Figure legends:
Figure 1 – RDR01752 rescues F508del-CFTR processing and PM
expression. (A) CFBE cells stably expressing F508del-CFTR were
incubated for 24 h with DMSO (negative control), VX-809 (3.7 µM), VX-661
(5 µM) or an increasing concentration of RDR01752. (B) CFTR
processing (C/B+C) was quantified and normalized to tubulin levels
(loading control). N = 4. Vs. DMSO: *P <0.05,
***P <0.001. Vs. VX-809:#P <0.05,##P <0.01. (C) CFBE stably
expressing mCherry-Flag-F508del-CFTR were incubated for 48 h with DMSO
(negative control), VX-809 (3.7 µM) or an increasing concentration of
RDR01752. (D) Immunostaining was performed and fluorescence
images of extracellularly exposed Flag-tags were quantified to determine
CFTR PM expression. Data are normalized to the negative control (DMSO).
N = 4. Vs. VX-809: #P <0.05,##P <0.01.
Figure 2 – RDR01752 rescues F508del-CFTR function. (A)Representative cell fluorescence recording acquired with a microplate
reader. CFBE cells stably co-expressing F508del-CFTR and the HS-YFP were
incubated for 24h with DMSO (vehicle), VX-809 (3.7 µM) or increasing
concentrations of RDR01752. Cells were then acutely (30 min) stimulated
with Fsk (20 µM) and Gen (50 µM). (B) CFTR activity was
quantified based on the rate of YFP quenching and normalized to the
negative control (DMSO, dashed line). N = 4. Vs. VX-809:#P <0.05. (C-F) Monolayers
of CFBE cells stably expressing F508del-CFTR were incubated for 24 h
with (C) DMSO (negative control), (D) VX-809 (3.7 µM),
or (E) RDR01752 (10 µM). Original Ussing chamber (open-circuit)
recordings depicting transepithelial voltage measurements
(Vte). There is an absence of response in cells treated
with DMSO, while negative deflections are observed in cells treated with
VX-809 or RDR01752 following the application of Fsk+IBMX and genistein,
which are reverted by application of Inh172.(F) Data are expressed as Isc calculated from voltage
deflections obtained for the responses to Fsk+IBMX+Gen. N = 3.Vs. DMSO: *P <0.05, **P <0.01.Vs. VX-809: #P <0.05.
Figure 3 – Intestinal organoids (F508del/F508del) respond
positively to RDR01752. (A) Bright-field images of organoids incubated
for 24 h with DMSO (negative control), VX-809 (3.7 µM), VX-661 (5 µM) or
RDR01752 (10 µM) and acutely stimulated with forskolin (Fsk, 0.128 µM)
with VX-770 (3 µM) or genistein (Gen, 50 µM). (B) Data of FIS
of organoids are expressed as the absolute area under the curve (AUC;
baseline = 100%, t = 60 min, 0.128 µM Fsk). Absence of bars
indicates there was no swelling (NS). N = 3. Vs. DMSO:
*P <0.05, **P <0.01,
***P <0.001.
Figure 4 – RDR01752 increases the rescue of F508del-CFTR PM
expression in low temperature and in cells expressing in cis the
genetic revertants G550E and 4RK, but not in R1070W. CFBE stably
expressing mCherry-Flag-CFTR (WT, F508del, DD/AA variants or carrying
G550E, R1070W, 4RK in cis with F508del) were incubated for 24 h
with DMSO (negative control), RDR01752 (10 µM), VX-809 (3.7 µM) or
VX-661 (5 µM) and then maintained for additional 24h at (A)37°C or (B) in low temperature (27°C). Data are represented as
heatmaps of the quantification from fluorescence images of
extracellularly exposed Flag-tags to determine CFTR PM expression. The
values for each cell line are normalized to DMSO at 37°C. N = 6.
Figure 5 – RDR01752 is not additive to VX-809 or VX-661 in
rescuing F508del-CFTR traffic. (A) CFBE stably expressing
mCherry-Flag-F508del-CFTR were incubated for 48 h with the following
compounds individually or in combination: DMSO (negative control),
RDR01752 (10 µM), VX-809 (3.7 µM), VX-661 (5 µM) and VX-770 (3 µM).(B) Immunostaining was performed and fluorescence images of
extracellularly exposed Flag-tags were quantified to determine CFTR PM
expression. Data are normalized to the negative control (DMSO). N = 4.Vs. single corrector (white bars): *P <0.05,
***P < 0.001. n.s.: no significant.
Figure 6 – Assessment of RDR01752 effects on rare CFTR mutants
by FLIPR membrane potential (FMP) assay. FRT cells stably expressing
CFTR variants (wt, G85E, R334W, T338I, R347P, F508del, V520F, S549F,
G551D, M1101K or N1303K) were incubated for 24 h with DMSO (negative
control), RDR01752 (10 µM) or VX-809 (1 µM). FMP assay was performed to
monitor membrane depolarization induced by stimulation with forskolin
(10 µM) plus genistein (50 µM) as a measurement of CFTR function. N = 3.Vs. DMSO (black bars): *P <0.05.
Figure 7 – Effect of RDR01752 and VX-809 individually or in
combination on functional rescue of CFTR carrying F508del, G85E, N1303K
or R334W. (A) Monolayers of FRT cells stably expressing CFTR variants
(F508del, G85E, N1303K or R334W) were incubated for 24 h with DMSO
(negative control), VX-809 (1 µM) and RDR01752 (10 µM) alone or
combined. Representative recordings of Isc measurements of Ussing
chamber for each CFTR mutant. CFTR currents were stimulated using
forskolin (FSK; 10 µM) and genistein (GST; 50 µM) and inhibited by
CFTRinh -172 (172; 10 µM). ATP (100µM) was added at the
end of each experiment as a positive control for viability. (B)Data are represented as mean increase in Isc induced by FSK+GST. N =
3-6. Vs. DMSO: *P < 0.05, **P <
0.01.