Journal: PLoS ONE

Article Title: Functional Study of NIPA2 Mutations Identified from the Patients with Childhood Absence Epilepsy

doi: 10.1371/journal.pone.0109749

Figure Lengend Snippet: CM: cytomembrane marked by DIO (a cytomembrane marker, green); NIPA2 was labeled by red fluorescence. MERGE: overlapped image. WT: wild-type. NIPA2-WT proteins (red) were distributed at cytomembrane, overlapped with DIO (green), but the three mutant proteins (I178F, N244S, N334_E335insD; red) were trapped in cytoplasm and showed no overlap with the plasma membrane. n = 4 experiments, scale bar: 5 µm.

Article Snippet: Herpes simplex virus (HSV) vectors with NIPA2 (wild-type, mutations or siRNA) were conducted by OrienGene Biotechnology Ltd. (Beijing, China).

Techniques: Marker, Labeling, Fluorescence, Mutagenesis, Clinical Proteomics, Membrane

Journal: PLoS ONE

Article Title: Functional Study of NIPA2 Mutations Identified from the Patients with Childhood Absence Epilepsy

doi: 10.1371/journal.pone.0109749

Figure Lengend Snippet: Cell viability was measured by MTT detection. The absorbance of formazan was proportional to metabolic activity of the cells. Relative cell viability was calculated as: (mean absorbance of transfected group - mean absorbance of basal temperature control)/(mean absorbance of naïve group - mean absorbance of basal temperature control). Naïve: the cultured neurons without transfection; Overexpression: neurons transfected with NIPA2 WT ; Ins: the small insertion (N334_E335insD); siRNA: neurons transfected with NIPA2 -siRNA. Neurons were co-transfected with the mutant and NIPA2 -siRNA in the groups (I178F+siRNA, N244S+siRNA and ins+siRNA). The group (WT+siRNA) represented the neurons transfected with NIPA2 WT and NIPA2 -siRNA. The mutant groups (I178F, N244S and Ins) represented the neurons transfected only with the mutant. n = 9 experiments. No significant differences between naïve group and any other condition were found.

Article Snippet: Herpes simplex virus (HSV) vectors with NIPA2 (wild-type, mutations or siRNA) were conducted by OrienGene Biotechnology Ltd. (Beijing, China).

Techniques: Activity Assay, Transfection, Control, Cell Culture, Over Expression, Mutagenesis

Journal: PLoS ONE

Article Title: Functional Study of NIPA2 Mutations Identified from the Patients with Childhood Absence Epilepsy

doi: 10.1371/journal.pone.0109749

Figure Lengend Snippet: There was no significant change of extracellular Mg 2+ concentration among the groups compared to naïve cells; the mutant N244S and Ins dramatically decreased the concentration of intracellular Mg 2+ , though the mutant I178F and the overexpression group were not different from the naïve group. Naïve: the cultured neurons without transfection; Overexpression: neurons transfected with NIPA2 (WT); Ins: N334_E335insD. siRNA: neurons transfected with NIPA2 -siRNA; the group (WT+siRNA): neurons transfected with NIPA2 WT and NIPA2 -siRNA; the mutant+siRNA group (I178F+siRANA, N244S+siRNA, Ins+siRNA): neurons transfected with the mutant and NIPA2 -siRNA. Relative concentration was calculated as: Mg 2+ concentration of the transfected group/Mg 2+ concentration of naïve group. n = 4 experiments.

Article Snippet: Herpes simplex virus (HSV) vectors with NIPA2 (wild-type, mutations or siRNA) were conducted by OrienGene Biotechnology Ltd. (Beijing, China).

