Abstract
This report provides data that are specifically related to the differential sialylation of nutrient deprived breast cancer cells to sialic acid supplementation in support of the research article entitled, “Nutrient-deprived cancer cells preferentially use sialic acid to maintain cell surface glycosylation" [1]. Particularly, breast cancer cells, when supplemented with sialic acid under nutrient deprivation, display sialylated glycans at the cell surface, but non-malignant mammary cells show sialylated glycans intracellularly. The impact of sialic acid supplementation under nutrient deprivation was demonstrated by measuring levels of expression and sialylation of two markers, EGFR1 and MUC1. This Data in Brief article complements the main manuscript by providing detailed instructions and representative results for cell-level imaging and Western blot analyses of changes in sialylation during nutrient deprivation and sialic acid supplementation. These methods can be readily generalized for the study of many types of glycosylation and various glycoprotein markers through the appropriate selection of fluorescently-labeled lectins.
Highlights
To further corroborate the increased sialylation of these cancer markers, equal amounts of immunoprecipitated MUC1 and EGFR were precipitated with MAL-I and subjected to Western blot analysis followed by immunodetection that again showed that the malignant cells migrated slower during SDS-PAGE consistent with increased sialylation (DIB Fig. 2B)
To confirm MAL-I specificity towards Neu5Acα2-3Gal, equal amounts of desialylated immunoprecipitated glycoproteins were precipitated with MAL-I and subjected to Western blot followed by immunodetection (DIB Fig. 2B)
RPMI1640 medium (ATCC modification), hydroxyethylpiperazine-N-2-ethane sulfonic acid (HEPES) buffer, fetal bovine serum (FBS), and TO-PRO-3 were purchased from Life Technologies Corporation (USA)
Summary
Western blot Imaging cells on DV elite imaging system from Applied Precision (Applied Precision, USA). Consistent with the flow cytometry data, FITC-conjugated WGA, SNA, and MAL-I lectins showed enhanced fluorescence signal for both normal and cancer cells after sialic acid treatment [1]. The higher levels of MUC1 and EGFR immunopurified by antibodies that recognize protein epitopes of these glycoproteins was consistent with qRT-PCR results where mRNA levels of these two proteins in nutrientdeprived cells supplemented with sialic acid were compared to the untreated cells To further corroborate the increased sialylation of these cancer markers, equal amounts of immunoprecipitated MUC1 and EGFR were precipitated with MAL-I and subjected to Western blot analysis followed by immunodetection that again showed that the malignant cells migrated slower during SDS-PAGE consistent with increased sialylation (DIB Fig. 2B). To confirm MAL-I specificity towards Neu5Acα2-3Gal, equal amounts of desialylated immunoprecipitated glycoproteins (asialoMUC1 and asialoEGFR) were precipitated with MAL-I and subjected to Western blot followed by immunodetection (DIB Fig. 2B)
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