Figure 2

CF MDMs have a different iron-related protein expression profile compared to non-CF MDMs. Densitometry calculations were performed on samples run on the same membrane, at the same exposure time for each protein. Short and long exposure times are depicted for HO-1, FTH1, and β Actin. Numbers and letters denote arbitrary subject identification. “X” indicates the lane was excluded because this subject was a repeat donor. Black arrows indicate molecular weight of the protein in cases where there are multiple bands. Blots were cropped for presentation. Full images of blots are in Supplementary Fig. S5a and b. (a) Representative images of western blots in CF (n = 7) pretreated with vehicle or modulators and non-CF (n = 6) MDMs pretreated with vehicle, followed by exposure to vehicle for 18 h. (b) Representative images of western blots in CF (n = 8) and non-CF (n = 5) MDMs pretreated with vehicle or modulators, followed by LPS for 18 h. (c–e) After log2 transformation of the densitometric values, protein was normalized to β Actin and analyzed. Graphs were generated with R version 3.6.0 (2019-04-26, https://www.R-project.org/) (c) Baseline differences between CF and non-CF protein expression and differences between LPS-treated CF and non-CF protein expression were analyzed using Wilcoxon rank sum tests (‘*’ indicates P < 0.05, ‘**’ indicates P < 0.01). Within the CF population, whether a patient was using in vivo ivacaftor/lumacaftor or ivacaftor/tezacaftor was denoted by color (yellow “iva/luma” and black “iva/teza”). (d) The impact of modulators (ivacaftor [30 nM] and lumacaftor [3 µM], VX) on CF MDM protein expression was analyzed using linear models with factors to account for exposure and subject (‘*’ indicates P < 0.05). (e) The impact of modulators (ivacaftor [30 nM] and lumacaftor [3 µM], VX) on CF MDM protein expression in the presence of LPS was analyzed using linear models (‘.’ indicates P = 0.07). (f) Hepcidin, (g) lactoferrin, and (h) transferrin ELISAs were performed on supernatants collected from CF (n = 7) and non-CF (n = 5) MDMs pretreated with vehicle or modulators, followed by exposure to vehicle or LPS for 24 h. No-LPS controls were analyzed using non-parametric Kruskal–Wallis test with Dunn’s test for multiple comparisons. LPS-treated non-CF and CF groups were compared using Mann–Whitney tests. LPS-treated CF and LPS-treated CF with modulator pretreatment were compared using paired Wilcoxon rank sum tests.