Techniques: Concentration Assay, Mutagenesis, Over Expression, Cell Culture, Transfection

Journal: Cell Reports

Article Title: De novo steroidogenesis in tumor cells drives bone metastasis and osteoclastogenesis

doi: 10.1016/j.celrep.2024.113936

Figure Lengend Snippet: Pregnenolone promotes human osteoclastogenesis and is secreted by human tumor cells (A–C) Representative images (A) and quantification (B and C) of TRAP-stained cell cultures of human blood mononuclear cell-derived OCs cultured for 2 days in the presence of 20 ng/mL recombinant human M-CSF and 20 ng/mL soluble human RANKL and then in the presence of the indicated concentration of pregnenolone or vehicle for 12 days. Scale bars represent 100 μm. (D) Levels of pregnenolone in the supernatants of A375, MDA-MB-435S, A549, NCI-H1299, NCI-H460, MCF-7, MDA-MB-231, DU145, LNCaP, PC-3, Hs895.T, SK-MEL-28, BEAS-2B, NCI-H128, NCI-H2126, HCC70, MCF-10A, MDA-PCa-2b, RWPE-1, and LASCPC-01 cells measured by ELISA. (E) Kaplan-Meier curve of breast-cancer-specific survival for 2,976 patients with high or low expression of Cyp11a1 in the primary tumor. Bar graphs show mean and SD of data from 3 independent experiments. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.002; n.s., not significant.

Article Snippet: Mouse Competitive ELISA Kit dihydrotestosterone , Elabscience Biotech. , Cat# E-EL-0031.

Techniques: Staining, Derivative Assay, Cell Culture, Recombinant, Concentration Assay, Enzyme-linked Immunosorbent Assay, Expressing

Journal: Cell Reports

Article Title: De novo steroidogenesis in tumor cells drives bone metastasis and osteoclastogenesis

doi: 10.1016/j.celrep.2024.113936

Figure Lengend Snippet:

Article Snippet: Mouse Competitive ELISA Kit dihydrotestosterone , Elabscience Biotech. , Cat# E-EL-0031.

Techniques: Virus, shRNA, Recombinant, Modification, Staining, RNA Library Preparation, TaqMan Assay, Competitive ELISA, RNA Sequencing, Plasmid Preparation, Software, Real-time Polymerase Chain Reaction

Journal: eLife

Article Title: NHE6 depletion corrects ApoE4-mediated synaptic impairments and reduces amyloid plaque load

doi: 10.7554/eLife.72034

Figure Lengend Snippet:

Article Snippet: Chemical compound, drug , γ-Secretase inhibitor L-685458 , Tocris Bioscience , 2627 , .

Techniques: Staining, Plasmid Preparation, Transfection, Construct, Expressing, shRNA, Recombinant, Sequencing, Software, Imaging

Journal: Journal of Bone and Mineral Research

Article Title: Effects of 25-Hydroxyvitamin D 3 on Proliferation and Osteoblast Differentiation of Human Marrow Stromal Cells Require CYP27B1/1α-Hydroxylase

doi: 10.1002/jbmr.298

Figure Lengend Snippet: Primer Sets Used for RT-PCR

Article Snippet: Transient transfection of siRNA into hMSCs hi-1α was performed by electroporation with the Human MSC Nucleofector Kit (Lonza/Amaxa Biosystems, Walkersville, MD, USA) with either CYP27B1 siRNA, nonsilencing control siRNA (a nonhomologous, scrambled sequence equivalent; Santa Cruz Biotechnology, Inc.), or PBS according to the manufacturer's instructions and as described previously. ( ) In brief, hMSCs hi-1α were harvested by trypsinization and resuspended at 10 6 cells in 100 μL of Nucleofector Solution (Lonza/Amaxa Biosystems) with 10 or 100 pmol of CYP27B1 siRNA.

Techniques: Sequencing

Journal: Neuron

Article Title: Genome-wide in vivo CNS Screening Identifies Genes that Modify CNS Neuronal Survival and mHTT Toxicity

doi: 10.1016/j.neuron.2020.01.004

Figure Lengend Snippet: Key Resources Table:

Article Snippet: Taqman gene expression Mouse Actb Mm02619580_g1 (VIC-MGB_PL) , ThermoFisher , CAT#4448892.

