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qPCR shows cholesterol biosynthesis is significantly increased in plasma cultured cells. ( A ) Main steps in cholesterol and fatty acid biosynthesis. Cholesterol biosynthesis begins with acetyl-CoA and proceeds via HMG-CoA reductase (HMGCR), forming mevalonate and downstream intermediates (mevalonate-PP, isopentenyl-PP, and squalene). Squalene epoxidase (SQLE) converts squalene to 2,3(S)-oxidosqualene, leading to lanosterol and cholesterol synthesis. Fatty acid biosynthesis also originates from acetyl-CoA, with acetyl-CoA carboxylase (ACC) producing malonyl-CoA, followed by fatty acid synthase (FASN) generating palmitate and complex fatty acids. SREBP1/2 transcription factors regulate these pathways, with SREBP1 driving fatty acid synthesis and SREBP2 controlling cholesterol biosynthesis. The LDL receptor (LDLR) mediates cholesterol uptake, maintaining lipid homeostasis. Made using Biorender. ( B ) mRNA levels of HMGCR (HMG-CoA reductase), SQLE (Squalene Epoxidase), LDLR (Low density lipoprotein receptor), SREBP1 and SREBP2 (Sterol regulatory element-binding protein 1 or 2) relative to GAPDH on day 8 of differentiation after CD34 + isolation. A parametric Brown-Forsythe and Welch test was performed to test for differences between groups. p < 0.05 was considered statistically significant ( n = 3, N = 3). ( C ) Example waterfall plots of cells the same donor on day 8 of erythroid differentiation, labelled against Band3 and <t>CD49d,</t> grown in medium containing AB serum, Plasma, or Plasma supplemented with CRL. Corresponding percentage of each stage (I-Proerythoblast, II-Basophilic, III-Polychromatic, IV-Orthochromatic, and V-Reticulocyte) is indicated next to the corresponding gate. D Shows percentage of erythroid cell types present on day 8 for each condition studied, quantified as displayed in panel above ( n = 3). Error bars represent the standard deviation.
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qPCR shows cholesterol biosynthesis is significantly increased in plasma cultured cells. ( A ) Main steps in cholesterol and fatty acid biosynthesis. Cholesterol biosynthesis begins with acetyl-CoA and proceeds via HMG-CoA reductase (HMGCR), forming mevalonate and downstream intermediates (mevalonate-PP, isopentenyl-PP, and squalene). Squalene epoxidase (SQLE) converts squalene to 2,3(S)-oxidosqualene, leading to lanosterol and cholesterol synthesis. Fatty acid biosynthesis also originates from acetyl-CoA, with acetyl-CoA carboxylase (ACC) producing malonyl-CoA, followed by fatty acid synthase (FASN) generating palmitate and complex fatty acids. SREBP1/2 transcription factors regulate these pathways, with SREBP1 driving fatty acid synthesis and SREBP2 controlling cholesterol biosynthesis. The LDL receptor (LDLR) mediates cholesterol uptake, maintaining lipid homeostasis. Made using Biorender. ( B ) mRNA levels of HMGCR (HMG-CoA reductase), SQLE (Squalene Epoxidase), LDLR (Low density lipoprotein receptor), SREBP1 and SREBP2 (Sterol regulatory element-binding protein 1 or 2) relative to GAPDH on day 8 of differentiation after CD34 + isolation. A parametric Brown-Forsythe and Welch test was performed to test for differences between groups. p < 0.05 was considered statistically significant ( n = 3, N = 3). ( C ) Example waterfall plots of cells the same donor on day 8 of erythroid differentiation, labelled against Band3 and <t>CD49d,</t> grown in medium containing AB serum, Plasma, or Plasma supplemented with CRL. Corresponding percentage of each stage (I-Proerythoblast, II-Basophilic, III-Polychromatic, IV-Orthochromatic, and V-Reticulocyte) is indicated next to the corresponding gate. D Shows percentage of erythroid cell types present on day 8 for each condition studied, quantified as displayed in panel above ( n = 3). Error bars represent the standard deviation.
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qPCR shows cholesterol biosynthesis is significantly increased in plasma cultured cells. ( A ) Main steps in cholesterol and fatty acid biosynthesis. Cholesterol biosynthesis begins with acetyl-CoA and proceeds via HMG-CoA reductase (HMGCR), forming mevalonate and downstream intermediates (mevalonate-PP, isopentenyl-PP, and squalene). Squalene epoxidase (SQLE) converts squalene to 2,3(S)-oxidosqualene, leading to lanosterol and cholesterol synthesis. Fatty acid biosynthesis also originates from acetyl-CoA, with acetyl-CoA carboxylase (ACC) producing malonyl-CoA, followed by fatty acid synthase (FASN) generating palmitate and complex fatty acids. SREBP1/2 transcription factors regulate these pathways, with SREBP1 driving fatty acid synthesis and SREBP2 controlling cholesterol biosynthesis. The LDL receptor (LDLR) mediates cholesterol uptake, maintaining lipid homeostasis. Made using Biorender. ( B ) mRNA