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. 2022 Jan 4;11(1):156. doi: 10.3390/cells11010156

Figure 2.

Figure 2

PNC is localized to the cell surface of myofibroblasts. Myofibroblasts from heart and lung tissues were stained and analyzed by flow cytometry using m-α-PNC mAb 10A10, excluding debris and dead cells via gating and 7AAD exclusion using Flowjo software. Myofibroblasts from fibrosis origins stain positive for cell surface PNC: (A) CF-DCM, cardiac myofibroblasts from dilated cardiomyopathy; Chi-Squared = 177.5; SE Dymax % Positive = 48.0. LL97A, IPF; Chi-Squared = 93.4; SE Dymax % Positive = 54.8, and LL29, IPF; Chi-Squared = 113.5; SE Dymax % Positive = 51.0. PNC was not detected on the surface of primary normal human cardiac myofibroblasts from healthy donor, NHCF; Chi-Squared = 3.47; SE Dymax % Positive = 8.54, primary normal human lung myofibroblasts from healthy donor, NHLF; Chi-Squared = 0; SE Dymax % Positive = 0, or immortalized CCD-16Lu lung myofibroblasts from healthy donor; Chi-Squared = 0; SE Dymax % Positive = 0. Mouse IgG1 isotype control (blue shaded) was compared to m-α-PNC mAb (unshaded). Results are representative of 3 independent experiments, n = 3. For all flow cytometry experiments, Chi-squared ≥ 4 is statistically significant. (B) Cell surface proteins were isolated from myofibroblasts and immunoblotted for N-cadherin, PNC, and Na,k-ATPase α-1 (ATP1A1) cell surface compartment loading control. PNC and N-Cadherin lysates were normalized to the ATP1A1 cell surface loading control for each sample and are reported as a relative value below each band.