Techniques: Virus, Genome Wide, shRNA, Control, Sequencing, Recombinant, Expressing, Multiplex Assay, Software

Journal: PLoS Genetics

Article Title: PPP2R5C Couples Hepatic Glucose and Lipid Homeostasis

doi: 10.1371/journal.pgen.1005561

Figure Lengend Snippet: (A-C) mRNA levels of PPP2R5C are nutritionally regulated in various mouse tissues. Quantitative RT-PCR of PPP2R5C (NCBI splice variant 4) from liver (A), abdominal white adipose tissue (B) and gastrocnemius muscle (C) of control C57BL/6 (“WT”) or diabetic leptin-receptor deficient (“db/db”) 8–10 week old male mice. Mice were first starved for 16 hours (“Fasting”) and then given normal chow diet for 6 hours (“Refed”). Error bars: std. dev. *p-value<0.05, **p-value<0.01, ***p-value<0.001 by student t-test (n ≥ 3).

Article Snippet: Human PPP2R5C mRNA expression was measured by quantitative real-time RT-PCR in a fluorescent temperature cycler using TaqMan assay-on-demand kits (Hs00604899_g1, Applied Biosystems, Darmstadt, Germany), and fluorescence was detected on an ABI PRISM 7000 sequence detector (Applied Biosystems, Darmstadt, Germany).

Techniques: Quantitative RT-PCR, Variant Assay, Control

Journal: PLoS Genetics

Article Title: PPP2R5C Couples Hepatic Glucose and Lipid Homeostasis

doi: 10.1371/journal.pgen.1005561

Figure Lengend Snippet: (A-C) Increased glucose uptake/tolerance in mice upon liver-specific knockdown of PPP2R5C. 8–10 week CL57BL/6 male mice tail-injected with adeno-associated virus containing miRNA targeting PPP2R5C (“PPP2R5C KD”) or a scrambled control miRNA (“Control KD”). 7 weeks after knockdown mice were starved for 16 hours (“Fasting”) or starved and then given normal chow diet for 6 hours (“Refed”) prior to sacrificing. Although blood glucose levels are not altered (A), serum insulin levels are significantly reduced (B) compared to controls. (C) Glucose tolerance test performed 4 weeks after virus injection shows significantly improved tolerance in knockdown mice (2g glucose/kg body weight injected intraperitoneally, n = 12). (D) Insulin signaling, detected via AKT and GSK3 beta phosphorylation, does not drop in all feeding regimes in PPP2R5C HepKD livers compared to controls, despite PPP2R5C HepKD serum insulin levels being lower (see panel B). (E) Insulin sensitivity is increased after PPP2R5C knockdown in liver. Control C57BL/6 mice and PPP2R5C HepKD mice were virus-injected as in Fig 2. Four weeks later, mice were fasted for 6 hours, then insulin was injected 1IU/kg and 10 minutes later mice were sacrified and liver samples were taken. (F-G) Glucose consumption and lactate production are increased in Hepa 1–6 cells upon PPP2R5C knockdown. Hepa 1–6 cells infected by adenovirus carrying shRNA targeting all mouse PPP2R5C isoforms (PPP2R5C KD) or a negative-control scramble shRNA (Control KD). After 48h, glucose consumption (F) and lactate production (G) were measured in the medium for 24 hours, and normalized to total cell protein. (n = 3) (H) Glycolytic flux measured as ECAR (extracellular acidification rate) using the glycolysis stress kit from Seahorse Bioscience on the extracellular flux analyser XF96. After addition of glucose to control or PPP2R5C knockdown Hepa 1–6 cells, oligomycin is added to inhibit respiration, thereby boosting glycolytic flux. 2-deoxy-glucose is added to compete with glucose and shut down glycolysis (n = 9). (I) Acute glucose uptake is increased in Hepa 1–6 cells upon PPP2R5C knockdown. Stably transfected Hepa1-6 cell-lines carrying two independent, inducible shRNAs (PPP2R5C KD1 and KD2) were induced with 30 μg/ml cumate for 3 days, starved overnight in serum-free DMEM, and uptake of fluorescent 2-deoxy-glucose analog 2-NBDG was quantified by FACS. (n = 3) Error bars: std. dev. *p-value<0.05, **p-value<0.01, ***p-value<0.001, †p-value<10 −4 by Wilcoxon signed-rank test (C) or student t-test (B, F-I).

