ST3GAL5-catalysed gangliosides inhibit TGF-β-induced epithelial-mesenchymal transition via TβRI degradation (2022)
Figure 5

Figure 5. ST3GAL5 inhibits TGF-β signaling and TGF-β-induced EMT.

EIC of GSL-glycans released from A549-VIM-RFP cells transfected with nontargeting, ST3GAL5 or B4GALNT1 siRNA. GT1b: internal normalization control.

Immunoblot analysis of p-SMAD2, t-SMAD2, and ST3GAL5 in A549-VIM-RFP cells with siRNA-mediated ST3GAL5 knockdown or transfection of nontargeting siRNA and treated with vehicle control or TGF-β for 1 h. Vinculin: loading control.

Quantification of the p-SMAD2 level in A549-VIM-RFP cells transfected with nontargeting or ST3GAL5 siRNA and treated with TGF-β as shown in (B).

Expression levels of ST3GAL5, the epithelial marker E-cadherin, and mesenchymal markers, including N-cadherin, vimentin, and SNAIL, in siRNA ST3GAL5-depleted and nontargeting siRNA-transfected A549-VIM-RFP cells treated with vehicle control or TGF-β for 48 h. Vinculin: loading control.

The time course of RFP-tagged vimentin expression was monitored with an IncuCyte system in siRNA ST3GAL5 knockdown and nontargeting siRNA-transfected A549-VIM-RFP cells treated with vehicle control or TGF-β for the indicated times. The red object intensity was normalized to the red intensity at 0 h.

Immunoblot analysis of p-SMAD2 and t-SMAD2 in A549-VIM-RFP cells transduced with empty vector (pLV-EV) or the ST3GAL5 expression construct and stimulated with vehicle control or TGF-β for 1 h. GAPDH: loading control.

Quantification of the p-SMAD2 level in A549-VIM-RFP cells transduced with pLV-EV or the ST3GAL5 overexpression construct and treated with TGF-β, as shown in (F).

A549-VIM-RFP cells transduced with the CAGA-GFP lentiviral vector and with the pLV-EV control or ST3GAL5 expression construct were treated with vehicle control or TGF-β for the indicated times. SMAD3/SMAD4-dependent (CAGA)12-mediated transcriptional GFP reporter expression levels were monitored with an IncuCyte system. The GFP object intensity was normalized to the green intensity at 0 h.

Immunoblot analysis of the epithelial marker E-cadherin and mesenchymal markers, including N-cadherin, vimentin, and SNAIL, in A549-VIM-RFP cells transduced with the pLV-EV control or ST3GAL5 expression construct and treated with vehicle control or TGF-β for 48 h. GAPDH: loading control.

Real-time expression of RFP-tagged vimentin was monitored with an IncuCyte system in A549-VIM-RFP cells transduced with pLV-EV or the ST3GAL5 expression construct and treated with vehicle control or TGF-β for the indicated times. The red object intensity was normalized to the red intensity at 0 h.

Alexa Fluor 488 phalloidin staining of F-actin (green) in pLV-EV control- or ST3GAL5-expressing A549-VIM-RFP cells after stimulation with vehicle control or TGF-β for 48 h. 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 (C, E, G, H, J), the data are expressed as the means ± SD values from three biological replicates (n=3). *, P ≤ 0.05; **, P < 0.01; ***, P < 0.001. In (C), statistical analysis was based on unpaired Student's t test. In (E, G, H, J), statistical analysis was based on two-way ANOVA.