Journal: eLife

Article Title: Unravelling druggable signalling networks that control F508del-CFTR proteostasis

doi: 10.7554/eLife.10365

Figure Lengend Snippet: ( A–D ) CFBE cells were treated with the indicated inhibitors of the MLK3 pathway or VX-809 for 48 h, and the rescue of F508del-CFTR from ERQC was monitored ( A ). MLK3 pathway inhibitors rescue F508del-CFTR to levels comparable to or more than that achieved by VX-809. Changes in the levels of band C ( B ), band B ( C ) and the band C/band B ratio ( D ) were quantitated and shown mean ± SEM (n > 3). Normalized concentration (abscissa in panels B–D ) refers to concentration (VX-809, JNKi IX and Oxozeaenol [1.25, 2.5, 5, 10 μM], JNKi II [6.25,12.5, 25, 50 μM], JNKi XI [3.12, 6.25,12.5, 25 μM]) values that were normalized to the maximum used concentrations of the respective drugs. Refer panel A for concentrations (µM) of the drugs used. ( E ) CFBE cells were treated with inhibitors of the MLK3 pathway and/or VX-809 (5 μM) for 48 hr and changes in band C levels monitored. The concentrations of the MLK3 pathway inhibitors used were: JNKi II (12.5 μM), JNKi IX (5 μM), JNKi XI (25 μM) and oxozeaenol (5 μM). Wild-type CFTR (wt-CFTR) was used as a control. ( F ) Quantitation of band C levels from ( E ) normalized to the levels of band C after VX-809 treatment are shown as mean ± SEM (n > 3). The results show that synergy obtained between the MLK3 pathway inhibitors and VX-809 brings the levels of band C to ~40% of the wild-type levels. DOI: http://dx.doi.org/10.7554/eLife.10365.016

Article Snippet: The reagents used include: VX-809 (Selleckchem, Germany), JNKi II (SP600125), JNKi IX and JNKi XI (Merck Millipore, Germany), oxozeaenol (Tocris Bioscience, UK), siRNAs (see ), lipofectamine 2000 (Thermo Fisher Scientific) and ECL (Luminata crescendo from Merck Millipore).

Techniques: Concentration Assay, Control, Quantitation Assay

Journal: eLife

Article Title: Unravelling druggable signalling networks that control F508del-CFTR proteostasis

doi: 10.7554/eLife.10365

Figure Lengend Snippet: ( A ) CFBE cells were treated with indicated JNK inhibitors for 24 hr and processed for western blotting. The levels of phospho-c-jun as a measure of JNK inhibition was monitored. MLK3 pathway inhibitors reduce phospho-c-jun levels efficiently indicating a strong reduction in the activity of JNK and hence presumably of the MLK3 pathway. ( B ) CFBE cells were treated with TAK1 or MLK3 siRNA as indicated and changes in F508del-CFTR proteostasis were monitored by western blotting. TAK1 does not regulate F508del-CFTR proteostasis, as evidenced by the absence of change in the levels of bands C or B. The fold change in the band C levels was quantitated and plotted as mean ± SD (n = 2). ( C ) CFBE cells were treated with 5 μM oxozeaenol for 48 hr, or with MLK3 siRNA, or with both, and the correction of the F508del-CFTR folding/trafficking defect was monitored by changes in the levels of band C. There was no additive effect observed with the combination of MLK3 downregulation and oxozeaenol treatment. The quantitated band C levels are expressed as mean ± SD (n > 3). ( D ) CFBE cells were treated with 5 μM oxozeaenol for 24 hr, and the activity of the JNK pathway was measured by western blotting for phospho c-jun levels and F508del-CFTR. The levels of phospho c-jun were reduced, suggesting that oxozeaenol leads to a reduction in the activity of JNK. The increase in band C levels of F508del-CFTR show that the reduction in the activity of JNK is accompanied by a rescue of F508del-CFTR from ERQC. ( E ) CFBE cells were treated with flunarizine (at concentrations 6.25–50 μM) targeting the CAMKK2 pathway for 48 hr and the effect on F508del-CFTR proteostasis measured by western blotting. Treatment with flunarizine increased the levels of band C of F508del-CFTR. Other small molecules known to inhibit the CAMKK2 pathway components (verapamil and STO-609) did not show any effect on correction of F508del-CFTR. ( F ) CFBE cells transiently transfected with the P-glycoprotein mutant (P-gp DY490), the NCC mutant (R948X), or the hERG mutant (G601S) were treated with JNKi II for 24 hr, and the effect of the drug on their proteostasis monitored by western blotting. While the trafficking of P-gp DY490 out of the ER was enhanced by this treatment (seen as an increase in the Golgi-associated band C, indicated by arrows), other mutants are subjected to enhanced degradation upon drug treatment, as shown by a decrease in the levels of both bands B and C. ( G ) CFBE cells were treated with JNKi II at indicated concentrations (25, 50 µM) for 48 hr and the changes in the proteostasis of non-mutant endogenous proteins like E-cadherin, IGF1Rß and EGFR were monitored by western blotting. There was no significant change in the proteostasis of these proteins. DOI: http://dx.doi.org/10.7554/eLife.10365.017

Article Snippet: The reagents used include: VX-809 (Selleckchem, Germany), JNKi II (SP600125), JNKi IX and JNKi XI (Merck Millipore, Germany), oxozeaenol (Tocris Bioscience, UK), siRNAs (see ), lipofectamine 2000 (Thermo Fisher Scientific) and ECL (Luminata crescendo from Merck Millipore).