Article Snippet: Human PPP2R5C mRNA expression was measured by quantitative real-time RT-PCR in a fluorescent temperature cycler using TaqMan assay-on-demand kits (Hs00604899_g1, Applied Biosystems, Darmstadt, Germany), and fluorescence was detected on an ABI PRISM 7000 sequence detector (Applied Biosystems, Darmstadt, Germany).

Techniques: Knockdown, Injection, Virus, Control, Phospho-proteomics, Infection, shRNA, Negative Control, Stable Transfection, Transfection, Significance Assay

Journal: PLoS Genetics

Article Title: PPP2R5C Couples Hepatic Glucose and Lipid Homeostasis

doi: 10.1371/journal.pgen.1005561

Figure Lengend Snippet: (A-C) Liver-specific knockdown of PPP2R5C leads to pro-anabolic changes including increased liver lipid synthesis and secretion and reduced glycogen breakdown. As in , 7 weeks post hepatocyte-specific knockdown, liver weight (A), glycogen (B) and triglycerides (C) were quantified. (n = 5 or 6) (D-E) Cellular triglyceride levels are increased in Hepa 1–6 (D) or mouse primary hepatocytes (E) upon PPP2R5C knockdown. Cells infected as in . (n = 3) ( F-G ) Liver-specific knockdown of PPP2R5C leads to elevated serum VLDL levels. 7 weeks post hepatocyte-specific knockdown, serum triglycerides were quantified either in aggregate (F), or when fractionated by FPLC to resolve lipoprotein particles of various densities (G). (n = 5 or 6) Error bars: std. dev. *p-value<0.05, **p-value<0.01 by one-way ANCOVA with liver NEFA as a covariate (C, “Random”) or student t-test (A-F).

Article Snippet: Human PPP2R5C mRNA expression was measured by quantitative real-time RT-PCR in a fluorescent temperature cycler using TaqMan assay-on-demand kits (Hs00604899_g1, Applied Biosystems, Darmstadt, Germany), and fluorescence was detected on an ABI PRISM 7000 sequence detector (Applied Biosystems, Darmstadt, Germany).

Techniques: Knockdown, Infection

Journal: PLoS Genetics

Article Title: PPP2R5C Couples Hepatic Glucose and Lipid Homeostasis

doi: 10.1371/journal.pgen.1005561

Figure Lengend Snippet: (A) Schematic diagram of “BioID”—proximity-dependent biotinylation of proteins—to identify PPP2R5C substrates interacting transiently with the phosphatase complex. Biotin ligase (BirA) was fused to either the N-terminus (Myc-BirA-PPP2R5C) or C-terminus (PPP2R5C-BirA-HA) of PPP2R5C (NCBI variant 1). As negative controls, only BirA-HA or Myc-BirA were used. A mutation in the active center of the catalytic domain was introduced (PP2A-C D85N) as a substrate-trapping mutation. Biotinylated proteins from Hepa 1–6 cells were purified by cell lysis and streptavidin pulldown, and subsequently detected by immunoblotting. (B-B’) BioID identifies AMPK beta 1, HIF1 α , STAT3 and S6K as PPP2R5C interacting proteins. Proteins interacting with PPP2R5C in vivo in Hepa 1–6 cells were biotinylated and purified as in (A), and probed by immunoblotting. LXR, PPAR alpha, SREBP-1, Actin, and Tubulin were included as negative control proteins which were not detected in the pulldowns. (Lysates shown at same exposure as biotin pulldowns.) (C) AMPK phosphorylation and activity increase upon PPP2R5C knockdown in Hepa 1–6 cells. PPP2R5C was knocked-down using two independent, inducible shRNAs as in . AMPK phosphorylation increases on T172 upon PPP2R5C knockdown. AMPK activity determined via phosphorylation of two substrates, ACC1 and TBC1D1. (D) HIF1α phosphorylation increases upon PPP2R5C knockdown in Hepa 1–6 cells. HIF1α phosphorylation is detected as reduced mobility (up) on a 25 μM PhosTag gel (8% gel), which is abolished upon treatment of cell lysates with calf intestinal phosphatase (CIP) prior to gel electrophoresis (right panel). PPP2R5C knockdown was performed by generating stable cell lines carrying two independent inducible shRNA constructs (KD1 and KD2) to exclude off-target effects. (E) Expression of HIF1 α target genes increases in primary hepatocytes upon PPP2R5C knockdown. (n = 4) Error bars: std. dev. *p-value<0.05, **p-value<0.01, ***p-value<0.001 by student t-test (E).

