marmoset iPSCs cultured without different inhibitors or hrLIF for 5–6 passages determined by relative quantitation qPCR. One of the lines cultured without any inhibitors and hrLIF was used as a reference. (Data are presented as mean + SEM. Statistically significant differences between experimental groups are indicated with asterisks as follows: * p
    Figure Legend Snippet: Role of small molecule inhibitors in maintaining pluripotency gene expression of marmoset iPSCs in long-term culture. ( A ) Marmoset iPSCs cultured either with full culture medium, with omission of individual factors (-IWR1, -CHIR99021, -CGP77675, -hrLIF, -Forskolin), or without any inhibitors and hrLIF. (All scale bars = 50 μm). ( B ) Pluripotency gene expression of marmoset iPSCs cultured without different inhibitors or hrLIF for 5–6 passages determined by relative quantitation qPCR. One of the lines cultured without any inhibitors and hrLIF was used as a reference. (Data are presented as mean + SEM. Statistically significant differences between experimental groups are indicated with asterisks as follows: * p

    Techniques Used: Expressing, Cell Culture, Quantitation Assay, Real-time Polymerase Chain Reaction

    Paradigm for two-step flexible reprogramming of marmoset somatic cells to pluripotency or to the neural lineage. Transfection of somatic cells with vector carrying reprogramming transcription factors and culture in medium containing CHIR99021 and SB431542 leads to generation of intermediate primary colonies. These colonies can be maintained as neural progenitors with the same culture medium, directed into the neural lineage by culture with bFGF and EGF, or further reprogrammed to iPSCs by culture with IWR1, CHIR99021, CGP77675, leukemia inhibitory factor (LIF), and Forskolin.
    Figure Legend Snippet: Paradigm for two-step flexible reprogramming of marmoset somatic cells to pluripotency or to the neural lineage. Transfection of somatic cells with vector carrying reprogramming transcription factors and culture in medium containing CHIR99021 and SB431542 leads to generation of intermediate primary colonies. These colonies can be maintained as neural progenitors with the same culture medium, directed into the neural lineage by culture with bFGF and EGF, or further reprogrammed to iPSCs by culture with IWR1, CHIR99021, CGP77675, leukemia inhibitory factor (LIF), and Forskolin.

    Techniques Used: Transfection, Plasmid Preparation

    20) Product Images from "Residual function of cystic fibrosis mutants predicts response to small molecule CFTR modulators"

    Article Title: Residual function of cystic fibrosis mutants predicts response to small molecule CFTR modulators

    Journal: JCI Insight

    doi: 10.1172/jci.insight.121159

    Lumacaftor response correlates with residual function. ( A ) Forskolin (10 μM) stimulated CFTR function of 45 missense variants expressed in CF bronchial epithelial (CFBE) cells treated for 24 hours with 6 μM lumacaftor compared with residual forskolin-stimulated (10 μM) CFTR function when incubated for 24 hours with an equal volume of DMSO. Each variant was measured n ≥ 3 and plotted as mean ± SEM on both axes. ( B ) Forskolin-stimulated (5 μM) CFTR function of 18 missense variants expressed in Fisher rat thyroid (FRT) cells treated for 48 hours with 3 μM lumacaftor compared with residual forskolin-stimulated (5 μM) CFTR function when incubated for 48 hours with an equal volume of DMSO. Each variant was measured n ≥ 3 and plotted as mean ± SEM on both axes. ( C ) Separation of variants based on their fold response to lumacaftor. Response of cell lines expressing G91R, E92K, L138ins, L145H, and L206W were designated as outliers by demonstrating fold response greater than 2 SD beyond the mean fold response of all variants studied in CFBE and FRT cells, and they are labeled as high-response variants. Intermediate-response variants were those that remained outliers when high-response variants were removed from the comparison. All remaining variants were classified as modest response. Lines through data points represent the mean value ± 1 SD for modest-response variants and mean of intermediate- and high-response variants. ( D ) Comparison of best fit functions for variants expressed in CFBE and FRT cells, which demonstrated modest response to lumacaftor. Correlation ( r ) values calculated using Pearson linear correlation.
    Figure Legend Snippet: Lumacaftor response correlates with residual function. ( A ) Forskolin (10 μM) stimulated CFTR function of 45 missense variants expressed in CF bronchial epithelial (CFBE) cells treated for 24 hours with 6 μM lumacaftor compared with residual forskolin-stimulated (10 μM) CFTR function when incubated for 24 hours with an equal volume of DMSO. Each variant was measured n ≥ 3 and plotted as mean ± SEM on both axes. ( B ) Forskolin-stimulated (5 μM) CFTR function of 18 missense variants expressed in Fisher rat thyroid (FRT) cells treated for 48 hours with 3 μM lumacaftor compared with residual forskolin-stimulated (5 μM) CFTR function when incubated for 48 hours with an equal volume of DMSO. Each variant was measured n ≥ 3 and plotted as mean ± SEM on both axes. ( C ) Separation of variants based on their fold response to lumacaftor. Response of cell lines expressing G91R, E92K, L138ins, L145H, and L206W were designated as outliers by demonstrating fold response greater than 2 SD beyond the mean fold response of all variants studied in CFBE and FRT cells, and they are labeled as high-response variants. Intermediate-response variants were those that remained outliers when high-response variants were removed from the comparison. All remaining variants were classified as modest response. Lines through data points represent the mean value ± 1 SD for modest-response variants and mean of intermediate- and high-response variants. ( D ) Comparison of best fit functions for variants expressed in CFBE and FRT cells, which demonstrated modest response to lumacaftor. Correlation ( r ) values calculated using Pearson linear correlation.

