Figure 4.
HERC3 and RNF5/185 facilitate ∆F508-CFTR retrotranslocation. (A) A schematic diagram of the HiBiT retrotranslocation assay, where ∆F508-CFTR-HiBiT(Ex) and cytosolic LgBiT were co-expressed. The luminescence signal generated by the interaction of LgBiT and the HiBiT tag exposed in the cytosol after retrotranslocation was measured in living cells during MG-132 treatment. (B) A typical measurement of ∆F508-CFTR-HiBiT(Ex) retrotranslocation in 293MSR cells. The luminescence signal was measured in living cells upon treatment with 10 µM MG-132, with or without 10 µM DBeQ. (C) Kinetic retrotranslocation of ∆F508-CFTR-HiBiT(Ex) in 293MSR cells treated with DMSO (0.3%) or Trikafta (3 µM VX-661, 3 µM VX-445, 1 µM VX-770) for 24 h at 37°C. Luminescence was continuously monitored in the presence of MG-132 with or without CHX. The signal increased by the MG-132 treatment was plotted as retrotranslocated CFTR. The retrotranslocation rate of ∆F508-CFTR-HiBiT(Ex) was calculated by linear fitting of the signal until 60 min (right, n = 4). Two-way RM ANOVA revealed a significant main effect of Trikafta or CHX, but no interaction between them (Pint > 0.05). (D) Kinetic retrotranslocation of ∆F508-CFTR-HiBiT(Ex) in 293MSR WT and RNF5/185 KO cells transfected with 50 nM siNT or siHERC3. Luminescence was continuously monitored over 60 min in the presence of MG-132. The signal increased by the MG-132 treatment was plotted as retrotranslocated CFTR. The retrotranslocation rate of ∆F508-CFTR-HiBiT(Ex) was calculated by linear fitting (right, n = 3). Two-way RM ANOVA revealed a significant main effect of HERC3 KD or RNF5/185 DKO, but no interaction between them (Pint > 0.05). (E) A schematic diagram of the HiBiT ER disappearance assay, where ∆F508-CFTR-HiBiT(Ex) and ER-luminal LgBiT (ER LgBiT) were coexpressed. The luminescence signal generated by the interaction of LgBiT and the HiBiT tag in the ER was measured in living cells during the CHX chase. (F) Kinetic ER disappearance of ∆F508-CFTR-HiBiT(Ex) in 293MSR WT and RNF5/185 KO cells transfected with 50 nM siNT or siHERC3. Luminescence was continuously monitored over 180 min in the presence of CHX and plotted normalized to the non-treated cells as remaining CFTR at the ER (%). The ER disappearance rate of ∆F508-CFTR-HiBiT(Ex) was calculated by fitting the kinetic ER disappearance curve (right, n = 3). Two-way RM ANOVA with Holm–Sidak multiple comparison tests revealed a significant main effect of RNF5/185 DKO and no interaction between HERC3 KD and RNF5/185 DKO (Pint > 0.05). Data distribution was assumed to be normal but was not formally tested. Each biological replicate (n) is color-coded: the averages from three or four technical replicates are shown in triangles (D and F). Data represent mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not significant.

HERC3 and RNF5/185 facilitate ∆F508-CFTR retrotranslocation. (A) A schematic diagram of the HiBiT retrotranslocation assay, where ∆F508-CFTR-HiBiT(Ex) and cytosolic LgBiT were co-expressed. The luminescence signal generated by the interaction of LgBiT and the HiBiT tag exposed in the cytosol after retrotranslocation was measured in living cells during MG-132 treatment. (B) A typical measurement of ∆F508-CFTR-HiBiT(Ex) retrotranslocation in 293MSR cells. The luminescence signal was measured in living cells upon treatment with 10 µM MG-132, with or without 10 µM DBeQ. (C) Kinetic retrotranslocation of ∆F508-CFTR-HiBiT(Ex) in 293MSR cells treated with DMSO (0.3%) or Trikafta (3 µM VX-661, 3 µM VX-445, 1 µM VX-770) for 24 h at 37°C. Luminescence was continuously monitored in the presence of MG-132 with or without CHX. The signal increased by the MG-132 treatment was plotted as retrotranslocated CFTR. The retrotranslocation rate of ∆F508-CFTR-HiBiT(Ex) was calculated by linear fitting of the signal until 60 min (right, n = 4). Two-way RM ANOVA revealed a significant main effect of Trikafta or CHX, but no interaction between them (Pint > 0.05). (D) Kinetic retrotranslocation of ∆F508-CFTR-HiBiT(Ex) in 293MSR WT and RNF5/185 KO cells transfected with 50 nM siNT or siHERC3. Luminescence was continuously monitored over 60 min in the presence of MG-132. The signal increased by the MG-132 treatment was plotted as retrotranslocated CFTR. The retrotranslocation rate of ∆F508-CFTR-HiBiT(Ex) was calculated by linear fitting (right, n = 3). Two-way RM ANOVA revealed a significant main effect of HERC3 KD or RNF5/185 DKO, but no interaction between them (Pint > 0.05). (E) A schematic diagram of the HiBiT ER disappearance assay, where ∆F508-CFTR-HiBiT(Ex) and ER-luminal LgBiT (ER LgBiT) were coexpressed. The luminescence signal generated by the interaction of LgBiT and the HiBiT tag in the ER was measured in living cells during the CHX chase. (F) Kinetic ER disappearance of ∆F508-CFTR-HiBiT(Ex) in 293MSR WT and RNF5/185 KO cells transfected with 50 nM siNT or siHERC3. Luminescence was continuously monitored over 180 min in the presence of CHX and plotted normalized to the non-treated cells as remaining CFTR at the ER (%). The ER disappearance rate of ∆F508-CFTR-HiBiT(Ex) was calculated by fitting the kinetic ER disappearance curve (right, n = 3). Two-way RM ANOVA with Holm–Sidak multiple comparison tests revealed a significant main effect of RNF5/185 DKO and no interaction between HERC3 KD and RNF5/185 DKO (Pint > 0.05). Data distribution was assumed to be normal but was not formally tested. Each biological replicate (n) is color-coded: the averages from three or four technical replicates are shown in triangles (D and F). Data represent mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not significant.

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