Figure 3. Silencing RPL12 enhances F508del-CFTR functional expression in primary HBE.

(A) Following RPL12 knockdown, endogenous expression of mature, fully glycosylated F508del protein (band C, white arrowhead) is compared with levels of immature glycoform (band B, black arrowhead). Amounts of band B, band C, and RPL12 are quantified on right. Data are represented as mean ± SEM obtained from siRPL12-treated cells normalized to NS siRNA controls (dotted line set to 100%) (n = 3). Asterisks represent statistical comparison between siRPL12 and NS siRNA. *P < 0.0167; **P < 0.01 (unequal variance t test on log₂-transformed data with post hoc Bonferroni’s correction; α = 0.0167). (B) Conversion ratio of band C to bands B and C (i.e., test of maturation efficiency) from A. Data include mean ± SEM (n = 3). ***P < 0.001 (2-sample t test). (C) F508del-mediated transepithelial ion transport (I_SC) is augmented by RPL12 depletion (n = 3). Asterisks represent statistical comparison of forskolin+genistein stimulation (i.e., total constitutive plus acutely potentiated CFTR function). ****P < 0.0001, 2-sample t test. Fsk, forskolin; Gen, genistein. 48, deidentified patient code. (D) siRPL12 application enhances levels of cAMP-dependent CFTR ion transport (i.e., ratio of forskolin- to forskolin+genistein). Data are represented as mean ± SEM (n = 3). **P < 0.01, 2-sample t test. NS, NS siRNA; siRPL12, RPL12-targeting siRNA; Forskolin (5 μM), activator of PKA; genistein (50 μM), stimulator of CFTR-gating activity (i.e., potentiator); Inh₁₇₂, 10 μM, inhibitor of CFTR. HBE were isolated from a CF individual with CFTR^{F508del/F508del} genotype, cultured at air-liquid interface, and transfected twice per week with siRPL12 or NS siRNA (100 nM) for 3 weeks.