    Techniques Used: Incubation, Variant Assay, Expressing, Labeling

    Variants located in the sixth transmembrane domain (TM6) show modest response to ivacaftor. ( A ) reveals heterogeneous response to 10 μM ivacaftor of partially or well-processed low–residual function missense variants (labeled). Filled green circles represent variants approved by FDA for ivacaftor treatment; G551D labeled in italics. ( B ) Western blot demonstrating that all TM6 variants produce mature C band CFTR protein when transiently expressed in HEK293 cells and representative I sc tracings of all TM6 variants stably expressed in CF bronchial epithelial (CFBE) cells demonstrating response to acute treatment with 10 μM ivacaftor, recorded as area-corrected current (μA/cm 2 ), over time, measured in minutes represented by tick marks in 1-minute intervals. Data are representative of n ≥ 3 for each variant. ( C ) Summary data for response of TM6 variants to acute treatment with 10 μM ivacaftor expressed as %WT function. Box plots divide the data by quartile, with the median value indicated by a horizontal line within the box and whiskers extended to minimum and maximum values. ( D ) Fold response for acute treatment with 10 μM ivacaftor calculated over residual function (10 μM forskolin) of TM6 variants compared with modest-responsive variants identified in this study. Box plots divide the data by quartile, with the median value indicated by a horizontal line within the box and whiskers extended to minimum and maximum values.
    Figure Legend Snippet: Variants located in the sixth transmembrane domain (TM6) show modest response to ivacaftor. ( A ) reveals heterogeneous response to 10 μM ivacaftor of partially or well-processed low–residual function missense variants (labeled). Filled green circles represent variants approved by FDA for ivacaftor treatment; G551D labeled in italics. ( B ) Western blot demonstrating that all TM6 variants produce mature C band CFTR protein when transiently expressed in HEK293 cells and representative I sc tracings of all TM6 variants stably expressed in CF bronchial epithelial (CFBE) cells demonstrating response to acute treatment with 10 μM ivacaftor, recorded as area-corrected current (μA/cm 2 ), over time, measured in minutes represented by tick marks in 1-minute intervals. Data are representative of n ≥ 3 for each variant. ( C ) Summary data for response of TM6 variants to acute treatment with 10 μM ivacaftor expressed as %WT function. Box plots divide the data by quartile, with the median value indicated by a horizontal line within the box and whiskers extended to minimum and maximum values. ( D ) Fold response for acute treatment with 10 μM ivacaftor calculated over residual function (10 μM forskolin) of TM6 variants compared with modest-responsive variants identified in this study. Box plots divide the data by quartile, with the median value indicated by a horizontal line within the box and whiskers extended to minimum and maximum values.

    Techniques Used: Labeling, Western Blot, Stable Transfection, Variant Assay

    Ivacaftor/lumacaftor (iva/lum) response correlates with residual function. ( A ) Ivacaftor (10 μM) enhanced CFTR function of 45 missense variants expressed in CFBE cells following for 24-hour incubation with 6 μM lumacaftor compared with residual forskolin-stimulated (10 μM) CFTR function when incubated for 24 hours with DMSO. Each variant was measured n ≥ 3 and plotted as mean ± SEM. ( B ) Ivacaftor (5 μM) enhanced CFTR function of 18 missense variants expressed in FRT cells following 24-hour incubation with 3 μM lumacaftor compared with residual forskolin-stimulated (5 μM) CFTR function when incubated for 24 hours with DMSO. Each variant was measured n ≥ 3 and plotted as mean ± SEM. ( C ) Separation of variants based on their fold response to lumacaftor. Response of cell lines expressing G91R, E92K, L138ins, L145H, and G551D (CFBE) were designated as outliers by demonstrating a fold response greater than 2 SD beyond the mean fold response of all variants studied in CFBE and FRT cells, and they are labeled as high-response variants. Intermediate-response variants were those that remained outliers when high-response variants were removed from the comparison. All remaining variants were classified as modest response. Lines through data points represent the mean value ± 1 SD for modest-response variants and mean of intermediate- and high-response variants. ( D ) Comparison of best fit functions for variants expressed in CFBE and FRT cells that demonstrated modest response to ivacaftor/lumacaftor combination treatment. Correlation ( r ) values calculated using Pearson linear correlation.
    Figure Legend Snippet: Ivacaftor/lumacaftor (iva/lum) response correlates with residual function. ( A ) Ivacaftor (10 μM) enhanced CFTR function of 45 missense variants expressed in CFBE cells following for 24-hour incubation with 6 μM lumacaftor compared with residual forskolin-stimulated (10 μM) CFTR function when incubated for 24 hours with DMSO. Each variant was measured n ≥ 3 and plotted as mean ± SEM. ( B ) Ivacaftor (5 μM) enhanced CFTR function of 18 missense variants expressed in FRT cells following 24-hour incubation with 3 μM lumacaftor compared with residual forskolin-stimulated (5 μM) CFTR function when incubated for 24 hours with DMSO. Each variant was measured n ≥ 3 and plotted as mean ± SEM. ( C ) Separation of variants based on their fold response to lumacaftor. Response of cell lines expressing G91R, E92K, L138ins, L145H, and G551D (CFBE) were designated as outliers by demonstrating a fold response greater than 2 SD beyond the mean fold response of all variants studied in CFBE and FRT cells, and they are labeled as high-response variants. Intermediate-response variants were those that remained outliers when high-response variants were removed from the comparison. All remaining variants were classified as modest response. Lines through data points represent the mean value ± 1 SD for modest-response variants and mean of intermediate- and high-response variants. ( D ) Comparison of best fit functions for variants expressed in CFBE and FRT cells that demonstrated modest response to ivacaftor/lumacaftor combination treatment. Correlation ( r ) values calculated using Pearson linear correlation.

    Techniques Used: Incubation, Variant Assay, Expressing, Labeling

    Summary of response of missense variants to CFTR modulators. ( A ) CFTR function for variants that were modest-response variants for ivacaftor and lumacaftor when treated with forskolin (baseline), ivacaftor, lumacaftor, or iva/lum combination. Error bars represent ± SEM. ( B ) Response to ivacaftor, lumacaftor, or ivacaftor/lumacaftor combination for variants designated as intermediate- or high-response to ivacaftor. ( C ) Response to ivacaftor, lumacaftor, or iva/lum combination for variants designated as intermediate or high response to lumacaftor.
    Figure Legend Snippet: Summary of response of missense variants to CFTR modulators. ( A ) CFTR function for variants that were modest-response variants for ivacaftor and lumacaftor when treated with forskolin (baseline), ivacaftor, lumacaftor, or iva/lum combination. Error bars represent ± SEM. ( B ) Response to ivacaftor, lumacaftor, or ivacaftor/lumacaftor combination for variants designated as intermediate- or high-response to ivacaftor. ( C ) Response to ivacaftor, lumacaftor, or iva/lum combination for variants designated as intermediate or high response to lumacaftor.