levels of HMGCR (HMG-CoA reductase), SQLE (Squalene Epoxidase), LDLR (Low density lipoprotein receptor), SREBP1 and SREBP2 (Sterol regulatory element-binding protein 1 or 2) relative to GAPDH on day 8 of differentiation after CD34 + isolation. A parametric Brown-Forsythe and Welch test was performed to test for differences between groups. p < 0.05 was considered statistically significant ( n = 3, N = 3). ( C ) Example waterfall plots of cells the same donor on day 8 of erythroid differentiation, labelled against Band3 and <t>CD49d,</t> grown in medium containing AB serum, Plasma, or Plasma supplemented with CRL. Corresponding percentage of each stage (I-Proerythoblast, II-Basophilic, III-Polychromatic, IV-Orthochromatic, and V-Reticulocyte) is indicated next to the corresponding gate. D Shows percentage of erythroid cell types present on day 8 for each condition studied, quantified as displayed in panel above ( n = 3). Error bars represent the standard deviation.
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qPCR shows cholesterol biosynthesis is significantly increased in plasma cultured cells. ( A ) Main steps in cholesterol and fatty acid biosynthesis. Cholesterol biosynthesis begins with acetyl-CoA and proceeds via HMG-CoA reductase (HMGCR), forming mevalonate and downstream intermediates (mevalonate-PP, isopentenyl-PP, and squalene). Squalene epoxidase (SQLE) converts squalene to 2,3(S)-oxidosqualene, leading to lanosterol and cholesterol synthesis. Fatty acid biosynthesis also originates from acetyl-CoA, with acetyl-CoA carboxylase (ACC) producing malonyl-CoA, followed by fatty acid synthase (FASN) generating palmitate and complex fatty acids. SREBP1/2 transcription factors regulate these pathways, with SREBP1 driving fatty acid synthesis and SREBP2 controlling cholesterol biosynthesis. The LDL receptor (LDLR) mediates cholesterol uptake, maintaining lipid homeostasis. Made using Biorender. ( B ) mRNA levels of HMGCR (HMG-CoA reductase), SQLE (Squalene Epoxidase), LDLR (Low density lipoprotein receptor), SREBP1 and SREBP2 (Sterol regulatory element-binding protein 1 or 2) relative to GAPDH on day 8 of differentiation after CD34 + isolation. A parametric Brown-Forsythe and Welch test was performed to test for differences between groups. p < 0.05 was considered statistically significant ( n = 3, N = 3). ( C ) Example waterfall plots of cells the same donor on day 8 of erythroid differentiation, labelled against Band3 and <t>CD49d,</t> grown in medium containing AB serum, Plasma, or Plasma supplemented with CRL. Corresponding percentage of each stage (I-Proerythoblast, II-Basophilic, III-Polychromatic, IV-Orthochromatic, and V-Reticulocyte) is indicated next to the corresponding gate. D Shows percentage of erythroid cell types present on day 8 for each condition studied, quantified as displayed in panel above ( n = 3). Error bars represent the standard deviation.
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qPCR shows cholesterol biosynthesis is significantly increased in plasma cultured cells. ( A ) Main steps in cholesterol and fatty acid biosynthesis. Cholesterol biosynthesis begins with acetyl-CoA and proceeds via HMG-CoA reductase (HMGCR), forming mevalonate and downstream intermediates (mevalonate-PP, isopentenyl-PP, and squalene). Squalene epoxidase (SQLE) converts squalene to 2,3(S)-oxidosqualene, leading to lanosterol and cholesterol synthesis. Fatty acid biosynthesis also originates from acetyl-CoA, with acetyl-CoA carboxylase (ACC) producing malonyl-CoA, followed by fatty acid synthase (FASN) generating palmitate and complex fatty acids. SREBP1/2 transcription factors regulate these pathways, with SREBP1 driving fatty acid synthesis and SREBP2 controlling cholesterol biosynthesis. The LDL receptor (LDLR) mediates cholesterol uptake, maintaining lipid homeostasis. Made using Biorender. ( B ) mRNA levels of HMGCR (HMG-CoA reductase), SQLE (Squalene Epoxidase), LDLR (Low density lipoprotein receptor), SREBP1 and SREBP2 (Sterol regulatory element-binding protein 1 or 2) relative to GAPDH on day 8 of differentiation after CD34 + isolation. A parametric Brown-Forsythe and Welch test was performed to test for differences between groups. p < 0.05 was considered statistically significant ( n = 3, N = 3). ( C ) Example waterfall plots of cells the same donor on day 8 of erythroid differentiation, labelled against Band3 and CD49d, grown in medium containing AB serum, Plasma, or Plasma supplemented with CRL. Corresponding percentage of each stage (I-Proerythoblast, II-Basophilic, III-Polychromatic, IV-Orthochromatic, and V-Reticulocyte) is indicated next to the corresponding gate. D Shows percentage of erythroid cell types present on day 8 for each condition studied, quantified as displayed in panel above ( n = 3). Error bars represent the standard deviation.

