pathscan phospho eif2α ser51 Search Results


phospho eif2α  (Cell Signaling Technology Inc)
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Cell Signaling Technology Inc phospho eif2α
Figure 2. Dependence of induced formation of SG-like G3BP1-positive granules by TC41 on <t>eIF2α</t> phosphorylation. (A) U2OS cells were exposed to increasing concentrations of TC41 (1,5,10 µM) for 24 h. Cell viability was determined using MTT assay (mean ± SEM; n = 3, * p < 0.05; *** p < 0.001 in Student’s t-test). (B) U2OS cells treated with 5 µM of TC41 for 2 h. Immunoblotting of the whole cell lysate was used to assess the phosphorylation statuses of eIF2a and S6K, using specific antibodies. Actin was a loading control for the assay. (C) Relative intensity of p-S6K, p-eIf2α in each condition (mean ± SEM; n = 3, * p < 0.05; ** p < 0.01, ns, non-significant in Student’s t-test). (D) U2OS cells exposed to either Tg (1 µM) or TC41 (5 µM) either alone or in combination with cycloheximide (CHX, at 2 µg/mL) or ISRIB (a p-eIF2α inhibitor, at 200 nM) for 2 h. G3BP1-positive granules were visualized through immunofluorescence staining (Scale bars: 10 µm). (E) The proportion of U2OS cells forming granules was calculated based on G3BP1 immunofluorescence (mean ± SEM; n ≥3, * p < 0.05; ** p < 0.01; *** p < 0.001, ns, non-significant, Student’s t-test). (F) G3BP1 positive granules in U2OS cells were quantified using the Granule index (granule area/cell area), derived from G3BP1 immunofluorescence. Results are presented in arbitrary units (a.u.) (mean ± SEM; n ≥3, ** p < 0.01; in Student’s t-test).
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antiphospho eif2α ser51  (Cell Signaling Technology Inc)
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Figure 2. Dependence of induced formation of SG-like G3BP1-positive granules by TC41 on <t>eIF2α</t> phosphorylation. (A) U2OS cells were exposed to increasing concentrations of TC41 (1,5,10 µM) for 24 h. Cell viability was determined using MTT assay (mean ± SEM; n = 3, * p < 0.05; *** p < 0.001 in Student’s t-test). (B) U2OS cells treated with 5 µM of TC41 for 2 h. Immunoblotting of the whole cell lysate was used to assess the phosphorylation statuses of eIF2a and S6K, using specific antibodies. Actin was a loading control for the assay. (C) Relative intensity of p-S6K, p-eIf2α in each condition (mean ± SEM; n = 3, * p < 0.05; ** p < 0.01, ns, non-significant in Student’s t-test). (D) U2OS cells exposed to either Tg (1 µM) or TC41 (5 µM) either alone or in combination with cycloheximide (CHX, at 2 µg/mL) or ISRIB (a p-eIF2α inhibitor, at 200 nM) for 2 h. G3BP1-positive granules were visualized through immunofluorescence staining (Scale bars: 10 µm). (E) The proportion of U2OS cells forming granules was calculated based on G3BP1 immunofluorescence (mean ± SEM; n ≥3, * p < 0.05; ** p < 0.01; *** p < 0.001, ns, non-significant, Student’s t-test). (F) G3BP1 positive granules in U2OS cells were quantified using the Granule index (granule area/cell area), derived from G3BP1 immunofluorescence. Results are presented in arbitrary units (a.u.) (mean ± SEM; n ≥3, ** p < 0.01; in Student’s t-test).
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polyclonal antibodies to the total and phosphorylated forms of erk (thr202/tyr204)  (Cell Signaling Technology Inc)
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Figure 2. Dependence of induced formation of SG-like G3BP1-positive granules by TC41 on <t>eIF2α</t> phosphorylation. (A) U2OS cells were exposed to increasing concentrations of TC41 (1,5,10 µM) for 24 h. Cell viability was determined using MTT assay (mean ± SEM; n = 3, * p < 0.05; *** p < 0.001 in Student’s t-test). (B) U2OS cells treated with 5 µM of TC41 for 2 h. Immunoblotting of the whole cell lysate was used to assess the phosphorylation statuses of eIF2a and S6K, using specific antibodies. Actin was a loading control for the assay. (C) Relative intensity of p-S6K, p-eIf2α in each condition (mean ± SEM; n = 3, * p < 0.05; ** p < 0.01, ns, non-significant in Student’s t-test). (D) U2OS cells exposed to either Tg (1 µM) or TC41 (5 µM) either alone or in combination with cycloheximide (CHX, at 2 µg/mL) or ISRIB (a p-eIF2α inhibitor, at 200 nM) for 2 h. G3BP1-positive granules were visualized through immunofluorescence staining (Scale bars: 10 µm). (E) The proportion of U2OS cells forming granules was calculated based on G3BP1 immunofluorescence (mean ± SEM; n ≥3, * p < 0.05; ** p < 0.01; *** p < 0.001, ns, non-significant, Student’s t-test). (F) G3BP1 positive granules in U2OS cells were quantified using the Granule index (granule area/cell area), derived from G3BP1 immunofluorescence. Results are presented in arbitrary units (a.u.) (mean ± SEM; n ≥3, ** p < 0.01; in Student’s t-test).