    Techniques Used:

    Ivacaftor response correlates with residual function. ( A ) Ivacaftor (10 μM) enhanced CFTR function compared with residual forskolin-stimulated (10 μM) CFTR function for 45 variants expressed in CF bronchial epithelial (CFBE) cells. Each variant measured n ≥ 3 and was plotted as mean ± SEM on both axes. ( B ) Ivacaftor (5 μM) enhanced CFTR function compared with residual forskolin-stimulated (5 μM) CFTR function for 18 variants expressed in Fisher rat thyroid (FRT) cells. Each variant measured n ≥ 3 and was plotted as mean ± SEM on both axes. ( C ) Separation of variants based on their fold response to ivacaftor. Response of cell lines expressing G551D (CFBE), G551D (FRT), S1159F, and S1159P were designated as outliers by demonstrating fold response greater than 2 SD beyond the mean fold response of all variants studied in CFBE and FRT cells and labeled as high-response variants. Intermediate-response variants were those that remained outliers when high-response variants were removed from the comparison. All remaining variants were classified as modest response. Lines through data points represent the mean value ± 1 SD for modest-response variants and mean of intermediate- and high-response variants. ( D ) of nongating variants plotted as in A and B . ( E ) Comparison of trend lines of modest-response variants identified in CFBE and FRT studies compared with nongating variants identified in FRT* cells. Correlation ( r ) values calculated using Pearson linear correlation.
    Figure Legend Snippet: Ivacaftor response correlates with residual function. ( A ) Ivacaftor (10 μM) enhanced CFTR function compared with residual forskolin-stimulated (10 μM) CFTR function for 45 variants expressed in CF bronchial epithelial (CFBE) cells. Each variant measured n ≥ 3 and was plotted as mean ± SEM on both axes. ( B ) Ivacaftor (5 μM) enhanced CFTR function compared with residual forskolin-stimulated (5 μM) CFTR function for 18 variants expressed in Fisher rat thyroid (FRT) cells. Each variant measured n ≥ 3 and was plotted as mean ± SEM on both axes. ( C ) Separation of variants based on their fold response to ivacaftor. Response of cell lines expressing G551D (CFBE), G551D (FRT), S1159F, and S1159P were designated as outliers by demonstrating fold response greater than 2 SD beyond the mean fold response of all variants studied in CFBE and FRT cells and labeled as high-response variants. Intermediate-response variants were those that remained outliers when high-response variants were removed from the comparison. All remaining variants were classified as modest response. Lines through data points represent the mean value ± 1 SD for modest-response variants and mean of intermediate- and high-response variants. ( D ) of nongating variants plotted as in A and B . ( E ) Comparison of trend lines of modest-response variants identified in CFBE and FRT studies compared with nongating variants identified in FRT* cells. Correlation ( r ) values calculated using Pearson linear correlation.

    Techniques Used: Variant Assay, Expressing, Labeling

    21) Product Images from "Evaluation of both exonic and intronic variants for effects on RNA splicing allows for accurate assessment of the effectiveness of precision therapies"

    Article Title: Evaluation of both exonic and intronic variants for effects on RNA splicing allows for accurate assessment of the effectiveness of precision therapies

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1009100

    CFTR intronic splice site variants with residual function are responsive to CFTR modulators. (A) Sashimi plots showing skipping of exon 16 caused by 2657+5G > A variant . RNA-seq was performed on the total RNA extracted from nasal cells of the individual harboring c.2657+5G > A/W1282X genotype. RNA from healthy individual served as a control. Per-base expression is plotted on y-axis of Sashimi plot. ‘E’ refers to exon locations on x-axis. +5 on the Sashimi plot indicates variant is located in intron 16 (not to scale). Numbers on the Sashimi plot indicate counts of reads spanning exon junctions. ɸ on the illustration indicates relative location of the variant c.2657+5 G > A. (B) Residual full-length CFTR transcript allows for modulator response in a c.2657+5G > A/W1282X individual . Representative short circuit current (I sc ) tracings on Human nasal epithelial (HNE) cells harvested from an individual harboring c.2657+5G > A/W1282X. CFTR channel function and CFTR modulator response were evaluated in well-differentiated airway liquid interface (ALI) culture. (C) Primary cells from an individual harboring c.3717+40A > G/F508del respond to modulator therapy. Representative short circuit current (I sc ) tracings on Human nasal epithelial (HNE) cells harvested from an individual harboring c.3717+40A > G/F508del. CFTR channel function and CFTR modulator response were evaluated in well- differentiated airway liquid interface (ALI) culture. (D) Intronic variants that allow for residual normal splicing respond to modulator therapies. Stacked bar graphs indicate effect of modulator treatment on CFBE stable cells expressing different CFTR intronic variants. Change in current (ΔI sc ) was defined as the current inhibited by Inh-172 after sustained I sc responses were achieved upon stimulation with forskolin alone or sequentially with ivacaftor. Data shown as mean±SEM (minimum of three independent measurements per condition). p value was determined by one-way ANOVA. ****( p ≤0.0001), n.s. (not significant, p > 0.05) when compared to DMSO treated vehicle control. Data underlying graphs in this figure reported in S5 Data .
    Figure Legend Snippet: CFTR intronic splice site variants with residual function are responsive to CFTR modulators. (A) Sashimi plots showing skipping of exon 16 caused by 2657+5G > A variant . RNA-seq was performed on the total RNA extracted from nasal cells of the individual harboring c.2657+5G > A/W1282X genotype. RNA from healthy individual served as a control. Per-base expression is plotted on y-axis of Sashimi plot. ‘E’ refers to exon locations on x-axis. +5 on the Sashimi plot indicates variant is located in intron 16 (not to scale). Numbers on the Sashimi plot indicate counts of reads spanning exon junctions. ɸ on the illustration indicates relative location of the variant c.2657+5 G > A. (B) Residual full-length CFTR transcript allows for modulator response in a c.2657+5G > A/W1282X individual . Representative short circuit current (I sc ) tracings on Human nasal epithelial (HNE) cells harvested from an individual harboring c.2657+5G > A/W1282X. CFTR channel function and CFTR modulator response were evaluated in well-differentiated airway liquid interface (ALI) culture. (C) Primary cells from an individual harboring c.3717+40A > G/F508del respond to modulator therapy. Representative short circuit current (I sc ) tracings on Human nasal epithelial (HNE) cells harvested from an individual harboring c.3717+40A > G/F508del. CFTR channel function and CFTR modulator response were evaluated in well- differentiated airway liquid interface (ALI) culture. (D) Intronic variants that allow for residual normal splicing respond to modulator therapies. Stacked bar graphs indicate effect of modulator treatment on CFBE stable cells expressing different CFTR intronic variants. Change in current (ΔI sc ) was defined as the current inhibited by Inh-172 after sustained I sc responses were achieved upon stimulation with forskolin alone or sequentially with ivacaftor. Data shown as mean±SEM (minimum of three independent measurements per condition). p value was determined by one-way ANOVA. ****( p ≤0.0001), n.s. (not significant, p > 0.05) when compared to DMSO treated vehicle control. Data underlying graphs in this figure reported in S5 Data .

