Journal: Biochimica et biophysica acta

Article Title: The soluble form of HFE protein regulates hephaestin mRNA expression in the duodenum through an endocytosis-dependent mechanism.

doi: 10.1016/j.bbadis.2014.07.017

Figure Lengend Snippet: Fig. 3. Effect of exogenous sHFE stimulus on the expression of iron metabolism-related genes in HuTu-80 cells. HEK293 cells were transfected with pcDNA empty vector (mock), pcDNA + HFE- Flag and pcDNA + sHFE-Flag constructs. Forty-eight hours post-transfection cell culture supernatants were collected and then used to stimulate previously cultured HuTu-80 cells. Endocytosis was inhibited with dynasore 40 μM (dyn) or chlorpromazine 10 μg/mL (CPZ). Four hours later, cells were harvested for RNA and the expression of (A, D) CYBRD1 and (B, E) HEPH was evaluated by RT-qPCR. (C, F) HuTu-80 cells were stimulated with medium containing both holo-Tf 20 μM and dynasore 40 μM or chlorpromazine 10 μg/mL, to control the inhibition of endocytosis mea- sured as the inhibition of TFR1 decrease by holo-Tf. Values were obtained by normalizing first the target gene expression to HPRT1 and then to the mock control. Bars represent the standard deviations from at least three independent experiments; *p b 0.05; **p b 0.001.

Article Snippet: HuTu-80 cells (human duodenal enterocyte cell line) and HEK293 cells (used for protein production) (DSMZ, Germany) were maintained in DMEMGlutaMAXTMmedium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% (v/v) fetal bovine serum (FBS), while THP-1 cells (human monocyte-derived cell line), Caco-2 and HT-29 cells (human epithelial colorectal adenocarcinoma cells) (DSMZ, Germany) were maintained in RPMI 1640 medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% (v/v) FBS, in a 37 °C/5% CO2 incubator.

Techniques: Expressing, Transfection, Plasmid Preparation, Construct, Cell Culture, Quantitative RT-PCR, Control, Inhibition, Targeted Gene Expression