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anti mouse eif2α  (Cell Signaling Technology Inc)
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Figure 2. Dependence of induced formation of SG-like G3BP1-positive granules by TC41 on <t>eIF2α</t> phosphorylation. (A) U2OS cells were exposed to increasing concentrations of TC41 (1,5,10 µM) for 24 h. Cell viability was determined using MTT assay (mean ± SEM; n = 3, * p < 0.05; *** p < 0.001 in Student’s t-test). (B) U2OS cells treated with 5 µM of TC41 for 2 h. Immunoblotting of the whole cell lysate was used to assess the phosphorylation statuses of eIF2a and S6K, using specific antibodies. Actin was a loading control for the assay. (C) Relative intensity of p-S6K, p-eIf2α in each condition (mean ± SEM; n = 3, * p < 0.05; ** p < 0.01, ns, non-significant in Student’s t-test). (D) U2OS cells exposed to either Tg (1 µM) or TC41 (5 µM) either alone or in combination with cycloheximide (CHX, at 2 µg/mL) or ISRIB (a p-eIF2α inhibitor, at 200 nM) for 2 h. G3BP1-positive granules were visualized through immunofluorescence staining (Scale bars: 10 µm). (E) The proportion of U2OS cells forming granules was calculated based on G3BP1 immunofluorescence (mean ± SEM; n ≥3, * p < 0.05; ** p < 0.01; *** p < 0.001, ns, non-significant, Student’s t-test). (F) G3BP1 positive granules in U2OS cells were quantified using the Granule index (granule area/cell area), derived from G3BP1 immunofluorescence. Results are presented in arbitrary units (a.u.) (mean ± SEM; n ≥3, ** p < 0.01; in Student’s t-test).
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p eif2α  (Cell Signaling Technology Inc)
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Figure 2. Dependence of induced formation of SG-like G3BP1-positive granules by TC41 on <t>eIF2α</t> phosphorylation. (A) U2OS cells were exposed to increasing concentrations of TC41 (1,5,10 µM) for 24 h. Cell viability was determined using MTT assay (mean ± SEM; n = 3, * p < 0.05; *** p < 0.001 in Student’s t-test). (B) U2OS cells treated with 5 µM of TC41 for 2 h. Immunoblotting of the whole cell lysate was used to assess the phosphorylation statuses of eIF2a and S6K, using specific antibodies. Actin was a loading control for the assay. (C) Relative intensity of p-S6K, p-eIf2α in each condition (mean ± SEM; n = 3, * p < 0.05; ** p < 0.01, ns, non-significant in Student’s t-test). (D) U2OS cells exposed to either Tg (1 µM) or TC41 (5 µM) either alone or in combination with cycloheximide (CHX, at 2 µg/mL) or ISRIB (a p-eIF2α inhibitor, at 200 nM) for 2 h. G3BP1-positive granules were visualized through immunofluorescence staining (Scale bars: 10 µm). (E) The proportion of U2OS cells forming granules was calculated based on G3BP1 immunofluorescence (mean ± SEM; n ≥3, * p < 0.05; ** p < 0.01; *** p < 0.001, ns, non-significant, Student’s t-test). (F) G3BP1 positive granules in U2OS cells were quantified using the Granule index (granule area/cell area), derived from G3BP1 immunofluorescence. Results are presented in arbitrary units (a.u.) (mean ± SEM; n ≥3, ** p < 0.01; in Student’s t-test).