    Techniques Used: Variant Assay, RNA Sequencing Assay, Expressing

    22) Product Images from "Efficient Gene Editing at Major CFTR Mutation Loci"

    Article Title: Efficient Gene Editing at Major CFTR Mutation Loci

    Journal: Molecular Therapy. Nucleic Acids

    doi: 10.1016/j.omtn.2019.02.006

    Restoration of CFTR Function in Gene-Corrected CFPAC-1 Cells and iPSC-Derived Proximal Lung Organoids (A) Correction efficiencies in patient-derived CFPAC-1 cells determined by deepseq. (B) Efficiency in establishing a heterozygously corrected (dF/CT) CFPAC-1 cell clone. (C) Iodide efflux assay using uncorrected CFPAC-1 (dF/dF) and heterozygously corrected (dF/CT) CF-PAC1 cells. Data are presented as mean ± SEM. (D) Forskolin-stimulated swelling assay using gene-corrected iPSC-derived proximal lung organoids. Data are presented as mean ± SEM. Left, bright-field images of pLOs; right, quantitative fold change of pLO size between CT/CT and dF/dF genotypes. Scale bar, 100 μm.
    Figure Legend Snippet: Restoration of CFTR Function in Gene-Corrected CFPAC-1 Cells and iPSC-Derived Proximal Lung Organoids (A) Correction efficiencies in patient-derived CFPAC-1 cells determined by deepseq. (B) Efficiency in establishing a heterozygously corrected (dF/CT) CFPAC-1 cell clone. (C) Iodide efflux assay using uncorrected CFPAC-1 (dF/dF) and heterozygously corrected (dF/CT) CF-PAC1 cells. Data are presented as mean ± SEM. (D) Forskolin-stimulated swelling assay using gene-corrected iPSC-derived proximal lung organoids. Data are presented as mean ± SEM. Left, bright-field images of pLOs; right, quantitative fold change of pLO size between CT/CT and dF/dF genotypes. Scale bar, 100 μm.

    Techniques Used: Derivative Assay

    23) Product Images from "Efficient and rapid conversion of human astrocytes and ALS mouse model spinal cord astrocytes into motor neuron-like cells by defined small molecules"

    Article Title: Efficient and rapid conversion of human astrocytes and ALS mouse model spinal cord astrocytes into motor neuron-like cells by defined small molecules

    Journal: Military Medical Research

    doi: 10.1186/s40779-020-00271-7

    Characterization of human astrocytes and the phenotypic conversion after chemical induction. a Immunostaining of cultured human astrocytes for the astrocyte markers glial fibrillary acidic protein (GFAP) and Vimentin. b Quantitative analysis of cultured human astrocytes for marker expression of astrocytes, neural progenitor cells (NPCs), neuronal cells, or oligodendrocytes. The data are presented as the means ± SEM from three independent experiments. c A schematic diagram showing the small molecule-based induction protocol. K, kenpaullone; F, forskolin; Y, Y-27632; P, purmorphamine; R, retinoic acid. d Microscopic images of the cell morphological changes after chemical induction. e Expression of class III β-tubulin 1 (TUJ1), microtubule-associated protein 2 (MAP2), and GFAP in the control group and the KFYPR-induced group. All scale bars = 50 μm
    Figure Legend Snippet: Characterization of human astrocytes and the phenotypic conversion after chemical induction. a Immunostaining of cultured human astrocytes for the astrocyte markers glial fibrillary acidic protein (GFAP) and Vimentin. b Quantitative analysis of cultured human astrocytes for marker expression of astrocytes, neural progenitor cells (NPCs), neuronal cells, or oligodendrocytes. The data are presented as the means ± SEM from three independent experiments. c A schematic diagram showing the small molecule-based induction protocol. K, kenpaullone; F, forskolin; Y, Y-27632; P, purmorphamine; R, retinoic acid. d Microscopic images of the cell morphological changes after chemical induction. e Expression of class III β-tubulin 1 (TUJ1), microtubule-associated protein 2 (MAP2), and GFAP in the control group and the KFYPR-induced group. All scale bars = 50 μm

    Techniques Used: Immunostaining, Cell Culture, Marker, Expressing

    24) Product Images from "ColXV promotes adipocyte differentiation via inhibiting DNA methylation and cAMP/PKA pathway in mice"

    Article Title: ColXV promotes adipocyte differentiation via inhibiting DNA methylation and cAMP/PKA pathway in mice

    Journal: Oncotarget

    doi: 10.18632/oncotarget.18550

    CREB attenuated ColXV transcription in adipocyte differentiation and lipolysis CREB was selected as candidate transcription regulator for ColXV according to analyzing results in Genomatix software. ( A ) The schematic diagram of CREB binding sites on ColXV promoter. Luciferase reporter gene fused ColXV promoter fragments ( B ) or mutated the CREB binding sites on ColXV promoter fragments ( C ) were transfected into cells with or without Forskolin treatment ( n = 6). Luciferase activity was detected and corrected with Renilla luciferase activity ( n = 6). To explore the function of CREB on ColXV expression, Forskolin was added in cells overexpressed or interfered ColXV, then relative ColXV mRNA level ( D ) were measured, together with protein levels of adipogenic genes, such as C/EBPβ, PPARγ, FABP4 and ATGL ( E ) were detected ( n = 6). Data represent the mean ± SEM of triplicate. *# P
    Figure Legend Snippet: CREB attenuated ColXV transcription in adipocyte differentiation and lipolysis CREB was selected as candidate transcription regulator for ColXV according to analyzing results in Genomatix software. ( A ) The schematic diagram of CREB binding sites on ColXV promoter. Luciferase reporter gene fused ColXV promoter fragments ( B ) or mutated the CREB binding sites on ColXV promoter fragments ( C ) were transfected into cells with or without Forskolin treatment ( n = 6). Luciferase activity was detected and corrected with Renilla luciferase activity ( n = 6). To explore the function of CREB on ColXV expression, Forskolin was added in cells overexpressed or interfered ColXV, then relative ColXV mRNA level ( D ) were measured, together with protein levels of adipogenic genes, such as C/EBPβ, PPARγ, FABP4 and ATGL ( E ) were detected ( n = 6). Data represent the mean ± SEM of triplicate. *# P

