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Fig. 7 | Cell Communication and Signaling

Fig. 7

From: NF-κB upregulates glutamine-fructose-6-phosphate transaminase 2 to promote migration in non-small cell lung cancer

Fig. 7

GFPT2 regulates mesenchymal cell migration in NSCLC. a-d Stable A549 cells expressing doxycycline-inducible shRNA GFPT2 (A549:shGFPT2) or scrambled control shRNA (A549:shControl) were stimulated with TNF and TGFβ with or without Doxycycline (Dox). a Doxycycline treated A549:shGFPT2 cells express significantly lower levels of basal and TNF/TGF-stimulated GFPT2 transcripts, relative to untreated cells as measured by RT-qPCR. Changes in mRNA expression were calculated relative to HPRT with mean and SD + shown; * p = < 0.05, ns = not significant, n = 3. b Knockdown of GFPT2 dampened TNF/TGF-induced increases in O-GlcNAc modified proteins detected by immunoblotting. Densitometric analysis of the bands indicated (*) relative to GAPDH were used to determine fold changes in O-GlcNAcylated proteins present in extracts after GFPT2 knockdown. c No significant fold change differences in mesenchymal protein marker were detected following GFPT2 knockdown, as demonstrated by densitometric analysis of the relative N-Cadherin expression compared to GAPDH loading control. d GFPT2 expression is required for cell migration and invasion through extracellular matrix as determined in transwell assays. Numbers of migrated and invaded cells without doxycycline treatment for each cell line were considered 100%. Data represents one of three independent experiments, *p = < 0.05, and **p = < 0.01. e-f Stable H1299 cell line expressing doxycycline-inducible Flag-tagged GFPT2 (H1299:iGFPT2) or luciferase control (H1299:iControl) were cultured in spheroids with or without TNF and TGFβ stimulation, and with or without the addition of doxycycline. e Immunoblot analysis confirms that the inducible expression of GFPT2 increases the abundance of O-GlcNAcylated proteins (*), relative to GAPDH. f Light microscopy photographs demonstrate that doxycycline-inducible expression of GFPT2 in H1299 cells increases cell migration as determine in scratch assays. The surface of the gap was measured using TScratch software (45) and data was plotted as percent closure. Data represents one of three independent experiments

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