Journal: Scientific Reports

Article Title: Choice of lipid supplementation for in vitro erythroid cell culture impacts reticulocyte yield and characteristics

doi: 10.1038/s41598-026-37229-z

Figure Lengend Snippet: qPCR shows cholesterol biosynthesis is significantly increased in plasma cultured cells. ( A ) Main steps in cholesterol and fatty acid biosynthesis. Cholesterol biosynthesis begins with acetyl-CoA and proceeds via HMG-CoA reductase (HMGCR), forming mevalonate and downstream intermediates (mevalonate-PP, isopentenyl-PP, and squalene). Squalene epoxidase (SQLE) converts squalene to 2,3(S)-oxidosqualene, leading to lanosterol and cholesterol synthesis. Fatty acid biosynthesis also originates from acetyl-CoA, with acetyl-CoA carboxylase (ACC) producing malonyl-CoA, followed by fatty acid synthase (FASN) generating palmitate and complex fatty acids. SREBP1/2 transcription factors regulate these pathways, with SREBP1 driving fatty acid synthesis and SREBP2 controlling cholesterol biosynthesis. The LDL receptor (LDLR) mediates cholesterol uptake, maintaining lipid homeostasis. Made using Biorender. ( B ) mRNA levels of HMGCR (HMG-CoA reductase), SQLE (Squalene Epoxidase), LDLR (Low density lipoprotein receptor), SREBP1 and SREBP2 (Sterol regulatory element-binding protein 1 or 2) relative to GAPDH on day 8 of differentiation after CD34 + isolation. A parametric Brown-Forsythe and Welch test was performed to test for differences between groups. p < 0.05 was considered statistically significant ( n = 3, N = 3). ( C ) Example waterfall plots of cells the same donor on day 8 of erythroid differentiation, labelled against Band3 and CD49d, grown in medium containing AB serum, Plasma, or Plasma supplemented with CRL. Corresponding percentage of each stage (I-Proerythoblast, II-Basophilic, III-Polychromatic, IV-Orthochromatic, and V-Reticulocyte) is indicated next to the corresponding gate. D Shows percentage of erythroid cell types present on day 8 for each condition studied, quantified as displayed in panel above ( n = 3). Error bars represent the standard deviation.

Article Snippet: CD49d was detected using a FITC-conjugated anti-human CD49d antibody (clone MZ18-24A9), a mouse IgG2b from Miltenyi Biotech.

Techniques: Clinical Proteomics, Cell Culture, Binding Assay, Isolation, Standard Deviation