Fig. 4: CYGB regulates HPaSteCs activation and collagen production.

A Representative contrast and fluorescent images of the double staining for CYGB (green) and αSMA (red) in HPaSteCs cultured in medium with (S+) or without (S−) supplement. Scale bars, 100 µm. Dapi was used to visualize nuclei. B Immunoblotting analysis of CYGB, COL1A1, and αSMA along with their quantifications (left and middle panel), and qRT-PCR analysis of CYGB, αSMA, COL1A1, COL3A1, and PDFGRβ (right panel) in HPaSteCs under S+ or S− condition. GAPDH was used as loading control. C Immunoblotting analysis of CYGB, COL1A1, and αSMA along with their quantifications (left and middle panel), and qRT-PCR analysis of CYGB, αSMA, COL1A1, COL3A1, PDFGRβ, and antioxidant related-genes (SOD1, HMOX1/2, HSP1A1) of HPaSteCs transfected with control lentiviral eGFP expression vector (GFP) or CYGB overexpression vector (CYGB) (right panel). GAPDH was used as loading control. D (Top-left panel) Determination of CYGB presence in cell lysate and cultured medium of HPaSteCs by immunoblotting assay. Entire medium (7.5 µL/lane) and total cell lysate (6 µg/lane) from HPaSteCs transfected with control lentiviral eGFP expression vector (GFP) or CYGB expression vector (CYGB) were loaded per lane. His-FLAG-tagged-CYGB (23.4 kDa) presented above endogenous CYGB (21 kDa) in the cell lysate. GAPDH was used as loading control. M molecular marker. (Bottom-left panel) Quantification for CYGB expression by immunoblotting assay. Loading amount: 7.5 µL of 100 time-condensed (100x) (lane 1, 2) or 1x medium (lane 3, 4) from lentiviral eGFP expression vector (lane 1, 2) or CYGB expression vector-transfected HPaSteCs (lane 3, 4) at S+ and S− conditions, along with a ladder of rhCYGB serving as standard samples (lane 5–9). Note that the secreted CYGB from HPaSteCs transfected with control vector (GFP) at S+ and S− conditions (lane 1, 2) were only found when the medium was 100x. (Right panel) A calibration curve was plotted from densities of rhCYGB standards (orange circles) and used to determine the endogenous CYGB content in the 100x medium from HPaSteCs transfected with Lentiviral eGFP vector at S+ (purple rhombus) and S− conditions (blue triangle), and secreted CYGB from entire medium of HPaSteCs transfected with lentiviral CYGB expression vector at S+ (green circle) and S− conditions (red square) (right-panel). Data were shown as mean ± SD from three independent experiments, n = 4. *p < 0.05, **p < 0.01, ***p < 0.001, Student’s t test, two-tailed.