Figure 4.
Rapamycin not nicotinamide riboside alleviates the erythroid differentiation arrest caused by PUS1 deletion by inhibiting global protein synthesis. (A) Frequencies of the iPSC-derived erythroblasts after 7 days of treatment with rapamycin under hypoxia conditions. n = 3. (B) Phosphorylation levels of ribosomal protein S6 (S6; left) and 4E-BP1 (right) were examined by western blot in iPSCs. Normalized to β-actin. (C-D) Phosphorylation levels of 4E-BP1 were measured by flow cytometry in iPSCs (C) and iPSC–derived HE cells (D; n = 3). (E) Pie chart representing the difference in TE of 94 TOP or TOP-like mRNAs between 2 iPSC lines. UP (purple) represents genes with increased TE in MLASA-iPSCs, whereas DOWN (azure) shows the decreased. NA (green) means the undetected genes, and NS (blue) means no significant difference. (F) Global protein synthesis was examined by puromycin incorporation in iPSCs. Western blot (left) and densitometry analyses (right) of the relative rate of protein synthesis are shown. Protein levels are normalized to β-tubulin (n = 3). (G) Protein synthesis rates monitored by OP-puro incorporation in HEs derived from iPSCs. The representative histogram (left) and MFI (right) of OP-puro are shown (n = 4). (H) Protein synthesis rates monitored by OP-puro incorporation in HE cells treated with rapamycin for 48 hours during erythroid differentiation from HEs. The representative histogram (left) and MFI (right) of OP-puro are shown. n = 3. (I-J) Activity of complex III in Normal-iPSCs with or without antimycin A (0.4 nM and 1.6 nM; n = 3) (I) and 4NQO (100 nM; n = 3) (J) treatment. (K-L) Phosphorylation levels of S6 (K) and 4E-BP1 (L) were examined by flow cytometry in Normal-iPSCs with or without antimycin A (0.4 nM and 1.6 nM). Representative graph (left) and frequency statistics (right) are shown (n = 4). (M-N) Phosphorylation levels of S6 (M) and 4E-BP1 (N) were examined by flow cytometry in Normal-iPSCs with or without 4NQO (100 nM). Representative graph (left) and gMFI (right) are shown. The gMFI was obtained using FlowJo 10.4 and the gMFI values of S6 and 4E-BP1 were normalized for each immunoglobulin G background (n = 3). Values in all panels denote mean ± SD (∗P < .05; ∗∗P < .01; ∗∗∗P < .001); unpaired Student t test (C, D, F, G, J, M, N); 1-way ANOVA, (H, I, K, L) or 2-way ANOVA (A). OP-puro, O-propargyl-puromycin; Rapa, rapamycin; TOP, 5′ terminal oligopyrimidine.

Rapamycin not nicotinamide riboside alleviates the erythroid differentiation arrest caused by PUS1 deletion by inhibiting global protein synthesis. (A) Frequencies of the iPSC-derived erythroblasts after 7 days of treatment with rapamycin under hypoxia conditions. n = 3. (B) Phosphorylation levels of ribosomal protein S6 (S6; left) and 4E-BP1 (right) were examined by western blot in iPSCs. Normalized to β-actin. (C-D) Phosphorylation levels of 4E-BP1 were measured by flow cytometry in iPSCs (C) and iPSC–derived HE cells (D; n = 3). (E) Pie chart representing the difference in TE of 94 TOP or TOP-like mRNAs between 2 iPSC lines. UP (purple) represents genes with increased TE in MLASA-iPSCs, whereas DOWN (azure) shows the decreased. NA (green) means the undetected genes, and NS (blue) means no significant difference. (F) Global protein synthesis was examined by puromycin incorporation in iPSCs. Western blot (left) and densitometry analyses (right) of the relative rate of protein synthesis are shown. Protein levels are normalized to β-tubulin (n = 3). (G) Protein synthesis rates monitored by OP-puro incorporation in HEs derived from iPSCs. The representative histogram (left) and MFI (right) of OP-puro are shown (n = 4). (H) Protein synthesis rates monitored by OP-puro incorporation in HE cells treated with rapamycin for 48 hours during erythroid differentiation from HEs. The representative histogram (left) and MFI (right) of OP-puro are shown. n = 3. (I-J) Activity of complex III in Normal-iPSCs with or without antimycin A (0.4 nM and 1.6 nM; n = 3) (I) and 4NQO (100 nM; n = 3) (J) treatment. (K-L) Phosphorylation levels of S6 (K) and 4E-BP1 (L) were examined by flow cytometry in Normal-iPSCs with or without antimycin A (0.4 nM and 1.6 nM). Representative graph (left) and frequency statistics (right) are shown (n = 4). (M-N) Phosphorylation levels of S6 (M) and 4E-BP1 (N) were examined by flow cytometry in Normal-iPSCs with or without 4NQO (100 nM). Representative graph (left) and gMFI (right) are shown. The gMFI was obtained using FlowJo 10.4 and the gMFI values of S6 and 4E-BP1 were normalized for each immunoglobulin G background (n = 3). Values in all panels denote mean ± SD (∗P < .05; ∗∗P < .01; ∗∗∗P < .001); unpaired Student t test (C, D, F, G, J, M, N); 1-way ANOVA, (H, I, K, L) or 2-way ANOVA (A). OP-puro, O-propargyl-puromycin; Rapa, rapamycin; TOP, 5′ terminal oligopyrimidine.

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