Article Snippet: Human PPP2R5C mRNA expression was measured by quantitative real-time RT-PCR in a fluorescent temperature cycler using TaqMan assay-on-demand kits (Hs00604899_g1, Applied Biosystems, Darmstadt, Germany), and fluorescence was detected on an ABI PRISM 7000 sequence detector (Applied Biosystems, Darmstadt, Germany).

Techniques: Variant Assay, Mutagenesis, Purification, Lysis, Western Blot, In Vivo, Negative Control, Phospho-proteomics, Activity Assay, Knockdown, Nucleic Acid Electrophoresis, Stable Transfection, shRNA, Construct, Expressing

Journal: PLoS Genetics

Article Title: PPP2R5C Couples Hepatic Glucose and Lipid Homeostasis

doi: 10.1371/journal.pgen.1005561

Figure Lengend Snippet: (A) PPP2R5C knockdown leads to upregulation of genes enriched for PPARA and SREBP-1 targets. Genes either up- or down-regulated upon PPP2R5C knockdown in mouse primary hepatocytes were analyzed using TFactS software to identify transcription factors putatively misregulated upon PPP2R5C knockdown. FDR (False Discovery Rate) rate was controlled using the Benjamini-Hochberg procedure. (B-C) Expression of bona-fide SREBP-1 target genes is increased upon PPP2R5C knockdown in primary hepatocytes in culture (B) or in mouse liver in vivo (C). PPP2R5C was knocked-down in mouse primary hepatocytes using adenovirus and in vivo using adeno-associated virus as in . SREBP-1 target genes quantified by Q-RT-PCR, normalized to TBP. (D) Upon PPP2R5C knockdown in liver, SREBP-1 protein levels are elevated. (E) Expression of ChREBP target genes is increased upon PPP2R5C knockdown in mouse liver in vivo. PPP2R5C was knocked-down in vivo using adeno-associated virus as in . ChREBP target genes quantified by Q-RT-PCR, normalized to TBP. (F) Graphical representation of the metabolic changes induced upon PPP2R5C knockdown in mouse liver. Livers with reduced PPP2R5C have increased glucose uptake, increased TAG synthesis, and increased VLDL secretion. Error bars: std. dev. *p-value<0.05, **p-value<0.01, ***p-value<0.001 by student t-test (B-C,E) (n = 4 for mouse primary hepatocytes, and 5 or 6 for mouse liver).

Article Snippet: Human PPP2R5C mRNA expression was measured by quantitative real-time RT-PCR in a fluorescent temperature cycler using TaqMan assay-on-demand kits (Hs00604899_g1, Applied Biosystems, Darmstadt, Germany), and fluorescence was detected on an ABI PRISM 7000 sequence detector (Applied Biosystems, Darmstadt, Germany).

Techniques: Knockdown, Software, Expressing, In Vivo, Virus, Reverse Transcription Polymerase Chain Reaction

Journal: PLoS Genetics

Article Title: PPP2R5C Couples Hepatic Glucose and Lipid Homeostasis

doi: 10.1371/journal.pgen.1005561

Figure Lengend Snippet: (A) Expression of PPP2R5C is elevated in livers of diabetic patients. Quantitative RT-PCR of Human PPP2R5C in liver of healthy controls (n = 40) or type 2 diabetic patients (n = 26), normalized to 18S rRNA. (B) Expression of PPP2R5C is elevated in non-diabetics with visceral obesity. People enrolled in the analysis were divided into 3 subgroups according to their adiposity: Lean (n = 12), Subcutaneous (“SC”) Obesity (n = 21) and Visceral (“VIS”) Obesity (n = 7). (C) Expression of PPP2R5C inversely correlates with insulin sensitivity. Pearson correlation analysis was done for PPP2R5C expression levels and glucose infusion rate (GIR) during hyperinsulemic-euglycemic clamp. **p-value<0.01, ***p-value<0.001 by student t-test (A-B).