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anti-eif-2α  (Cell Signaling Technology Inc)
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Figure 2. Dependence of induced formation of SG-like G3BP1-positive granules by TC41 on <t>eIF2α</t> phosphorylation. (A) U2OS cells were exposed to increasing concentrations of TC41 (1,5,10 µM) for 24 h. Cell viability was determined using MTT assay (mean ± SEM; n = 3, * p < 0.05; *** p < 0.001 in Student’s t-test). (B) U2OS cells treated with 5 µM of TC41 for 2 h. Immunoblotting of the whole cell lysate was used to assess the phosphorylation statuses of eIF2a and S6K, using specific antibodies. Actin was a loading control for the assay. (C) Relative intensity of p-S6K, p-eIf2α in each condition (mean ± SEM; n = 3, * p < 0.05; ** p < 0.01, ns, non-significant in Student’s t-test). (D) U2OS cells exposed to either Tg (1 µM) or TC41 (5 µM) either alone or in combination with cycloheximide (CHX, at 2 µg/mL) or ISRIB (a p-eIF2α inhibitor, at 200 nM) for 2 h. G3BP1-positive granules were visualized through immunofluorescence staining (Scale bars: 10 µm). (E) The proportion of U2OS cells forming granules was calculated based on G3BP1 immunofluorescence (mean ± SEM; n ≥3, * p < 0.05; ** p < 0.01; *** p < 0.001, ns, non-significant, Student’s t-test). (F) G3BP1 positive granules in U2OS cells were quantified using the Granule index (granule area/cell area), derived from G3BP1 immunofluorescence. Results are presented in arbitrary units (a.u.) (mean ± SEM; n ≥3, ** p < 0.01; in Student’s t-test).
Anti Eif 2α, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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phospho-eif2α ser51 antibody  (Danaher Inc)
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Figure 2. Dependence of induced formation of SG-like G3BP1-positive granules by TC41 on <t>eIF2α</t> phosphorylation. (A) U2OS cells were exposed to increasing concentrations of TC41 (1,5,10 µM) for 24 h. Cell viability was determined using MTT assay (mean ± SEM; n = 3, * p < 0.05; *** p < 0.001 in Student’s t-test). (B) U2OS cells treated with 5 µM of TC41 for 2 h. Immunoblotting of the whole cell lysate was used to assess the phosphorylation statuses of eIF2a and S6K, using specific antibodies. Actin was a loading control for the assay. (C) Relative intensity of p-S6K, p-eIf2α in each condition (mean ± SEM; n = 3, * p < 0.05; ** p < 0.01, ns, non-significant in Student’s t-test). (D) U2OS cells exposed to either Tg (1 µM) or TC41 (5 µM) either alone or in combination with cycloheximide (CHX, at 2 µg/mL) or ISRIB (a p-eIF2α inhibitor, at 200 nM) for 2 h. G3BP1-positive granules were visualized through immunofluorescence staining (Scale bars: 10 µm). (E) The proportion of U2OS cells forming granules was calculated based on G3BP1 immunofluorescence (mean ± SEM; n ≥3, * p < 0.05; ** p < 0.01; *** p < 0.001, ns, non-significant, Student’s t-test). (F) G3BP1 positive granules in U2OS cells were quantified using the Granule index (granule area/cell area), derived from G3BP1 immunofluorescence. Results are presented in arbitrary units (a.u.) (mean ± SEM; n ≥3, ** p < 0.01; in Student’s t-test).
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antibodies for chop  (Santa Cruz Biotechnology)
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Figure 2. Dependence of induced formation of SG-like G3BP1-positive granules by TC41 on <t>eIF2α</t> phosphorylation. (A) U2OS cells were exposed to increasing concentrations of TC41 (1,5,10 µM) for 24 h. Cell viability was determined using MTT assay (mean ± SEM; n = 3, * p < 0.05; *** p < 0.001 in Student’s t-test). (B) U2OS cells treated with 5 µM of TC41 for 2 h. Immunoblotting of the whole cell lysate was used to assess the phosphorylation statuses of eIF2a and S6K, using specific antibodies. Actin was a loading control for the assay. (C) Relative intensity of p-S6K, p-eIf2α in each condition (mean ± SEM; n = 3, * p < 0.05; ** p < 0.01, ns, non-significant in Student’s t-test). (D) U2OS cells exposed to either Tg (1 µM) or TC41 (5 µM) either alone or in combination with cycloheximide (CHX, at 2 µg/mL) or ISRIB (a p-eIF2α inhibitor, at 200 nM) for 2 h. G3BP1-positive granules were visualized through immunofluorescence staining (Scale bars: 10 µm). (E) The proportion of U2OS cells forming granules was calculated based on G3BP1 immunofluorescence (mean ± SEM; n ≥3, * p < 0.05; ** p < 0.01; *** p < 0.001, ns, non-significant, Student’s t-test). (F) G3BP1 positive granules in U2OS cells were quantified using the Granule index (granule area/cell area), derived from G3BP1 immunofluorescence. Results are presented in arbitrary units (a.u.) (mean ± SEM; n ≥3, ** p < 0.01; in Student’s t-test).
