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. 2022 Jun 8;29(11):1662–1675. doi: 10.1038/s41417-022-00485-y

Fig. 3. Natively expressing ST6GAL1 is involved in breast cancer cell growth and invasiveness.

Fig. 3

B Mouse breast cancer bone metastatic 4T1.2 cells were transfected with a validated shST6GAL1 #1 and shControl for 24 h with Lipofectamine P3000 reagents (Invitrogen), according to the company’s instruction. 5000 cells transfected with shControl and shST6GAL1 were cultured in the 24-well tissue culture plate in the medium containing 5% serum for another 48 h and 72 h, and cell proliferation was determined with WST-8 reagent. Normalized A450 nm readings were plotted (n = 5). Dara are means ± s.e., Student’s t-test, ***p < 0.001. A duplicate 48 h culture of 4T1.2 cells was used for Crystal Violet staining (B, lower panels). Representative live-cell images are shown for shControl and shST6GAL1. A Duplicate culture of 4T1.2 cells was used for SYBR-Green-qPCR and protein analyses for the ST6GAL1 gene. ST6GAL1 mRNA levels from the shControl and shST6GAL1 samples were normalized with the house-keeping gene GAPDH, and the normalized ST6GAL1 levels were calculated using the Delta-Delta Ct method, N = 3, data are means ± s.e., Student’s t-test, p < 0.001. A representative blot was shown for ST6GAL1 western blot analysis (N = 3), and GAPDH was used for housekeeping control for equal loading (A, right panels). (C, upper panels) 20,000 4T1.2 cells transfected with shST6GAL1 vs. shControl were plated on 12-well low-adherent tissue culture plates mixed with growth factor reduced Matrigel for 72 h to obtain aggressive tumor-cell Invasion in a 3D cell culture setting. The experiments were repeated three times, and representative phase-contrast images (×10 magnification) of tumor-cell invasions are shown for each condition; the yellow line indicates the invasion area on the spheroid body. (C, lower panel) Histograms represent the invading area’s quantification and the protrusion’s average length (N = 10 per condition, scale bar = 250 µm, data are ±s.e., Student’s t-test, p < 0.001. D–F Human TNBC BT-549 cells were transfected with shControl and shST6GAL1 #1 (Sigma Cat# TRCN0000035432) or shST6GAL1 #2 (Sigma Cat# TRCN0000035429), as mentioned before. 3D cell invasion assays (F, upper panels), quantification of cell invasion (F, lower panel), and cell proliferation (E) assays were performed. Duplicate cultures were used for qPCR and Western blot analysis of ST6GAL1 (D). For qPCR analysis, gene levels were normalized by GAPDH, N = 3, data are ±s.e., Student’s t-test, p < 0.05. Experiments were repeated at least three times, and representative blots and images were shown. For Western blotting, tubulin as a loading control for equal transfer. Histograms data for invasion assays are means ± s.e., scale bar 250 µm, one-way ANOVA, and post-hoc test for pair-wise comparison, p < 0.001.