Figure 2. KO of Ugcg, a key enzyme in the biosynthesis of GSLs, promotes TGF‐β signaling and TGF‐β‐induced EMT in NMuMG cells.

1. Combined extracted ion chromatography of GSL glycans in Ugcg KO and control NMuMG cells. GT1b: internal normalization control.
2. Mouse TGF‐β gene signatures were enriched in Cas9 control versus Ugcg KO NMuMG cells, as shown by GSEA. Normalized enrichment score (NES) = −1.27, P = 0.036.
3. GSEA of mouse EMT gene signatures in Cas9 control versus Ugcg KO NMuMG cells. NES = −2.17, P = 0.000.
4. Immunoblot analysis of p‐SMAD2 and total SMAD2 (t‐SMAD2) in NMuMG cells with or without Ugcg deficiency and treated with vehicle control or TGF‐β for 1 h. Tubulin: loading control.
5. qRT–PCR analysis of TGF‐β target genes, including Serpine1 and Smad7, in Ugcg KO NMuMG or control NMuMG cells treated with vehicle control or TGF‐β for 6 h.
6. Immunoblot analysis of E‐cadherin and N‐cadherin in Ugcg KO NMuMG or control NMuMG cells treated with vehicle control or TGF‐β for 48 h. Tubulin: loading control.
7. Cdh1 and Cdh2 mRNA levels in Ugcg KO or control NMuMG cells after vehicle control or TGF‐β treatment for 48 h.
8. Immunofluorescence analysis of the expression and localization of E‐cadherin (red) and the formation of F‐actin (green) after treatment with vehicle control or TGF‐β for 2 days. Nuclei were counterstained with DAPI (blue). Images were acquired with confocal microscopy. Scale bar = 50 μm.

Data information: TGF‐β was applied at a final concentration of 2.5 ng/ml. In (E, G), the data are expressed as the mean ± SD values from three biological replicates (n = 3). **P < 0.01; ****P < 0.0001 based on unpaired Student's t‐test.