Article Figures & Data
Figures
- Figure 1.
The determination of optimal cordycepin concentration for use in cordycepin-induced HaCaT secretome (CHS) production and effect of CHS on human dermal fibroblast (HDF) proliferation. A: 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) cell viability assay and microscopic images of HaCaT cells in response to cordycepin. Cordycepin at 2.5 μM was the optimal concentration since it promoted HaCaT proliferation without any morphological alteration. Scale bar=200 μm. Significantly different from the control (0 μM) at: *p<0.05 and **p<0.01. B: MTT cell viability assay of HDFs in response to normal HaCaT secretome (NHS) and CHS. While NHS had no impact on HDF proliferation, CHS promoted HDF proliferation in a dose-dependent manner. Significantly different from the control (0%\) at: *p<0.05 and **p<0.01. C: Densitometric analysis for mRNA expression of MKI67. It was highly up-regulated in NHS- and CHS-treated HDFs compared to those without any secretome. Notably, CHS-treated HDFs showed the highest MKI67 expression. MKI67 expression in CHS-treated HDFs was also significantly higher than that of NHS-treated HDFs. D: Representative fluorescence-microscopy images showing the highest MKI67 fluorescence intensity was in CHS-treated HDFs. Scale bar=100 μm. Significantly different at: **p<0.01 from control, non-treated HDFs; #p<0.05 and ##p<0.01. The experiments were performed in triplicate.
- Figure 2.
Cordycepin-induced HaCaT secretome (CHS) led to up-regulation of intracellular reactive oxygen species (ROS)-scavenging pathways. A: Quantitative analysis of dichloro-dihydro-fluorescein diacetate assay of normal HaCaT secretome (NHS)- and CHS-treated human dermal fibroblasts (HDFs) showing ROS-scavenging capability comparable to that of the antioxidant N-acetyl cysteine (NAC). Significantly different at p<0.01 from: **control, non-treated HDFs; ##non-treated HDFs with H2O2 stimulation. B: Densitometric analysis for the expression of genes involved in intracellular ROS-scavenging pathways. CHS promoted the expression of superoxide dismutase 1 (SOD1), glutathione peroxidase 1 (GPX1) and catalase (CAT). Significantly different from the control (0%) at: *p<0.05 and **p<0.01. The experiments were performed in triplicate.
- Figure 3.
Cordycepin-induced HaCaT secretome (CHS) promotes wound healing in human dermal fibroblasts (HDFs) compared to normal HaCaT secretome (NHS) and control without any secretome. A: Representative images of wound-healing assays. Scale bar=200 μm. B: Quantitative analysis of migrating HDF cells in the scratched area in response to NHS and CHS treatment at 0, 24 and 48 h post wounding. Significantly different at: **p<0.01 from control, non-treated HDFs; #p<0.05 and ##p<0.01. The experiments were performed in triplicate.
- Figure 4.
Cordycepin-induced HaCaT secretome (CHS) regulated the expression of extracellular matrix-related elements at both the transcriptional and translational levels. A: Densitometric analysis for mRNA expression of extracellular matrix-related genes. The mRNA expression of collagen type I alpha 1 chain (COL1A1), collagen type I alpha 2 chain (COL1A2), collagen type III alpha 1 chain (COL3A1) and elastin (ELN) were up-regulated. In contrast, the mRNA expression of matrix metalloproteinases MMP1 and MMP3 was down-regulated. Expression of most genes was dose-dependent manner. Significantly different from control, non-treated HDFs at: *p<0.05 and **p<0.01. B. Representative fluorescence-microscopy images showing fluorescence of COL1A1 protein. Scale bar=100 μm. DAPI: 4′,6-Diamidino-2-phenylindole. The greatest fluorescence was present in CHS-treated HDFs. However, the difference between NHS- and CHS-treated HDFs was not statistically significant. **Significantly different from the control (0%) at p<0.01. The experiments were performed in triplicate.
- Figure 5.
Cordycepin-induced HaCaT secretome (CHS) activated the expression of autophagy-related genes in human dermal fibroblasts (HDFs) at both the transcriptional and translational levels. A: Densitometric analysis for mRNA expression of autophagy-related genes indicated the up-regulation of sequestosome 1 (SQSTM1), autophagy related 5 (ATG5), beclin 1 (BECN1) and microtubule-associated protein 1 light chain 3 alpha (MAP1LC3A). The most obvious up-regulation was found in LC3 expression. Significantly different from the control (0%) at: *p<0.05 and **p<0.01. B: Representative fluorescence-microscopy images showing the highest fluorescence intensity of MAP1LC3A protein was in CHS-treated HDFs. Scale bar=100 μm. Significantly different at: **p<0.01 from control, non-treated HDFs; #p<0.05 and ##p<0.01. The experiments were performed in triplicate.
- Figure 6.
Densitometric analysis for the determination and comparison of cytokine components of normal HaCaT secretome (NHS) and cordycepin-induced HaCaT secretome (CHS). 1: C-C motif chemokine ligand 2 (CCL2); 2: chemokine C-X-C motif ligand 1 (CXCL1); 3: interleukin 1 receptor antagonist (IL1RN); 4: interleukin 6 (IL6); 5: CXCL8; 6: macrophage migration-inhibitory factor (MIF); 7: serpin family E member 1 (SERPINE1). All were found in both NHS and CHS but among these, CXCL1, IL1RN, ICXCL8, MIF and SERPINE1 were found to be at higher levels in CHS.
- Figure 7.
Schematic illustration hypothesizing the possible biological activity of cordycepin-induced HaCaT secretome (CHS) in promoting skin regeneration. ATG5: Autophagy-related 5; BECN1: beclin 1; CAT: catalase; CCL2: C-C motif chemokine ligand 2; COL1A1: collagen type I alpha 1 chain; COL1A2: collagen type I alpha 2 chain; COL3A1: collagen type III alpha 1 chain; CXCL: chemokine C-X-C motif ligand; ELN: elastin; GPX1: glutathione peroxidase 1; IL: interleukin; IL1RN: interleukin 1 receptor antagonist; MAP1LC3A: microtubule-associated protein 1 light chain 3 alpha; MIF: macrophage migration-inhibitory factor; MMP: matrix metalloproteinase; SERPINE1: serpin family E member 1; SOD1: superoxide dismutase 1; SQSTM1: sequestosome 1. ↑ Up-regulation; ↓ down-regulation.