Article Snippet: Human PPP2R5C mRNA expression was measured by quantitative real-time RT-PCR in a fluorescent temperature cycler using TaqMan assay-on-demand kits (Hs00604899_g1, Applied Biosystems, Darmstadt, Germany), and fluorescence was detected on an ABI PRISM 7000 sequence detector (Applied Biosystems, Darmstadt, Germany).

Techniques: Expressing, Quantitative RT-PCR

Journal: PLoS Genetics

Article Title: PPP2R5C Couples Hepatic Glucose and Lipid Homeostasis

doi: 10.1371/journal.pgen.1005561

Figure Lengend Snippet: (A) Hyperglycemia in db/db mice is decreased upon hypatocyte-specific PPP2R5C knockdown with adeno-associated virus. After 5 weeks knockdown, db/db mice were sacrificed under ad libitum feeding with normal chow diet. Blood glucose was monitored at each week, week 0 was one day before virus injection (n = 6). (B) Insulin tolerance test shows improved insulin sensitivity in PPP2R5C knockdown db/db mice at week 4 after virus injection (1.5IU/kg insulin was tail-injected after 6 hour fasting) (n = 6). (C-F) PPP2R5C HepKD in db/db mice increases body weight (C), whole body fat content (D), and liver weight (E), without changing abdominal adipose tissue weight (Abd.WAT) (F) (n = 6). Error bars: std. dev. *p-value<0.05 and **p-value<0.01 by student t-test (D-E). p-value in the (A-C) was calculated by two-way ANOVA.

Article Snippet: Human PPP2R5C mRNA expression was measured by quantitative real-time RT-PCR in a fluorescent temperature cycler using TaqMan assay-on-demand kits (Hs00604899_g1, Applied Biosystems, Darmstadt, Germany), and fluorescence was detected on an ABI PRISM 7000 sequence detector (Applied Biosystems, Darmstadt, Germany).

Techniques: Knockdown, Virus, Injection

Journal: EMBO Reports

Article Title: Pyruvate metabolism guides definitive lineage specification during hematopoietic emergence