    Techniques Used: Software, Binding Assay, Luciferase, Transfection, Activity Assay, Expressing

    cAMP/PKA signal pathway was essential for effects of ColXV on adipocyte differentiation and lipolysis Adipocytes were pre-infected with Ad-Col15α1 and sh-Col15α1 (Ad-GFP as control) for 72 h. Part of those cells were incubated with 10 μM Forskolin for 1 h, then cAMP level ( A ) protein level of CREB, p-PKA/PKA ( B ) and C/EBPβ, FABP4, ATGL, p-ATGL Ser406 ( D ) were detected ( n = 6). Another part of those cells were treated with 10 μM H89 for 2 h, followed by cAMP level ( C ) protein level of CREB, p-PKA and PKA ( E ) and C/EBPβ, FABP4, ATGL, p-ATGL Ser406 ( F ) evaluation ( n = 6). Data represent the mean ± SEM of three independent experiments. *# P
    Figure Legend Snippet: cAMP/PKA signal pathway was essential for effects of ColXV on adipocyte differentiation and lipolysis Adipocytes were pre-infected with Ad-Col15α1 and sh-Col15α1 (Ad-GFP as control) for 72 h. Part of those cells were incubated with 10 μM Forskolin for 1 h, then cAMP level ( A ) protein level of CREB, p-PKA/PKA ( B ) and C/EBPβ, FABP4, ATGL, p-ATGL Ser406 ( D ) were detected ( n = 6). Another part of those cells were treated with 10 μM H89 for 2 h, followed by cAMP level ( C ) protein level of CREB, p-PKA and PKA ( E ) and C/EBPβ, FABP4, ATGL, p-ATGL Ser406 ( F ) evaluation ( n = 6). Data represent the mean ± SEM of three independent experiments. *# P

    Techniques Used: Infection, Incubation

    25) Product Images from "Functional Assays Are Essential for Interpretation of Missense Variants Associated with Variable Expressivity"

    Article Title: Functional Assays Are Essential for Interpretation of Missense Variants Associated with Variable Expressivity

    Journal: American Journal of Human Genetics

    doi: 10.1016/j.ajhg.2018.04.003

    CFTR mRNA, Protein Quantity, and Function Are Variable but Correlated (A) Mean and standard deviations of CFTR mRNA transcript quantity relative to that of HPRT1 (n = 3 for each cell line) for ten independent cell lines expressing WT-CFTR. (B) Immunoblot detecting varying quantities of mature (band C) CFTR from whole-cell lysates of ten cell lines expressing WT-CFTR. Controls include cell lines expressing the CF-causing variants F508del, which causes a folding defect (band B only), and G551D (band C); non-transfected CFBE cells (no signal); and HEK293 cells transiently expressing WT-CFTR (band C). Loading controls for protein quantity (Na + /K+ ATPase) are shown below. The plot on the right shows the mean and standard deviations of CFTR quantities for each cell line relative to the WT-CFTR 5 cell line assessed from at least three immunoblots. (C) Representative recordings of CFTR function measured by I sc for ten WT-CFTR cell lines. Forskolin (10 μM) activates CFTR chloride current, and the amount of current inhibited by the CFTR-specific inhibitor inh-172 (10 μM) determines the level of CFTR function. The plot on the right shows the mean and standard deviations for I sc derived from at least three measurements for the ten WT-CFTR cell lines. (D) Correlations of the quantity of CFTR mRNA with quantity of mature CFTR (left), quantity of mature CFTR with CFTR function (center), and quantity of CFTR mRNA with CFTR function (right) for ten independent cell lines expressing WT-CFTR.
    Figure Legend Snippet: CFTR mRNA, Protein Quantity, and Function Are Variable but Correlated (A) Mean and standard deviations of CFTR mRNA transcript quantity relative to that of HPRT1 (n = 3 for each cell line) for ten independent cell lines expressing WT-CFTR. (B) Immunoblot detecting varying quantities of mature (band C) CFTR from whole-cell lysates of ten cell lines expressing WT-CFTR. Controls include cell lines expressing the CF-causing variants F508del, which causes a folding defect (band B only), and G551D (band C); non-transfected CFBE cells (no signal); and HEK293 cells transiently expressing WT-CFTR (band C). Loading controls for protein quantity (Na + /K+ ATPase) are shown below. The plot on the right shows the mean and standard deviations of CFTR quantities for each cell line relative to the WT-CFTR 5 cell line assessed from at least three immunoblots. (C) Representative recordings of CFTR function measured by I sc for ten WT-CFTR cell lines. Forskolin (10 μM) activates CFTR chloride current, and the amount of current inhibited by the CFTR-specific inhibitor inh-172 (10 μM) determines the level of CFTR function. The plot on the right shows the mean and standard deviations for I sc derived from at least three measurements for the ten WT-CFTR cell lines. (D) Correlations of the quantity of CFTR mRNA with quantity of mature CFTR (left), quantity of mature CFTR with CFTR function (center), and quantity of CFTR mRNA with CFTR function (right) for ten independent cell lines expressing WT-CFTR.

    Techniques Used: Expressing, Transfection, Western Blot, Derivative Assay

    26) Product Images from "Generation of liver bipotential organoids with a small-molecule cocktail"

    Article Title: Generation of liver bipotential organoids with a small-molecule cocktail

    Journal: Journal of Molecular Cell Biology

    doi: 10.1093/jmcb/mjaa010

    Hepatocyte differentiation potential assay of liver bipotential organoids. ( A ) Bright-filed images (left) and qRT-PCR analysis (right) of GF-Orgs switched to small-molecule culture. Scale bar, 200 μm. ( B ) qRT-PCR analysis of hepatocyte differentiation markers in GF-Orgs and Chem-Orgs (G, growth factors; C, CHIR-99021; B, blebbistatin; F, forskolin; A, A83-01) with or without hepatocyte differentiation inducers (D, differentiation inducers). ( C ) qRT-PCR analysis of hepatocyte differentiation markers in Chem-Orgs and after Hnf4α overexpressing treatment. n = 3 independent experiments, mean ± SEM; * P
    Figure Legend Snippet: Hepatocyte differentiation potential assay of liver bipotential organoids. ( A ) Bright-filed images (left) and qRT-PCR analysis (right) of GF-Orgs switched to small-molecule culture. Scale bar, 200 μm. ( B ) qRT-PCR analysis of hepatocyte differentiation markers in GF-Orgs and Chem-Orgs (G, growth factors; C, CHIR-99021; B, blebbistatin; F, forskolin; A, A83-01) with or without hepatocyte differentiation inducers (D, differentiation inducers). ( C ) qRT-PCR analysis of hepatocyte differentiation markers in Chem-Orgs and after Hnf4α overexpressing treatment. n = 3 independent experiments, mean ± SEM; * P

