*2.1. Removal of Short Regulatory Extension (RES) Alone or with Regulatory Insertion (RI) has No Impact on 508del-CFTR Processing*

Given the controversy in defining both RI and RE, we chose to generate two versions of these regions because of the respective structural implications. Indeed, RI<sup>L</sup> and RE<sup>L</sup> are the complete regions which are absent in other ABC transporters (Figure S1), whereas, their shorter versions appeared as structurally meaningful to be removed: RI<sup>S</sup> is strictly the region described as destructured in the crystal structure and RE<sup>S</sup> is the core of RE, i.e., just the β-strand [19]. The impact of deleting RES—short RE (∆ <sup>654</sup>Ser-Gly673, Figure S1)—was first assessed, either alone or together with RI in its short and long variants (∆RI<sup>S</sup> and ∆RIL), on the in vivo processing of wt- and F508del-CFTR by Western blot (WB) of Baby Hamster Kidney (BHK) cells stably expressing such variants (Figure 1B,E,F and Table 1). Processing of CFTR was assessed by WB in terms of its fully-glycosylated form (also termed band C)

corresponding to post-Golgi forms, assumedly at the PM. Unprocessed CFTR, in turn corresponds to its core-glycosylated, ER-specific form (also termed band B). Removal of RE<sup>S</sup> had no impact on processing of either F508del-CFTR or wt-CFTR (Figure 1B, lanes 5,10; Table 1), despite that the immature form of ∆RES-F508del-CFTR appeared consistently at increased levels vs. those of F508del-CFTR (Figure 1B, lanes 5,2).

**Figure 1.** Effect of removal of short regulatory extension (∆RES) on processing of wt- and F508del-Cystic Fibrosis (CF) transmembrane conductance regulator (CFTR). (**A**) Structural features of the CFTR nucleotide binding domain 1 (NBD1) domain. Protein Data Bank identifier (PDB ID): 2BBO. The main structural regions are color coded. F508 is represented as red spheres. G550 is represented as brown spheres. ATP is shown as sticks and Mg2<sup>+</sup> as a pink sphere. Regulatory insertion (RI) is unstructured in the crystal structure and represented as a dotted red line. The G550E mutation in this structure was reverted in silico. (**B**–**D**) Samples from Baby Hamster Kidney (BHK) cell lines stably expressing different CFTR variants of ∆RE<sup>S</sup> and ∆RI, the latter either in its short "S" or long "L" versions (see Materials and Methods) and alone (**B**) or jointly with (**C**,**D**) genetic revertants, as indicated, were analyzed for CFTR protein expression by Western blot (WB) with anti-CFTR 596 Ab and also anti-calnexin (CNX) as loading control. All samples were incubated with 3 µM dimethyl sulfoxide (DMSO) for 48 h, as controls for experiments with corrector VX-809 (see Figure 3). Summary of data of CFTR variants on the wt- (**E**) or F508del- (**F**) CFTR backgrounds, expressed as normalized ratios (band C/(band C + band B)) and as a percentage to the respective ratio for wt-CFTR and as mean ± standard error of the mean (SEM). (n) indicates nr. of independent experiments. "\*" and "#" indicate significantly different (*p* < 0.05) from wt-CFTR and F508del-CFTR, respectively.


**Table 1.** Summary of Western blot quantification of original data in Figures 1–3 for different CFTR variants in the presence of VX-809 or DMSO control. Data are expressed as normalized ratios (band C/ band (B + C)) for each variant and as a percentage to wt-CFTR ratio.

**Figure 2.** Effect of removal of helix H9 in combination with ∆RE<sup>L</sup> and VX-809 on processing of wt- and

F508del-CFTR. (**A**,**B**) WB analysis of samples from BHK cell lines stably expressing wt- or F508del-CFTR variants with ∆REL, ∆H9, or ∆REL-∆H9. Cells were incubated with 3 µM VX-809 or DMSO (control) for 48 h at 37 ◦C. CFTR protein expression was analyzed by WB with the anti-CFTR 596 and anti-CNX Abs. (**C**) Summary of data expressed as normalized ratios (Band C / (Band C + B)) and as a percentage to the respective ratios on the wt- or F508del-CFTR backgrounds and shown as mean ± SEM. (n) indicates number of independent experiments. "#" indicates significantly different from the respective variant treated with DMSO. "\*" and "\*\*" indicate significantly different from wt- or F508del-CFTR, respectively.

**Figure 3.** Effect of VX-809 on processing of wt- and F508del-CFTR variants without regulatory extension.

(**A**–**C**) WB of samples from BHK cell lines stably expressing different CFTR variants of ∆RE and ∆RI, as indicated, alone (A) or jointly with (**B**,**C**) genetic revertants (see Materials and Methods). CFTR protein expression was analyzed by WB with anti-CFTR 596 and anti-CNX Abs as in Figure 1. All samples were incubated with 3 µM VX-809 for 48 h. (D, E) Summary of data of variants on the wt- (**D**) or F508del- (**E**) CFTR backgrounds, expressed as normalized ratios (band C/(band C + band B)) and as a percentage to the respective ratio for wt-CFTR and as mean ± SEM. (n) indicates number of independent experiments. "\*" indicates significantly different (*p* < 0.05) from respective variant without VX-809 (as shown in Figure 1 and indicated here by dashed bars).

Removal of RE<sup>S</sup> together with RIL—long RI (∆ <sup>404</sup>Gly-Leu435, Figure S1) led to an increase in processing of F508del-CFTR from 3 ± 2% to 71 ± 3% (vs. wt-CFTR levels), similarly to what had been previously reported by Alexandrov et al. for ∆RI<sup>L</sup> alone [13]. In contrast, removal of RIS—short RI (∆ <sup>412</sup>Ala-Leu428, Figure S1) had no impact on ∆RES-F508del-CFTR processing (Figure 1B, lanes 6,7; Figure 1F; Table 1). So in summary, removal of RE<sup>S</sup> from the ∆RIS- or ∆RIL-F508del-CFTR variants had no further effect on their processing, despite that processing of ∆RES-F508del-CFTR is different upon removal of RI<sup>S</sup> or RIL, but this difference remains equivalent to removal RI<sup>S</sup> or RI<sup>L</sup> alone. Despite this lack of impact on processing, removal of RE<sup>S</sup> does affect total protein expression levels of ∆RIS- or ∆RIL-F508del-CFTR variants leading to a decrease in RIS-F508del-CFTR (Figure 1B, lanes 3,6) and an increase in RIL-F508del-CFTR (Figure 1B, lanes 4,7). Interestingly, when RE<sup>S</sup> and RIS, were jointly removed from wt-CFTR, its processing was significantly reduced to 54 <sup>±</sup> 9%, while <sup>∆</sup>RE<sup>S</sup> jointly with ∆RI<sup>L</sup> caused no impact (Figure 1B, lanes11,12, respectively; Figure 1E; Table 1). Again, these data were equivalent to removal of RI<sup>S</sup> or RI<sup>L</sup> alone on wt-CFTR processing (Figure 1B, lanes 8,9; Figure 1F; Table 1).

The differential effect caused by removal of RI<sup>S</sup> vs RI<sup>L</sup> on F508del- and wt-CFTR emphasize the importance of those 8 N-term (404Gly-Lys411) and 7 C-term (Phe429-Leu435) amino acid residues that differ between the two RI regions for the folding and processing of CFTR.

Overall, removal of RE<sup>S</sup> alone or with RI has no impact on F508del-CFTR processing efficiency.
