Journal: JCI Insight

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

doi: 10.1172/jci.insight.121159

Figure Lengend Snippet: (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) Previously published data collected from FRT* cells (18, 19) 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.

Article Snippet: Cotton for identification of the L145H variant, M.J. Welsh for isogenic FRT cell lines, and F. Van Goor and Vertex Pharmaceuticals for access to ivacaftor response data generated using FRT cells.

Techniques: Variant Assay, Expressing, Labeling, Comparison

Journal: JCI Insight

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

doi: 10.1172/jci.insight.121159

Figure Lengend Snippet: (A) Plot of CFTR processing versus residual function for 54 variants previously expressed in FRT* cells (18) 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 Isc 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/cm2), 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.

Article Snippet: Cotton for identification of the L145H variant, M.J. Welsh for isogenic FRT cell lines, and F. Van Goor and Vertex Pharmaceuticals for access to ivacaftor response data generated using FRT cells.

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

Journal: JCI Insight

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

doi: 10.1172/jci.insight.121159

Figure Lengend Snippet: (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.

Article Snippet: Cotton for identification of the L145H variant, M.J. Welsh for isogenic FRT cell lines, and F. Van Goor and Vertex Pharmaceuticals for access to ivacaftor response data generated using FRT cells.

Techniques: Incubation, Variant Assay, Expressing, Labeling, Comparison

Journal: JCI Insight

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

doi: 10.1172/jci.insight.121159

Figure Lengend Snippet: (A) Comparison of response trends for ivacaftor (left), lumacaftor (middle), and ivacaftor/lumacaftor combination (right) of all variants studied in CF bronchial epithelial (CFBE) and Fisher rat thyroid (FRT) cell lines. (B) Comparison of response trends for all 3 treatment strategies for modest-response variants (left), intermediate-response variants (middle), and high-response variants (right).

Article Snippet: Cotton for identification of the L145H variant, M.J. Welsh for isogenic FRT cell lines, and F. Van Goor and Vertex Pharmaceuticals for access to ivacaftor response data generated using FRT cells.

Techniques: Comparison

Journal: Molecular Biology of the Cell

Article Title: The human 18S rRNA base methyltransferases DIMT1L and WBSCR22-TRMT112 but not rRNA modification are required for ribosome biogenesis

doi: 10.1091/mbc.E15-02-0073

Figure Lengend Snippet: The methyltransferase function of DIMT1L is not required for pre-rRNA processing. Protein levels and pre-rRNA processing in HEK293 cells stably expressing, via an siRNA-resistant mRNA (siRNAr), a construct encoding the wild-type or a catalytically deficient (Y131G) Flag-tagged version of DIMT1L in the presence or absence of endogenous DIMT1L. The allele encoding catalytically deficient DIMT1L and, as controls, the wild-type construct and the empty plasmid were integrated at the same genomic locus. Expression of the recombinant constructs was under the control of an inducible Tet promoter (bent arrow) and was induced by incubating the cells in 0.2 μg/ml tetracycline. Expression of the endogenous DIMT1L gene was suppressed by incubating the cells for 3 d with siRNA DIMT1L#2 (see Supplemental Table S3). As control, a nontargeting siRNA (SCR, scramble) was used. (A) Western blot analysis with an antibody against DIMT1L. The antibody detects the endogenous DIMT1L protein, displaying the expected molecular weight of 35 kDa, and the Flag-tagged recombinant variants, detected at 38 kDa. β-Actin was used as loading control. The anti-DIMT1L antibody detects a faint nonspecific band (asterisk) above Flag-tagged DIMT1L. Hybridizing the membrane with an anti-Flag antibody revealed only recombinant proteins of the expected size (unpublished data). (B) Northern blot analysis. Total RNA treated as described in was probed with oligonucleotide LD1827. The abundances of the various RNAs detected were established by Phosphorimager quantification and normalized with respect to the nontargeting SCR control and are presented as a heatmap with the color code indicated to the right.

Article Snippet: Cells were grown at 37°C in a humidified incubator under 5% CO 2 in the following media: DMEM (D6429; Sigma-Aldrich)/10% fetal bovine serum (FBS; A&E Scientific) for HeLa (AIDS Ref. 153; National Institutes of Health), HEK293 (LifeTech), and FIB364 cells (HeLa cells stably expressing fibrillarin-GFP); MEM (30-2003; American Type Culture Collection [ATCC]/10% FBS for WI-38 cells (CCL-75; ATCC); and McCoy's 5a modified (Sigma-Aldrich)/10% FBS for HCT116 cells (CCL-247; ATCC).