    Techniques Used: Quantitative RT-PCR

    Related Articles

    Fluorescence:

    Article Title: Orkambi® and amplifier co‐therapy improves function from a rare CFTR mutation in gene‐edited cells and patient tissue
    Article Snippet: Briefly, HEK‐293 cells overexpressing CFTR constructs were grown to 100% confluence in 24‐well plates (Costar) and washed with PBS, and the blue membrane potential dye (dissolved in chloride‐free buffer containing 136 mM sodium gluconate, 3 mM potassium gluconate, 10 mM glucose, 20 mM HEPES, pH 7.35, 300 mOsm, at a concentration of 0.5 mg/ml; Molecular devices), which can detect changes in transmembrane potential, was added to the cells for 1 h at 27 or 37°C as required. .. The plate was then read in a fluorescence plate reader (Molecular devices—Gemini EM) at the required temperature, and after reading the baseline fluorescence, CFTR was stimulated using the cAMP agonist forskolin (10 μM; Sigma‐Aldrich) or forskolin with VX‐770 (1 μM; Selleck Chemicals). .. CFTR‐mediated depolarization of the membrane was detected as an increase in fluorescence and repolarization or hyperpolarization as a decrease (Maitra et al, ).

    Cell Culture:

    Article Title: cAMP regulates 11β-hydroxysteroid dehydrogenase-2 and Sp1 expression in MLO-Y4/MC3T3-E1 cells
    Article Snippet: .. Cells in the exponential phase of growth were seeded into 6-well plates with 2x105 cells per well and cultured at 37˚C for 24 h. Subsequently, cells were divided into three groups: i) The NC group (negative control group), which was incubated with 100 µmol PBS; ii) the activator group, which was incubated with 100 µmol forskolin (Selleck Chemicals); and iii) the inhibitor group, which was incubated with 100 µmol SQ22536 (Selleck Chemicals). ..

    Negative Control:

    Article Title: cAMP regulates 11β-hydroxysteroid dehydrogenase-2 and Sp1 expression in MLO-Y4/MC3T3-E1 cells
    Article Snippet: .. Cells in the exponential phase of growth were seeded into 6-well plates with 2x105 cells per well and cultured at 37˚C for 24 h. Subsequently, cells were divided into three groups: i) The NC group (negative control group), which was incubated with 100 µmol PBS; ii) the activator group, which was incubated with 100 µmol forskolin (Selleck Chemicals); and iii) the inhibitor group, which was incubated with 100 µmol SQ22536 (Selleck Chemicals). ..

    Incubation:

    Article Title: cAMP regulates 11β-hydroxysteroid dehydrogenase-2 and Sp1 expression in MLO-Y4/MC3T3-E1 cells
    Article Snippet: .. Cells in the exponential phase of growth were seeded into 6-well plates with 2x105 cells per well and cultured at 37˚C for 24 h. Subsequently, cells were divided into three groups: i) The NC group (negative control group), which was incubated with 100 µmol PBS; ii) the activator group, which was incubated with 100 µmol forskolin (Selleck Chemicals); and iii) the inhibitor group, which was incubated with 100 µmol SQ22536 (Selleck Chemicals). ..

    Article Title: Efficient Gene Editing at Major CFTR Mutation Loci
    Article Snippet: .. In the pLO swelling assay, organoids were incubated with 10 μM Forskolin (S2449, Selleck Chemicals) for 24 h, and they were monitored by time-lapse microscopy. ..

    Article Title: Forskolin exerts anticancer roles in non-Hodgkin’s lymphomas via regulating Axin/β-catenin signaling pathway
    Article Snippet: Toledo, Farage, and RL cells were cultured in RPMI-1640 medium containing 10% FBS (Gibco, CA, USA); NK-92 cells were cultured in Alpha Minimum Essential Medium with ribonucleosides and deoxyribonucleosides free while with 2 mM L-glutamine and 1.5 g/L sodium bicarbonate, 0.2 mM inositol, 0.1 mM 2-mercaptoethanol, 0.02 mM folic acid, 100–200 U/mL recombinant IL-2, 12.5% horse serum (Sigma-Aldrich Co., St Louis, MO, USA), and 12.5% FBS. .. Toledo or NK-92 cells were incubated with different concentrations of forskolin (0, 10, 20, 40, 80, or 160 μM; cAMP analog 8-pCPT-2′-OMe-cAMP; Sigma-Aldrich Co) for the indicated times, and 20 μM of SP600125 (Selleck Chemicals, Shanghai, China), an inhibitor of c-Jun N-terminal kinase (JNK) for 24 hours. .. Cell transfection Small interference (si) RNA targeting human Axin (No. SR305433, Origene, CA, USA) was used to downregulate the expression of Axin in Toledo and NK-92 cells using Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA).

    Recombinant:

    Article Title: Fasting and Systemic Insulin Signaling Regulate Phosphorylation of Brain Proteins That Modulate Cell Morphology and Link to Neurological Disorders *
    Article Snippet: .. Materials Recombinant human insulin was from Novo Nordisk (Bagsvaerd, Denmark), forskolin and H-89 from Selleckchem (Shanghai, China), and microcystin-LR from Enzo Life Sciences (Farmingdale, NY). .. Precast NuPAGE® bis-Tris gels were from Thermo Fisher Scientific and protein G-Sepharose and glutathione-Sepharose 4B from GE Healthcare.

    Time-lapse Microscopy:

    Article Title: Efficient Gene Editing at Major CFTR Mutation Loci
    Article Snippet: .. In the pLO swelling assay, organoids were incubated with 10 μM Forskolin (S2449, Selleck Chemicals) for 24 h, and they were monitored by time-lapse microscopy. ..

    Western Blot:

    Article Title: ColXV promotes adipocyte differentiation via inhibiting DNA methylation and cAMP/PKA pathway in mice
    Article Snippet: Gene expressions of adipocyte differentiation and lipolysis makers were detected by Quantitative Real-Time PCR or Western blot. .. When testing the impact of ColXV on cAMP/PKA signal pathway activated by Forskolin (10 μM, Selleck, Houston, USA), differentiated adipocytes were treated with H89 (10 μM, Selleck, Houston, USA) or DMSO (Promega, Madison, USA) 1 h prior to Forskolin (10 μM), and samples were assessed by Western blot. .. Bodipy staining Bodipy (Invitrogen, Carlsbad, USA) with 513 nm green florescent was diluted with DMSO to a concentration of 1 mm as work solution.