doi: 10.15252/embr.202154384

Figure Lengend Snippet: A FACS‐sorted HE, EHT, or HSC‐like cells were subcultured with or without UK5099 (10 µM). Subculture day 3 CD43 + /GPA + cell frequencies ± SEM relative to the controls for all populations are presented (HE, n = 5 biological replicates; EHT, n = 6 biological replicates; HSC‐like, n = 4 biological replicates; paired t ‐tests). B FACS‐sorted HE cells were subcultured with or without 1‐AA (4 mM). Subculture day 3 CD43 + GPA + cell frequencies ± SEM relative to the control are shown ( n = 4 biological replicates, paired t ‐test). C Fold change of expression of MPC1 and MPC2 ± SEM relative to HPRT1 in shRNA‐transduced cells compared to shScrambled (shScr) is shown ( n = 3 biological replicates, unpaired t ‐tests). Untr, untransduced. D HE cells were transduced with shScrambled (shScr), shMPC1, shMPC2, or both the day after the sort and day 3 CD43 + /GPA + cell frequencies ± SEM relative to shScr are presented ( n = 4 biological replicates; one‐way ANOVA test). Untr, untransduced. E FACS‐sorted HE cells were stained with CTV and fluorescence was assessed by flow cytometry for GPA + cells at day 3 of subculture with or without UK5099 (10 µM). Representative of n = 3. F, G FACS‐sorted HE, EHT, or HSC‐like cells were subcultured with or without UK5099 (10 µM). Subculture day 6 CD43 + (F) and CD43 + CD45 + (G) cell frequencies ± SEM relative to the control for all populations are presented (HE, n = 7 biological replicates; EHT, n = 7 biological replicates; HSC‐like, n = 4 biological replicates; paired t ‐tests). H FACS‐sorted HE cells were subcultured with or without 1‐AA (4 mM). Subculture day 6 CD43 + and CD43 + CD45 + cell frequencies ± SEM relative to the control are shown ( n = 3 biological replicates, paired t ‐test). I, J FACS‐sorted HE cells were subcultured for 3 days with or without UK5099 (10 µM). CTV for HE‐derived CD45 + cells (I) and HE‐derived HSC‐like cell frequencies ± SEM relative to the control ( n = 7 biological replicates, paired t ‐test) (J) are shown. K–N FACS‐sorted HE and EHT cells were subcultured with or without DCA (3 mM). Subculture day 3 (K, n = 3 biological replicates) and day 6 (L, HE, n = 5 biological replicates; EHT, n = 4 biological replicates) CD43 + GPA + cell frequencies ± SEM relative to the controls are shown (paired t ‐test). (M) CTV for HE‐derived GPA + cells at subculture day 3 is shown. Representative of n = 3. (N) Subculture day 6 CD43 + CD45 + cell frequencies ± SEM relative to the controls for both populations are shown (HE, n = 5 biological replicates; EHT, n = 4 biological replicates; paired t ‐tests). O Fold change of expression of PDK1 , PDK2 , PDK3 , and PDK4 ± SEM relative to HPRT1 in shRNA‐transduced cells compared to shScrambled (shScr) is shown ( n = 3 biological replicates, paired t ‐tests). P HE cells were transduced with shScrambled (shScr) or shPDK1, shPDK2, shPDK3, or shPDK4 the day after the sort and day 6 CD43 + /CD45 + cell frequencies ± SEM relative to shScr are presented ( n = 3 biological replicates; one‐way ANOVA tests). Q CTV for HE‐derived CD45 + cells are shown. Representative of n = 3. R HE‐derived HSC‐like cell frequencies ± SEM relative to the control ( n = 4 biological replicates, paired t ‐test) are shown. S EdU incorporation ± SEM into HE cells was assessed by flow cytometry after a 24‐h pulse at days 1 and 2 of subculture with or without UK5099 (10 µM) or DCA (3 mM) ( n = 3 biological replicates). T, U Percentages of CFU assay colony types ± SEM obtained from HE cells subcultured with the indicated compounds for 3 days (U) ( n = 3 biological replicates, two‐way ANOVA test) or 6 days (T) ( n = 5 biological replicates, two‐way ANOVA test). CFU, colony‐forming unit; E, erythroid; M, macrophage; G, granulocyte; GEMM, mixed. V EryD and EryP CFU‐Es obtained from HE cells subcultured with the indicated compounds for 3 days. Scale bars, 100 µm. W Fold change in the expression of HBA1‐2 transcripts ± SEM normalized to KLF1 in CFUs obtained from HE cells treated with UK5099 (10 µM) or DCA (3 mM) relative to non‐treated cells ( n = 3 biological replicates, paired t ‐test). X Plots showing percentages of CD45 + CD56 + cells obtained following 35‐day co‐culture of 3‐day subcultured HE cells with OP9‐DL1 stroma. During the 3‐day subculture, HE cells were treated with the indicated compounds. Data information: ns, not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: HBA1/2 , Thermo Fisher Scientific , Hs00361191_g1.

Techniques: Control, Expressing, shRNA, Transduction, Staining, Fluorescence, Flow Cytometry, Derivative Assay, Colony-forming Unit Assay, Co-Culture Assay

Journal: EMBO Reports

Article Title: Pyruvate metabolism guides definitive lineage specification during hematopoietic emergence

doi: 10.15252/embr.202154384

Figure Lengend Snippet:

Article Snippet: HBA1/2 , Thermo Fisher Scientific , Hs00361191_g1.

Techniques: Recombinant, Saline, Flow Cytometry, Gene Expression, RNA Sequencing, Software