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. 2021 Sep 10;11:716055. doi: 10.3389/fonc.2021.716055

Figure 2.

Figure 2

Fibroblast-secreted galectin-1 (Gal-1) significantly promotes multiple CIC features in CRC cells. (A) Expression of endogenous Gal-1 in MRC-5 and WS1 fibroblasts as detected through Western blot (top panel; recombinant human Gal-1 protein (rhGal-1) used as positive control) and ELISA (bottom panel). (B) Invasion capacity of KM12C with the addition of increasing doses of rhGal-1. (C) qPCR analysis for the gene expression of Twist1 and E-cadherin and (D) Western blot for protein expression of Slug and E-cadherin in KM12C with the addition of increasing doses of rhGal-1. (E) IF staining of E-cadherin (green fluorescence) in KM12C with the addition of increasing doses of rhGal-1 for 48 hours. Nuclei were stained with Hoechst 33342 (blue fluorescence); scale bar, 10 μm. (F) Sphere formation capacity of KM12C with the addition of increasing doses of rhGal-1; quantitative results (top panel) and representative images (bottom panel) are shown; scale bar, 30 μm. (G) Drug resistance capacity of KM12C to cisplatin (25 µM) after pretreatment with increasing dosages of rhGal-1 for 24 hours. Cell viability was assessed 48 hours after drug treatment. (H) Left panel: Validation of Gal-1 knockdown using small interfering RNA (siRNA) specific for Gal-1 (siRNA-I & siRNA-II) in WS1 fibroblasts. Non-target siRNA was used as a negative control. After 48 hours, CM from siRNA-I, siRNA-II, and siC were removed and analyzed by ELISA. Right panel: Invasion capacity of KM12C after culturing in siGal-WS1-CM compared to siC-WS1-CM for 48 hours. (I) Sphere formation capacity of KM12C cultured in KM12C-CM, siC-WS1-CM, or siGal-WS1-CM for 72 hours; quantitative results (top panel) and representative images (bottom panel) are shown; scale bar, 30 μm. (J) Drug resistance capacity of KM12C to cisplatin (25 µM) after pretreatment with KM12C-CM, siGal-WS1-CM, or siC-WS1-CM for 24 hours. Cell viability was assessed 48 hours after drug treatment; control, KM12C without cisplatin treatment. All results are shown as the mean ± SEM of three independent experiments. *p < 0.05; **p < 0.01, and ***p < 0.005 compared to the control.