Journal: American Journal of Physiology - Cell Physiology
Article Title: A fluorescent reporter assay of differential gene expression response to insulin in hepatocytes
doi: 10.1152/ajpcell.00504.2018
Figure Lengend Snippet: Cells harboring nuclear forkhead box O (Foxo) 1 are more sensitive to stimulation by insulin. A: representative immunoblot (top) with quantification (bottom) of cultured FAStimerS Hepa1c1c7 cell populations sorted from FACS [timer early (TE), timer middle (TM), timer late (TL), and nonfluorescent (N)] after insulin treatment. Graph represents average of 5 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 versus respective controls. Within insulin treatment: ###P < 0.001 versus N, $$$$P < 0.0001 versus TL, and ^^P < 0.01 versus TM. B: fluorescent images of Hepa1c1c7 cells cotransfected with Foxo1Timer (red) and pClover (green) 24 h before start of overnight (O/N) insulin treatment. DAPI counterstains nuclei. Arrows, cell coexpressing Foxo1 and pClover. C: quantification of Foxo1Timer subcellular localization. ****P < 0.0001 versus control. D: immunofluorescence for Akt phosphorylation (pAkt, green) after insulin treatment in Hepa1c1c7 cells transfected with Fox red fluorescent protein (RFP, red). DAPI counterstains nuclei. Arrow, cells coexpressing FoxRFP and pAkt. E: quantification of FoxRFP nuclear localization. All images taken with ×20 objective, and the scale bar is 100 μM. ****P < 0.0001 versus control. F: quantification of pAkt+ cells. ****P < 0.0001 versus respective controls. Within insulin treatment: ####P < 0.0001 versus FoxRFP− and $$$$P < 0.0001 versus Foxo1 cyto+.
Article Snippet: TimerS (pSlow-FT-N1; Addgene plasmid no. 31912) was a gift from Vladislav Verkhusha ( 36 ), pClover (pLenti-Foxo1-Clover; Addgene plasmid no. 67759) was a gift from Peter Rotwein ( 16 ), Foxo1 fused to a red fluorescent protein (FoxRFP) plasmid was previously generated in laboratory (Addgene plasmid no. 34678), and superfolder green fluorescent protein (GFP) was a gift from Erik Snapp ( 3 ).
Techniques: Western Blot, Cell Culture, Control, Immunofluorescence, Phospho-proteomics, Transfection
Addgene inc is a verified supplier 
![Cells harboring nuclear forkhead box O (Foxo) 1 are more sensitive to stimulation by insulin. A: representative immunoblot (top) with quantification (bottom) of cultured FAStimerS Hepa1c1c7 cell populations sorted from FACS [timer early (TE), timer middle (TM), timer late (TL), and nonfluorescent (N)] after insulin treatment. Graph represents average of 5 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 versus respective controls. Within insulin treatment: ###P < 0.001 versus N, $$$$P < 0.0001 versus TL, and ^^P < 0.01 versus TM. B: fluorescent images of Hepa1c1c7 cells cotransfected with Foxo1Timer (red) and <t>pClover</t> (green) 24 h before start of overnight (O/N) insulin treatment. DAPI counterstains nuclei. Arrows, cell coexpressing <t>Foxo1</t> and pClover. C: quantification of Foxo1Timer subcellular localization. ****P < 0.0001 versus control. D: immunofluorescence for Akt phosphorylation (pAkt, green) after insulin treatment in Hepa1c1c7 cells transfected with Fox red fluorescent protein (RFP, red). DAPI counterstains nuclei. Arrow, cells coexpressing FoxRFP and pAkt. E: quantification of FoxRFP nuclear localization. All images taken with ×20 objective, and the scale bar is 100 μM. ****P < 0.0001 versus control. F: quantification of pAkt+ cells. ****P < 0.0001 versus respective controls. Within insulin treatment: ####P < 0.0001 versus FoxRFP− and $$$$P < 0.0001 versus Foxo1 cyto+.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_9749/pmc06689749/pmc06689749__zh00071984700007.jpg)