Fig 1: Direct interaction between CFTR and DNAJB9. (A) Illustration of ProtoArray Chip. The more CFTR captured, the brighter the spot, the higher signal intensity. (B) Known CFTR-interacting proteins were revealed by ProtoArray. (C) Different binding of DNAJB family members with CFTR was determined by protoArray. Data presented as mean of duplicate. (D,E) Western blotting of co-immunoprecipitation. HEK293 cell lines stably expressing WT- (D) and ?F508- (E) CFTR were transiently transfected with empty vector or DNAJB9-3HA vector. Forty-eight hours after transfection, immunoprecipitation was performed using anti-FLAG antibody beads (Left) and anti-HA antibody beads (Right). Precipitate and total cell lysate (input) were then subjected to western Blotting using anti-CFTR, anti-HA and anti-GAPDH antibodies. Data were representative of three independent experiments. Full-length blots are presented in Fig. S9. (F) Proximity Ligation Assay (PLA) using T84 cells. PLA assay was performed according to the manufacturer’s protocol using T84 cells which are known to endogenously express CFTR. From top to bottom, samples were treated with antibodies against both CFTR and DNAJB9, CFTR only, DNAJB9 only, and no primary ab. The interaction between CFTR and DNAJB9 was shown by red fluorescent; blue is DAPI staining. Data was representative of three independent experiments. Scale bar represents 20 µm.
Fig 2: Knockdown DNAJB9 rescues both WT- and ?F508-CFTR. (A–C) Knockdown of DNAJB9 by siRNA increased CFTR surface and functional expression in HEK293 cells. HEK293 cell lines stably expressing WT- or ?F508-CFTR were transiently transfected with empty vector (Ø) or siRNA against DNAJB9 (siRNA) for 36–48 hours before assays. Parental cells that do not express CFTR were recruited as a negative control. All data were representative of three independent experiments. (A) WES analysis of CFTR and Vinculin (loading control). Total cell lysate was analyzed by WES system using anti-CFTR and anti-Vinculin antibodies. Each sample was analysed in triplicate. Student’s t-test was performed to determine the statistical significance. (**P < 0.01; ***P < 0.005). (B) In-Cell western to determine surface CFTR levels. Cells were reverse transfected by siRNA against DNAJB9 and RNF5 in 96-well plate. Forty-eight hours after transfection, cells were then fixed and surface CFTR was probed by anti-FLAG antibody followed by IRDye 800CW conjugated Goat anti-Rabbit 2nd antibody. CellTag 700 was used as cell quantity control. Samples without anti-FLAG antibody incubation were used as background control. Representative images were shown on the top and quantification was shown at the bottom. Each condition was done in triplicates. Student’s t-test was performed to determine the statistical significance. (*P < 0.05; **P < 0.01). (C) SPQ assay to assess CFTR function. Cells were seeded into 96-well plate 24 hours after transfection and then subjected to SPQ assay described in “Materials and Methods” section. A representative graph is shown for WT-CFTR (top) and ?F508-CFTR (bottom). Each condition was performed in triplicate. (D) Knockdown DNAJB9 increased CFTR expression. Immunoblotting of mouse Jejunum membrane fraction from different genotypes, DNAJB9+/+ (WT), DNAJB9+/- (Het), and DNAJB9-/- (KO). CFTR and Na/K ATPase was probed using anti-CFTR and anti-Na/K ATPase antibodies, and Na/K ATPase was served as a membrane marker and loading control. Protein levels was quantified and ratio of KO/WT was determined. Full-length blots are presented in Fig. S11.
Fig 3: DNAJB9 may be a limiting factor for ERAD of ?F508-CFTR. (A) DNAJB9 heterozygosity rescued CFTR-dependent fluid secretion in ?F508-CFTR intestinal organoid. Intestinal organoid fluid secretion as described in “Material and Method” was performed to compare DNAJB9+/+CFTR?F508/?F508 with DNAJB9+/-CFTR?F508/?F508. Representative images before and after FSK stimulation were shown on the top. Quantification of organoid fluid secretion was shown at the bottom. Each symbol accounts for one organoid. (B) DNAJB9 heterozygosity enhanced CFTR-dependent cholera toxin (CTX)-induced fluid secretion in in vivo closed loop experiments. In vivo closed loop experiment was performed as described in “Materials and Methods” to compare DNAJB9+/+CFTR?F508/?F508 with DNAJB9+/-CFTR?F508/?F508. Representative images of intestinal loop were shown on the top upon two different-doses of CTX treatment. Quantification of secreted fluid were shown at the bottom. Student t-test was performed to determine the statistical significance. (*P < 0.05; **P < 0.01).
Fig 4: Genetic deficiency of DNAJB9 increased CFTR function in mouse intestinal organoids. Intestinal crypts from mouse in both WT-CFTR (A,B) and ?F508-CFTR (C,D) background were isolated and cultured in matrigel at 37 °C to form intestinal organoids. Organoid fluid secretion as described in “Material and Method” section was performed in the absence or presence of FSK. (A) Representative organoid images, in both resting and FSK-stimulated states, were shown for comparison between WT and DNAJB9 KO mice. (B) Quantification of (A). Each symbol represents one organoid. Data were representative of three independent experiments. (C) Representative organoid images, in both resting and FSK-stimulated states, were shown for comparison between DNAJB9+/+CFTR?F508/?F508 and DNAJB9-/-CFTR?F508/?F508 mice. (B) Quantification of (C). Each symbol represents one organoid. Data were representative of three independent experiments. Student’s t-test was performed to determine the statistical significance. (**P < 0.01; ***P < 0.001; ****P < 0.0001).
Fig 5: DNAJB9 heterozygosity improved ?F508 CF mice development. Mice body weight was compared between DNAJB9+/+CFTR?F508/?F508 and DNAJB9+/-CFTR?F508/?F508 from 5 independent sibling pairs as indicated by connected line. The body weight was determined at different ages. Blue represented male and pink represent female. (*P < 0.05).
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