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    Selleck Chemicals forskolin
    Restoration of CFTR Function in Gene-Corrected CFPAC-1 Cells and iPSC-Derived Proximal Lung Organoids (A) Correction efficiencies in patient-derived CFPAC-1 cells determined by deepseq. (B) Efficiency in establishing a heterozygously corrected (dF/CT) CFPAC-1 cell clone. (C) Iodide efflux assay using uncorrected CFPAC-1 (dF/dF) and heterozygously corrected (dF/CT) CF-PAC1 cells. Data are presented as mean ± SEM. (D) <t>Forskolin-stimulated</t> swelling assay using gene-corrected iPSC-derived proximal lung organoids. Data are presented as mean ± SEM. Left, bright-field images of pLOs; right, quantitative fold change of pLO size between CT/CT and dF/dF genotypes. Scale bar, 100 μm.
    Forskolin, supplied by Selleck Chemicals, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/forskolin/product/Selleck Chemicals
    Average 94 stars, based on 1 article reviews
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    Selleck Chemicals agonist forskolin
    cAMP/PKA is required for the pH-dependent regulation of osteogenesis and YAP activity. BMSCs were incubated in osteogenic media for 21 days and then transferred to low pH (pH = 7.0) basal media for different times (t = 0, 5, 10, 20, or 40 min). The phosphorylation of MYPT1 and CREB was assessed by western blotting (A) . BMSCs were incubated in osteogenic media at different pHs containing a cAMP activator <t>(forskolin)</t> or a PKA inhibitor (H 89 2 HCl) for 21 days. The calcium deposits in the differentiated BMSCs were evaluated by Alizarin red S staining (B) , and the expressions of IBSP and BGLAP were examined by qRT-PCR analysis (C). * P
    Agonist Forskolin, supplied by Selleck Chemicals, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Restoration of CFTR Function in Gene-Corrected CFPAC-1 Cells and iPSC-Derived Proximal Lung Organoids (A) Correction efficiencies in patient-derived CFPAC-1 cells determined by deepseq. (B) Efficiency in establishing a heterozygously corrected (dF/CT) CFPAC-1 cell clone. (C) Iodide efflux assay using uncorrected CFPAC-1 (dF/dF) and heterozygously corrected (dF/CT) CF-PAC1 cells. Data are presented as mean ± SEM. (D) Forskolin-stimulated swelling assay using gene-corrected iPSC-derived proximal lung organoids. Data are presented as mean ± SEM. Left, bright-field images of pLOs; right, quantitative fold change of pLO size between CT/CT and dF/dF genotypes. Scale bar, 100 μm.

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Efficient Gene Editing at Major CFTR Mutation Loci

    doi: 10.1016/j.omtn.2019.02.006

    Figure Lengend Snippet: Restoration of CFTR Function in Gene-Corrected CFPAC-1 Cells and iPSC-Derived Proximal Lung Organoids (A) Correction efficiencies in patient-derived CFPAC-1 cells determined by deepseq. (B) Efficiency in establishing a heterozygously corrected (dF/CT) CFPAC-1 cell clone. (C) Iodide efflux assay using uncorrected CFPAC-1 (dF/dF) and heterozygously corrected (dF/CT) CF-PAC1 cells. Data are presented as mean ± SEM. (D) Forskolin-stimulated swelling assay using gene-corrected iPSC-derived proximal lung organoids. Data are presented as mean ± SEM. Left, bright-field images of pLOs; right, quantitative fold change of pLO size between CT/CT and dF/dF genotypes. Scale bar, 100 μm.

    Article Snippet: In the pLO swelling assay, organoids were incubated with 10 μM Forskolin (S2449, Selleck Chemicals) for 24 h, and they were monitored by time-lapse microscopy.

    Techniques: Derivative Assay

    Characterization of human astrocytes and the phenotypic conversion after chemical induction. a Immunostaining of cultured human astrocytes for the astrocyte markers glial fibrillary acidic protein (GFAP) and Vimentin. b Quantitative analysis of cultured human astrocytes for marker expression of astrocytes, neural progenitor cells (NPCs), neuronal cells, or oligodendrocytes. The data are presented as the means ± SEM from three independent experiments. c A schematic diagram showing the small molecule-based induction protocol. K, kenpaullone; F, forskolin; Y, Y-27632; P, purmorphamine; R, retinoic acid. d Microscopic images of the cell morphological changes after chemical induction. e Expression of class III β-tubulin 1 (TUJ1), microtubule-associated protein 2 (MAP2), and GFAP in the control group and the KFYPR-induced group. All scale bars = 50 μm

    Journal: Military Medical Research

    Article Title: Efficient and rapid conversion of human astrocytes and ALS mouse model spinal cord astrocytes into motor neuron-like cells by defined small molecules

    doi: 10.1186/s40779-020-00271-7

    Figure Lengend Snippet: Characterization of human astrocytes and the phenotypic conversion after chemical induction. a Immunostaining of cultured human astrocytes for the astrocyte markers glial fibrillary acidic protein (GFAP) and Vimentin. b Quantitative analysis of cultured human astrocytes for marker expression of astrocytes, neural progenitor cells (NPCs), neuronal cells, or oligodendrocytes. The data are presented as the means ± SEM from three independent experiments. c A schematic diagram showing the small molecule-based induction protocol. K, kenpaullone; F, forskolin; Y, Y-27632; P, purmorphamine; R, retinoic acid. d Microscopic images of the cell morphological changes after chemical induction. e Expression of class III β-tubulin 1 (TUJ1), microtubule-associated protein 2 (MAP2), and GFAP in the control group and the KFYPR-induced group. All scale bars = 50 μm

    Article Snippet: The following small molecules were used at the indicated concentrations: 5 μM kenpaullone (MedChem Express #HY-12302), 10 μM forskolin (Selleck #S2449), 10 μM Y-27632 (Selleck #S1049), 2 μM purmorphamine (MedChem Express #HY-15108), and 2 μM retinoic acid (Sigma #R2625).

