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. 2022 Feb 7;18(4):1677–1694. doi: 10.7150/ijbs.69178

Figure 4.

Figure 4

CRC cells derived TSG-6 reprograms NFs into CAFs. (A) Schematic representation of co-culture of cancer cells and GFP-labeled normal fibroblasts, cancer cells were transfected by mCherry-Vector plasmids or mCherry-TSG-6 overexpressing plasmids. (B-C) High Content Screening assay to monitor the speed, movement distance (B) and cell size (C) of NFs coculturing with CRC cells. (D) Western blot evaluation of α-SMA and FAP expression in NFs treated with Vec-CM or T6-CM. (E) Immunostaining of α-SMA in NFs treated with Vec-CM or T6-CM, detected by confocal microscopy (Magnification: 200×). Scale bars: 10 µm. (F) Wound healing assay to determine the migration of NFs cultured with T6-CM and Vec-CM. TSG-6 neutralizing antibody A38 could abolish the effect of T6-CM. (G) Western blot evaluation of α-SMA and FAP expression in NFs treated with or without rhTSG-6. (H) Immunostaining of α-SMA in NFs treated with or without rhTSG-6, detected by confocal microscopy (Magnification: 200×). Scale bars: 10 µm. (I) Wound healing assay to determine the migration of NFs treated with or without rhTSG-6. (J-K) High Content Screening assay to monitor the movement speed (J), cell size and F-actin intensity (K) of NFs treated with rhTSG-6. F-actin stained with phalloidin (red) and nuclei stained with DAPI (blue). Scale bars: 20 µm. Data information: Statistical analysis was performed using two tailed unpaired Student's t-test (B, C, F, I, J, K). *p<0.05, **p<0.01.