tf1 human erythroleukemic cell line  (ATCC)

Journal: International Journal of Molecular Sciences

Article Title: P2 Receptor Antagonists Rescue Defective Heme Content in an In Vitro SLC25A38-Associated Congenital Sideroblastic Anemia Cell Model

doi: 10.3390/ijms252413314

Figure Lengend Snippet: ( A ) Expression level of SLC25A38 in A38-low and K562 WT cell lines. Total cellular proteins (50 µg) from the indicated cells were probed by a Western blot analysis for SLC25A38 expression. β-ATP synthase and free staining gel served as loading control. ( B ) Heme content in K562 cell lines. Heme content was determined in K562 cell lines from wild type (WT) (white), A38-low (blue) and A38-low cells transfected with pcDNA3.1-SLC25A38 plasmid (pA38-low) (grey) (** p < 0.01, *** p < 0.001, Student’s t test; n = 5). ( C ) Mitochondrial LIP in K562 cell lines. A38-low and WT cells were stained with 5 µM Mito-FerroGreen and analyzed using an Attune Acoustic Focusing Cytometer. ( D ) ROS levels in K562 WT and A38-low cells. Representative histograms illustrating the cellular and mitochondrial ROS levels were determined by cytofluorimetric analysis using 2′,7′-dichlorodihydrofluorescein diacetate (DCF-DA) and dihydrorhodamine 123 (DHR), respectively. The short black line in the figure highlights the populations with elevated oxidative stress. ( E ) PPIX contents in K562 WT (white) and A38-low cells (blue). PPIX was determined in total cellular extracts by a fluorescence method . The fluorescence values were normalized to mg/mL of each sample. PPIX content is expressed as the control-related fold change (*** p < 0.001, Student’s t test; n = 4). ( F ) Oxidative stress sensitivity. Cell viability of indicated cells, assessed through the resazurin assay, was determined following a 48 h treatment with H 2 O 2 at the indicated concentrations. The data are presented as a percentage of the control group (untreated), (** p < 0.01; *** p < 0.001, two-way ANOVA test; n = 3).

Article Snippet: K562 human erythroleukemic cell lines (ATCC, Manassas, VA, USA, Cat# CCL243) were cultured in Roswell Park Memorial Institute medium (RPMI 1640) supplemented with 2 mM glutamine, 16% fetal bovine serum, 100 U/mL penicillin, and 0.1 mg/mL streptomycin [ ].

Techniques: Expressing, Western Blot, Staining, Control, Transfection, Plasmid Preparation, Cytometry, Fluorescence, Resazurin Assay

Journal: International Journal of Molecular Sciences

Article Title: P2 Receptor Antagonists Rescue Defective Heme Content in an In Vitro SLC25A38-Associated Congenital Sideroblastic Anemia Cell Model

doi: 10.3390/ijms252413314

Figure Lengend Snippet: Effect of folic acid with glycine and B6 vitamers on A38-low cells. ( A ) Heme content, assessed in K562 WT, as well as in the A38-low cell line untreated (NT) or treated for 48 h with a mixture of 1 mM folic acid and 100 mM glycine (F/G), or with pyridoxal 5′-phosphate (PLP), pyridoxine (PN), pyridoxal (PL), and pyridoxamine (PM) at the concentrations of 0.1 mM. The values are expressed as the fold change related to the control cells. Number of replicates: 4 or 5. ( B ) Cellular and mitochondrial ROS levels in A38-low cells untreated (−) or treated with 0.1 mM PLP (+), expressed as the fold change in median fluorescence intensity as determined by cytofluorimetric analysis, using 2′,7′-dichlorodihydrofluorescein diacetate (DCF-DA) and dihydrorhodamine 123 (DHR), respectively. Number of replicates: 3. ( C ) State III respiration, in the presence of malate, pyruvate, glutamate, succinate and ADP, assessed in WT and in A38-low cell lines. Additionally, A38-low cells were treated with 0.1 mM PLP for 48 h. Comparative measurements were conducted in a pairwise manner against their respective control counterpart (* p < 0.05; ** p < 0.01; *** p < 0.001; Student’s t -test; n = 4 or 5).

Article Snippet: K562 human erythroleukemic cell lines (ATCC, Manassas, VA, USA, Cat# CCL243) were cultured in Roswell Park Memorial Institute medium (RPMI 1640) supplemented with 2 mM glutamine, 16% fetal bovine serum, 100 U/mL penicillin, and 0.1 mg/mL streptomycin [ ].

Techniques: Control, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: P2 Receptor Antagonists Rescue Defective Heme Content in an In Vitro SLC25A38-Associated Congenital Sideroblastic Anemia Cell Model

doi: 10.3390/ijms252413314

Figure Lengend Snippet: Pyridoxal 5′-phosphate content in WT and A38-low cell lines. ( A ) Representative histograms illustrating the intracellular pyridoxal 5′-phosphate (PLP) content, determined by cytofluorimetric analysis using the AcRAB6 probe, were obtained for the K562 WT and A38-low cell lines. ( B ) Epifluorescence images showing untreated K562 WT and A38-low cells (NT), along with A38-low cells treated with 50 µM pyridoxine (PN), pyridoxamine (PM), and pyridoxal (PL), and stained with AcRAB6 for 12 h as reported .

Article Snippet: K562 human erythroleukemic cell lines (ATCC, Manassas, VA, USA, Cat# CCL243) were cultured in Roswell Park Memorial Institute medium (RPMI 1640) supplemented with 2 mM glutamine, 16% fetal bovine serum, 100 U/mL penicillin, and 0.1 mg/mL streptomycin [ ].

Techniques: Staining

Journal: International Journal of Molecular Sciences

Article Title: P2 Receptor Antagonists Rescue Defective Heme Content in an In Vitro SLC25A38-Associated Congenital Sideroblastic Anemia Cell Model

doi: 10.3390/ijms252413314

Figure Lengend Snippet: Involvement of the P2 receptor in the PLP-mediated effect. Heme content was determined in total cell extracts from K562 WT (white circle) and A38-low cells (blue circle) after treatment with pyridoxal 5′-phosphate (PLP), pyridoxal (PL), pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS), and suramin (SUR) at the indicated concentrations (µM) for 48 h. Data were indicated as the control-related ratio (* p < 0.05; ** p < 0.01; Student’s t -test untreated-related; n = 4 or 5).

Article Snippet: K562 human erythroleukemic cell lines (ATCC, Manassas, VA, USA, Cat# CCL243) were cultured in Roswell Park Memorial Institute medium (RPMI 1640) supplemented with 2 mM glutamine, 16% fetal bovine serum, 100 U/mL penicillin, and 0.1 mg/mL streptomycin [ ].

Techniques: Control