    Techniques: Immunostaining, Cell Culture, Marker, Expressing

    CREB attenuated ColXV transcription in adipocyte differentiation and lipolysis CREB was selected as candidate transcription regulator for ColXV according to analyzing results in Genomatix software. ( A ) The schematic diagram of CREB binding sites on ColXV promoter. Luciferase reporter gene fused ColXV promoter fragments ( B ) or mutated the CREB binding sites on ColXV promoter fragments ( C ) were transfected into cells with or without Forskolin treatment ( n = 6). Luciferase activity was detected and corrected with Renilla luciferase activity ( n = 6). To explore the function of CREB on ColXV expression, Forskolin was added in cells overexpressed or interfered ColXV, then relative ColXV mRNA level ( D ) were measured, together with protein levels of adipogenic genes, such as C/EBPβ, PPARγ, FABP4 and ATGL ( E ) were detected ( n = 6). Data represent the mean ± SEM of triplicate. *# P

    Journal: Oncotarget

    Article Title: ColXV promotes adipocyte differentiation via inhibiting DNA methylation and cAMP/PKA pathway in mice

    doi: 10.18632/oncotarget.18550

    Figure Lengend Snippet: CREB attenuated ColXV transcription in adipocyte differentiation and lipolysis CREB was selected as candidate transcription regulator for ColXV according to analyzing results in Genomatix software. ( A ) The schematic diagram of CREB binding sites on ColXV promoter. Luciferase reporter gene fused ColXV promoter fragments ( B ) or mutated the CREB binding sites on ColXV promoter fragments ( C ) were transfected into cells with or without Forskolin treatment ( n = 6). Luciferase activity was detected and corrected with Renilla luciferase activity ( n = 6). To explore the function of CREB on ColXV expression, Forskolin was added in cells overexpressed or interfered ColXV, then relative ColXV mRNA level ( D ) were measured, together with protein levels of adipogenic genes, such as C/EBPβ, PPARγ, FABP4 and ATGL ( E ) were detected ( n = 6). Data represent the mean ± SEM of triplicate. *# P

    Article Snippet: When testing the impact of ColXV on cAMP/PKA signal pathway activated by Forskolin (10 μM, Selleck, Houston, USA), differentiated adipocytes were treated with H89 (10 μM, Selleck, Houston, USA) or DMSO (Promega, Madison, USA) 1 h prior to Forskolin (10 μM), and samples were assessed by Western blot.

    Techniques: Software, Binding Assay, Luciferase, Transfection, Activity Assay, Expressing

    cAMP/PKA signal pathway was essential for effects of ColXV on adipocyte differentiation and lipolysis Adipocytes were pre-infected with Ad-Col15α1 and sh-Col15α1 (Ad-GFP as control) for 72 h. Part of those cells were incubated with 10 μM Forskolin for 1 h, then cAMP level ( A ) protein level of CREB, p-PKA/PKA ( B ) and C/EBPβ, FABP4, ATGL, p-ATGL Ser406 ( D ) were detected ( n = 6). Another part of those cells were treated with 10 μM H89 for 2 h, followed by cAMP level ( C ) protein level of CREB, p-PKA and PKA ( E ) and C/EBPβ, FABP4, ATGL, p-ATGL Ser406 ( F ) evaluation ( n = 6). Data represent the mean ± SEM of three independent experiments. *# P

    Journal: Oncotarget

    Article Title: ColXV promotes adipocyte differentiation via inhibiting DNA methylation and cAMP/PKA pathway in mice

    doi: 10.18632/oncotarget.18550

    Figure Lengend Snippet: cAMP/PKA signal pathway was essential for effects of ColXV on adipocyte differentiation and lipolysis Adipocytes were pre-infected with Ad-Col15α1 and sh-Col15α1 (Ad-GFP as control) for 72 h. Part of those cells were incubated with 10 μM Forskolin for 1 h, then cAMP level ( A ) protein level of CREB, p-PKA/PKA ( B ) and C/EBPβ, FABP4, ATGL, p-ATGL Ser406 ( D ) were detected ( n = 6). Another part of those cells were treated with 10 μM H89 for 2 h, followed by cAMP level ( C ) protein level of CREB, p-PKA and PKA ( E ) and C/EBPβ, FABP4, ATGL, p-ATGL Ser406 ( F ) evaluation ( n = 6). Data represent the mean ± SEM of three independent experiments. *# P

    Article Snippet: When testing the impact of ColXV on cAMP/PKA signal pathway activated by Forskolin (10 μM, Selleck, Houston, USA), differentiated adipocytes were treated with H89 (10 μM, Selleck, Houston, USA) or DMSO (Promega, Madison, USA) 1 h prior to Forskolin (10 μM), and samples were assessed by Western blot.

    Techniques: Infection, Incubation

    cAMP/PKA is required for the pH-dependent regulation of osteogenesis and YAP activity. BMSCs were incubated in osteogenic media for 21 days and then transferred to low pH (pH = 7.0) basal media for different times (t = 0, 5, 10, 20, or 40 min). The phosphorylation of MYPT1 and CREB was assessed by western blotting (A) . BMSCs were incubated in osteogenic media at different pHs containing a cAMP activator (forskolin) or a PKA inhibitor (H 89 2 HCl) for 21 days. The calcium deposits in the differentiated BMSCs were evaluated by Alizarin red S staining (B) , and the expressions of IBSP and BGLAP were examined by qRT-PCR analysis (C). * P

    Journal: Scientific Reports

    Article Title: Decreased extracellular pH inhibits osteogenesis through proton-sensing GPR4-mediated suppression of yes-associated protein

    doi: 10.1038/srep26835

    Figure Lengend Snippet: cAMP/PKA is required for the pH-dependent regulation of osteogenesis and YAP activity. BMSCs were incubated in osteogenic media for 21 days and then transferred to low pH (pH = 7.0) basal media for different times (t = 0, 5, 10, 20, or 40 min). The phosphorylation of MYPT1 and CREB was assessed by western blotting (A) . BMSCs were incubated in osteogenic media at different pHs containing a cAMP activator (forskolin) or a PKA inhibitor (H 89 2 HCl) for 21 days. The calcium deposits in the differentiated BMSCs were evaluated by Alizarin red S staining (B) , and the expressions of IBSP and BGLAP were examined by qRT-PCR analysis (C). * P

    Article Snippet: Inhibitor and agonist Forskolin and H 89 2HCl (H89) were obtained from Selleck (Houston, TX, USA), and recombinant human CTGF protein was purchased from Pepro Tech Inc. (Rocky Hill, NJ, USA).

    Techniques: Activity Assay, Incubation, Western Blot, Staining, Quantitative RT-PCR