Fig 1: FAM13A protein expression in human lung tissue. (A) Expression of FAM13A in single cell RNA sequencing data of airway epithelial cells. The figure in the upper left corner shows the different clusters of airway epithelial cells. The red dots on the right figures indicate the expression of FAM13A in the different clusters of the lung cells. Figures in the lower panel show the non-epithelial cell clusters and the FAM13A expression among these clusters. (B) FAM13A expression in whole lung tissue and in airway epithelial cells was assessed in 9 non-COPD controls (top) and 12 COPD patients (bottom) by immunohistochemistry (IHC). Representative images are shown. (C) Intensity of FAM13A in the whole slide and in the epithelium of every airway in the tissue. The average intensity of all airways per donor is presented. Each dot represents one subject. Data are shown as mean. Significant differences were assessed by Unpaired t-test and p-value is indicated.
Fig 2: Significantly more rapid building-up of electrical resistance upon FAM13A overexpression in 16HBE14o- cells. 16HBE14o- cells were transfected with FAM13A overexpression plasmid or empty vector (pCMV6). After 24 h, the cells were trypsinized and seeded in duplicates into ECIS arrays. The high-frequency capacitance and low-frequency resistance were monitored over 72 h after seeding. (A) High-frequency capacitance and (B) low-frequency resistance values of a representative ECIS experiment. The vertical line indicates the time when cells reached confluence as indicated by the stabilization of high-frequency capacitance. Data are shown as mean ± SEM of two duplicates in the ECIS array. (C) The area under the curve (AUC) of resistance values from 0 to 72 h was calculated and depicted as mean and individual experiments (n = 8). Significant differences were assessed by the Wilcoxon signed rank test and p-value is indicated.
Fig 3: Expression of adherens junction protein E-cadherin is increased upon overexpression of FAM13A in 16HBE14o- cells and correlates with FAM13A in human lung tissue. 16HBE14o- cells were seeded in duplicate, transfected with FAM13A overexpression plasmid or empty vector (pCMV6) and harvested at 6 and 24 h after transfection. (A) E-cadherin protein expression levels were measured using western blot, quantified by densitometry and normalized to ß-actin as loading control (mean ± SEM, n = 6). Total ß-catenin (B) and active/non-phospho-ß-catenin expression levels were assessed by western blot, quantified by densitometry and normalized to ß-actin as loading control. (C) The ratio of active/non-phospho-ß-catenin to total ß-catenin is depicted. Representative blots are shown. Significant differences were assessed by the Wilcoxon signed rank test and the p-value between significant different conditions is as indicated. (D) The expression of E-cadherin in human lung tissue was assessed by immunohistochemistry comparable to FAM13A as described above. The correlation between the expression of FAM13A and E-cadherin in the same airway was tested using the Pearson’s correlation test. Each dot represents one airway. Number of data pairs = 77, r = 0.2812, p = 0.0132.
Fig 4: FAM13A expression in primary human airway epithelial cells. (A) Control and COPD-derived AECs were seeded in duplicate, grown to confluence, and incubated with hormone-free media for 24 h. FAM13A levels were detected by western blot in total cell lysates, quantified by densitometry and normalized to β-actin as the loading control. Medians are indicated, control n = 10, COPD n = 7. A representative blot is shown. Significant differences were assessed by the Mann-Whitney test. (B) Control and COPD-derived AECs were cultured and hormonally deprived as described above, and incubated with or without 20% cigarette smoke extract (CSE) for 24 h. FAM13A levels were detected and quantified as for panel A. Each dot represents one subject, control n = 10, COPD n = 7. Representative blots are shown. Significant differences were assessed by the Wilcoxon signed rank test and p-value is indicated.
Fig 5: Suppressive effect of FAM13A on CSE-induced CXCL8 secretion in 16HBE14o- cells. 16HBE14o- cells were seeded in duplicate, transfected with FAM13A overexpression plasmid or empty vector (pCMV6), serum deprived overnight and exposed to 10% CSE for 24 h. CXCL8 secretion levels were measured using ELISA. (A) Absolute CXCL8 levels in pCMV6 transfected cells upon CSE exposure. (B) CXCL8 secretion levels of FAM13A overexpressing cells were measured as above and normalized to the levels of pCMV6 cells of each CSE concentration, respectively, (n = 8). Data are shown as mean ± SEM (n = 8). Significant differences were assessed by the Wilcoxon signed rank test and the p-value is indicated.
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