Techniques: Stable Transfection, Expressing, Construct, Plasmid Preparation, Recombinant, Western Blot, Molecular Weight, Northern Blot

Journal: Molecular Biology of the Cell

Article Title: The human 18S rRNA base methyltransferases DIMT1L and WBSCR22-TRMT112 but not rRNA modification are required for ribosome biogenesis

doi: 10.1091/mbc.E15-02-0073

Figure Lengend Snippet: The methyltransferase function of WBSCR22 is not required for pre-rRNA processing. Protein levels and pre-rRNA processing in HEK293 cells stably expressing, via an siRNA-resistant mRNA (siRNAr), a construct encoding the wild-type or a catalytically deficient (G63E, D82K, and D63E/D82K) Flag-tagged version of WBSCR22 in the presence or absence of endogenous WBSCR22. The constructs encoding catalytically deficient WBSCR22 and, as controls, the wild-type construct and the empty plasmid were integrated at the same genomic locus. Expression of the recombinant constructs was under the control of an inducible Tet promoter (bent arrow) and was induced by incubating the cells in 0.2 μg/ml tetracycline. Expression of the endogenous WBSCR22 gene was suppressed by incubating the cells for 3 d with siRNA WBSCR22#1 (see Supplemental Table S3). As control, a nontargeting siRNA (SCR, scramble) was used. (A) Western blot analysis with an antibody against WBSCR22. The antibody detects the endogenous WBSCR22 protein, displaying the expected molecular weight of 32 kDa, and the Flag-tagged recombinant variants, detected at 35 kDa. β-Actin was used as loading control. Hybridizing the membrane with an anti-Flag antibody revealed only the recombinant variants of the expected size (unpublished data). (B) Northern blot analysis. Total RNA treated as described in was probed with oligonucleotide LD1827. The abundances of the various RNAs detected were established by Phosphorimager quantification and normalized with respect to the nontargeting SCR control and are presented as a heatmap with the color code indicated to the right.

Article Snippet: Cells were grown at 37°C in a humidified incubator under 5% CO 2 in the following media: DMEM (D6429; Sigma-Aldrich)/10% fetal bovine serum (FBS; A&E Scientific) for HeLa (AIDS Ref. 153; National Institutes of Health), HEK293 (LifeTech), and FIB364 cells (HeLa cells stably expressing fibrillarin-GFP); MEM (30-2003; American Type Culture Collection [ATCC]/10% FBS for WI-38 cells (CCL-75; ATCC); and McCoy's 5a modified (Sigma-Aldrich)/10% FBS for HCT116 cells (CCL-247; ATCC).

Techniques: Stable Transfection, Expressing, Construct, Plasmid Preparation, Recombinant, Western Blot, Molecular Weight, Northern Blot

Journal: PLoS ONE

Article Title: Inhibition of ANO1/TMEM16A Chloride Channel by Idebenone and Its Cytotoxicity to Cancer Cell Lines

doi: 10.1371/journal.pone.0133656

Figure Lengend Snippet: (A) Apical membrane currents were measured in FRT-ANO1 cells. Idebenone was added 10 min prior to ANO1 activation by 100 μM ATP. (B) Summary of dose-response (mean ± S.E., n = 3–4). (C) Intracellular calcium concentration was measured using Fluo-4 in HT-29 and FRT cells. 30 μM idebenone (IDE), miconazole (MCZ) and plumbagin (PLB) were pretreated for 20min and then 100 μM ATP was applied. (D) Effect of idebenone on CFTR chloride channel activity was measured in FRT cells expressing human wild-type CFTR. CFTR was activated by 20 μM forskolin and inhibited by 10 μM CFTR inh -172. (E) Effect of idebenone on mouse ANO2 (mANO2) was measured in FRT-mANO2 cells. (F) Effect of Coenzyme Q10 (CoQ10) on ANO1 channel activity was observed in FRT-ANO1 cells. 100 μM CoQ10 was pretreated for 20min and then 100 μM ATP was applied. (right) Summary of peak current (mean ± S.E., n = 3–4). (G) Effect of idebenone on ANO1 activation by E act in ANO1-expressing FRT cells. 100 μM idebenone (gray line) was pretreated for 20min and ANO1 was activated by 10 μM E act . The remaining ANO1 currents were inhibited by T16A inh -A01. (right) Summary of peak current (mean ± S.E., n = 3). (H) Idebenone reversibility. After vanishment of 100 μM ATP-induced ANO1 current, the cells were washed three times for 5 min each and then ANO1 was activated by 10 μM E act . (right) Summary of peak current (mean ± S.E., n = 3). **P < 0.01, ***P < 0.001, Students’ unpaired t-test.