Techniques: Western Blot, Inhibition, Activity Assay, Transfection, Mutagenesis

Journal: eLife

Article Title: Unravelling druggable signalling networks that control F508del-CFTR proteostasis

doi: 10.7554/eLife.10365

Figure Lengend Snippet: ( A–D ) CFBE cells were treated with the indicated inhibitors of the MLK3 pathway or VX-809 for 48 h, and the rescue of F508del-CFTR from ERQC was monitored ( A ). MLK3 pathway inhibitors rescue F508del-CFTR to levels comparable to or more than that achieved by VX-809. Changes in the levels of band C ( B ), band B ( C ) and the band C/band B ratio ( D ) were quantitated and shown mean ± SEM (n > 3). Normalized concentration (abscissa in panels B–D ) refers to concentration (VX-809, JNKi IX and Oxozeaenol [1.25, 2.5, 5, 10 μM], JNKi II [6.25,12.5, 25, 50 μM], JNKi XI [3.12, 6.25,12.5, 25 μM]) values that were normalized to the maximum used concentrations of the respective drugs. Refer panel A for concentrations (µM) of the drugs used. ( E ) CFBE cells were treated with inhibitors of the MLK3 pathway and/or VX-809 (5 μM) for 48 hr and changes in band C levels monitored. The concentrations of the MLK3 pathway inhibitors used were: JNKi II (12.5 μM), JNKi IX (5 μM), JNKi XI (25 μM) and oxozeaenol (5 μM). Wild-type CFTR (wt-CFTR) was used as a control. ( F ) Quantitation of band C levels from ( E ) normalized to the levels of band C after VX-809 treatment are shown as mean ± SEM (n > 3). The results show that synergy obtained between the MLK3 pathway inhibitors and VX-809 brings the levels of band C to ~40% of the wild-type levels. DOI: http://dx.doi.org/10.7554/eLife.10365.016

Article Snippet: The reagents used include: VX-809 (Selleckchem, Germany), JNKi II (SP600125), JNKi IX and JNKi XI (Merck Millipore, Germany), oxozeaenol (Tocris Bioscience, UK), siRNAs (see ), lipofectamine 2000 (Thermo Fisher Scientific) and ECL (Luminata crescendo from Merck Millipore).

Techniques: Concentration Assay, Control, Quantitation Assay

Journal: eLife

Article Title: Unravelling druggable signalling networks that control F508del-CFTR proteostasis

doi: 10.7554/eLife.10365

Figure Lengend Snippet: ( A ) CFBE cells were treated with indicated JNK inhibitors for 24 hr and processed for western blotting. The levels of phospho-c-jun as a measure of JNK inhibition was monitored. MLK3 pathway inhibitors reduce phospho-c-jun levels efficiently indicating a strong reduction in the activity of JNK and hence presumably of the MLK3 pathway. ( B ) CFBE cells were treated with TAK1 or MLK3 siRNA as indicated and changes in F508del-CFTR proteostasis were monitored by western blotting. TAK1 does not regulate F508del-CFTR proteostasis, as evidenced by the absence of change in the levels of bands C or B. The fold change in the band C levels was quantitated and plotted as mean ± SD (n = 2). ( C ) CFBE cells were treated with 5 μM oxozeaenol for 48 hr, or with MLK3 siRNA, or with both, and the correction of the F508del-CFTR folding/trafficking defect was monitored by changes in the levels of band C. There was no additive effect observed with the combination of MLK3 downregulation and oxozeaenol treatment. The quantitated band C levels are expressed as mean ± SD (n > 3). ( D ) CFBE cells were treated with 5 μM oxozeaenol for 24 hr, and the activity of the JNK pathway was measured by western blotting for phospho c-jun levels and F508del-CFTR. The levels of phospho c-jun were reduced, suggesting that oxozeaenol leads to a reduction in the activity of JNK. The increase in band C levels of F508del-CFTR show that the reduction in the activity of JNK is accompanied by a rescue of F508del-CFTR from ERQC. ( E ) CFBE cells were treated with flunarizine (at concentrations 6.25–50 μM) targeting the CAMKK2 pathway for 48 hr and the effect on F508del-CFTR proteostasis measured by western blotting. Treatment with flunarizine increased the levels of band C of F508del-CFTR. Other small molecules known to inhibit the CAMKK2 pathway components (verapamil and STO-609) did not show any effect on correction of F508del-CFTR. ( F ) CFBE cells transiently transfected with the P-glycoprotein mutant (P-gp DY490), the NCC mutant (R948X), or the hERG mutant (G601S) were treated with JNKi II for 24 hr, and the effect of the drug on their proteostasis monitored by western blotting. While the trafficking of P-gp DY490 out of the ER was enhanced by this treatment (seen as an increase in the Golgi-associated band C, indicated by arrows), other mutants are subjected to enhanced degradation upon drug treatment, as shown by a decrease in the levels of both bands B and C. ( G ) CFBE cells were treated with JNKi II at indicated concentrations (25, 50 µM) for 48 hr and the changes in the proteostasis of non-mutant endogenous proteins like E-cadherin, IGF1Rß and EGFR were monitored by western blotting. There was no significant change in the proteostasis of these proteins. DOI: http://dx.doi.org/10.7554/eLife.10365.017

Article Snippet: The reagents used include: VX-809 (Selleckchem, Germany), JNKi II (SP600125), JNKi IX and JNKi XI (Merck Millipore, Germany), oxozeaenol (Tocris Bioscience, UK), siRNAs (see ), lipofectamine 2000 (Thermo Fisher Scientific) and ECL (Luminata crescendo from Merck Millipore).

Techniques: Western Blot, Inhibition, Activity Assay, Transfection, Mutagenesis