Fig 2: Knockdown of CLEC3B could significantly attenuate the fibrogenesis of ASCs from lymphedema patients.a Schematic representation of the experimental procedure. ASCs from patients (n = 3) were transfected with CLEC3B siRNA or scramble siRNA (negative control). The cells were harvested 48 h and 96 h after transfection for mRNA and protein analysis, respectively. b Images showing the ASCs after 96 h of transfection. Scale bar: 100 μm. c Network plot showing the downregulated pathways in ASCs with siRNA-mediated knockdown of CLEC3B versus the negative control. Functional enrichment analysis was performed on the differential expressed genes detected by bulk RNA-seq. Adjusted P value < 0.05, the hypergeometric test. d Significantly reduced mRNA expression of c3 markers and fibrosis-related genes in ASCs with CLEC3B knockdown versus the negative control (determined by bulk RNA-seq). The significance threshold was set to be a P value < 0.05 of the sleuth differential expression test. e Significantly reduced mRNA expression of c3 markers and fibrosis-related genes in ASCs with CLEC3B knockdown versus the negative control (determined by qPCR). The significance threshold was set to be a P value < 0.05 of the one-tailed paired t-test. f Immunoblot assays showing the decreased protein expression of CLEC3B, Collagen-1, SMAD2/3, and phosphorylated SMAD2/3 in ASCs with CLEC3B knockdown versus the negative control (n = 3). g Densitometry analysis of the immunoblot results. The significance threshold was set to be a P value < 0.05 of the one-tailed paired t-test. h Immunofluorescent staining of Collagen-1 in ASCs transfected with CLEC3B siRNA or scramble siRNA. Scale bar: 50 μm. Representative fields of view of ASCs from the patient L21 are shown. i Collagen-1 staining fluorescence intensity comparison. Each value represents the mean fluorescence intensity of three representative fields of view. Two-tailed paired t-test.

Fig 3: RF started to receive CAF phenotype after interaction with tumor cells. (a) The mRNA expression of α-SMA, CLEC3B, CCRL1, TGM2 and MGP in PFs and RFs co-cultured with AN, M14, UM-SCC19 or UM-SCC47. (b) α-SMA was stained in PF-FTMs and RF-FTMs with primary keratinocytes, AN, M14, UM-SCC19 or UM-SCC47 epidermis. Arrows indicate the expression of the biomarker. Data was obtained from three independent experiments and is presented as mean ± standard deviation (SD); * p < 0.05, ** p < 0.01, *** p < 0.001.

Fig 4: The protein interaction network of 32 bone metastasis-related proteins of prostate cancer analyzed by STRING online tool. Only the F5, SEPP1, THBS1, HRG, SERPINA4, and CLEC3B were shown to interact with each other in the whole network. No other DEPs formed an interaction network.