Article Snippet: Snapwell inserts containing ANO1- or CFTR-expressing FRT cells were mounted in Ussing chambers (Physiologic Instruments, San Diego, CA).

Techniques: Activation Assay, Concentration Assay, Activity Assay, Expressing

Journal: PLoS ONE

Article Title: Inhibition of ANO1/TMEM16A Chloride Channel by Idebenone and Its Cytotoxicity to Cancer Cell Lines

doi: 10.1371/journal.pone.0133656

Figure Lengend Snippet: (A) Immunoblot of ANO1 protein in FRT-ANO1, PC3, CFPAC-1 and A549 cells. Representatives of three sets of studies are shown. (B) Effect of idebenone on CaCCs was measured in CFPAC-1 cells expressing halide sensitive mutant YFP. CaCCs were activated by 100 μM ATP. (C) PC3 and A549 cells were treated with idebenone (30 μM), miconazole (30 μM) and plumbagin (30 μM), and cell proliferation was measured after 2 days using MTS assays (mean ± S.E., n = 6). (D) Wound healing assay in PC3 cells. The cells were treated with T16A inh -A01 (30 μM) and idebenone. (left) The wound closure was quantified at every 2 h post-wound (mean ± S.E., n = 5). (right) Representative images taken at 0 h and 48 h post wounding (× 10). **P < 0.01, Students’ unpaired t-test.

Article Snippet: Snapwell inserts containing ANO1- or CFTR-expressing FRT cells were mounted in Ussing chambers (Physiologic Instruments, San Diego, CA).

Techniques: Western Blot, Expressing, Mutagenesis, Wound Healing Assay

Journal: JCI Insight

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

doi: 10.1172/jci.insight.121159

Figure Lengend Snippet: (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) Previously published data collected from FRT* cells (18, 19) 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.

Article Snippet: We next evaluated previously published ivacaftor response data generated by 54 CFTR variants stably expressed in a separate FRT cell line established by Vertex Pharmaceuticals (noted here as FRT*) that overlapped with the 16 variants expressed in FRT cells in this study ( 18 ).

Techniques: Variant Assay, Expressing, Labeling, Comparison

Journal: JCI Insight

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

doi: 10.1172/jci.insight.121159

Figure Lengend Snippet: (A) Plot of CFTR processing versus residual function for 54 variants previously expressed in FRT* cells (18) 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 Isc 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/cm2), 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.

Article Snippet: We next evaluated previously published ivacaftor response data generated by 54 CFTR variants stably expressed in a separate FRT cell line established by Vertex Pharmaceuticals (noted here as FRT*) that overlapped with the 16 variants expressed in FRT cells in this study ( 18 ).

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

Journal: JCI Insight

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

doi: 10.1172/jci.insight.121159

Figure Lengend Snippet: (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.

Article Snippet: We next evaluated previously published ivacaftor response data generated by 54 CFTR variants stably expressed in a separate FRT cell line established by Vertex Pharmaceuticals (noted here as FRT*) that overlapped with the 16 variants expressed in FRT cells in this study ( 18 ).

Techniques: Incubation, Variant Assay, Expressing, Labeling, Comparison

Journal: JCI Insight

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

doi: 10.1172/jci.insight.121159

Figure Lengend Snippet: (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.

Article Snippet: We next evaluated previously published ivacaftor response data generated by 54 CFTR variants stably expressed in a separate FRT cell line established by Vertex Pharmaceuticals (noted here as FRT*) that overlapped with the 16 variants expressed in FRT cells in this study ( 18 ).

Techniques: Incubation, Variant Assay, Expressing, Labeling, Comparison

Journal: JCI Insight

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

doi: 10.1172/jci.insight.121159

Figure Lengend Snippet: (A) Comparison of response trends for ivacaftor (left), lumacaftor (middle), and ivacaftor/lumacaftor combination (right) of all variants studied in CF bronchial epithelial (CFBE) and Fisher rat thyroid (FRT) cell lines. (B) Comparison of response trends for all 3 treatment strategies for modest-response variants (left), intermediate-response variants (middle), and high-response variants (right).

Article Snippet: We next evaluated previously published ivacaftor response data generated by 54 CFTR variants stably expressed in a separate FRT cell line established by Vertex Pharmaceuticals (noted here as FRT*) that overlapped with the 16 variants expressed in FRT cells in this study ( 18 ).

Techniques: Comparison