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antibodies against phospho-p53 (ser15)  (Cell Signaling Technology Inc)
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Figure 2. Dependence of induced formation of SG-like G3BP1-positive granules by TC41 on <t>eIF2α</t> phosphorylation. (A) U2OS cells were exposed to increasing concentrations of TC41 (1,5,10 µM) for 24 h. Cell viability was determined using MTT assay (mean ± SEM; n = 3, * p < 0.05; *** p < 0.001 in Student’s t-test). (B) U2OS cells treated with 5 µM of TC41 for 2 h. Immunoblotting of the whole cell lysate was used to assess the phosphorylation statuses of eIF2a and S6K, using specific antibodies. Actin was a loading control for the assay. (C) Relative intensity of p-S6K, p-eIf2α in each condition (mean ± SEM; n = 3, * p < 0.05; ** p < 0.01, ns, non-significant in Student’s t-test). (D) U2OS cells exposed to either Tg (1 µM) or TC41 (5 µM) either alone or in combination with cycloheximide (CHX, at 2 µg/mL) or ISRIB (a p-eIF2α inhibitor, at 200 nM) for 2 h. G3BP1-positive granules were visualized through immunofluorescence staining (Scale bars: 10 µm). (E) The proportion of U2OS cells forming granules was calculated based on G3BP1 immunofluorescence (mean ± SEM; n ≥3, * p < 0.05; ** p < 0.01; *** p < 0.001, ns, non-significant, Student’s t-test). (F) G3BP1 positive granules in U2OS cells were quantified using the Granule index (granule area/cell area), derived from G3BP1 immunofluorescence. Results are presented in arbitrary units (a.u.) (mean ± SEM; n ≥3, ** p < 0.01; in Student’s t-test).
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phosphoplus eif2α ser51 antibody duet  (Cell Signaling Technology Inc)
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Fig. 2 APOE deficiency alters neural differentiation in cerebral organoids by activating the <t>EIF2</t> signaling pathway. a–h DEGs in cell clusters were identified through the scRNA-seq and subjected to the Ingenuity pathway analysis. Volcano plot and gene ontology analyses for DEGs (APOE−/− vs. Con) in total excitatory neuron clusters (a, b), excitatory neuron cluster 3 (c, d), radial glia clusters (e, f), and astrocyte clusters (g, h) are shown. Genes significant at the P value ≤ 0.05 and fold change ≥ 1.2 are denoted in purple. Selective DEGs are labeled in the volcano plots. i Phosphorylation levels of eIF2α in the control and APOE−/− cerebral organoids at Day 90 were quantified by Western blotting. Three cerebral organoids were pooled and analyzed as one sample (n = 3 samples/genotype). j APOE−/− cerebral organoids at Day 60 were treated with DMSO or ISRIB (100 nM) for 30 days. Phosphorylation level of eIF2α in the cerebral organoids was quantified by Western blotting. Three cerebral organoids were pooled and analyzed as one sample (n = 6 samples/genotype). k The mRNA levels of cerebral layer markers (DCX, SLC17A7, TBR1 and CTIP2) and astrocytic markers (S100B and GFAP) were quantified by RT-qPCR. Three cerebral organoids were pooled and analyzed as one sample. All data are expressed as mean ± SEM (n = 4–5 samples/genotype). l Representative microscopy images of cerebral organoids stained with neuronal layer V marker CTIP2 and DAPI. The immunoreactivity of CTIP2 was quantified and normalized by DAPI fluorescent intensity (n = 6 organoids/genotype). Scale bar: 50 μm. Experiments were repeated in two independently differentiated batches (i–l). All data are expressed as mean ± SEM. Student’s t tests were performed to determine statistical significance. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. For raw data see Additional file 1: Figs. S7 and S8
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rabbit anti phospho eif2α ser51 polyclonal antibody  (Cell Signaling Technology Inc)
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Fig. 2 APOE deficiency alters neural differentiation in cerebral organoids by activating the <t>EIF2</t> signaling pathway. a–h DEGs in cell clusters were identified through the scRNA-seq and subjected to the Ingenuity pathway analysis. Volcano plot and gene ontology analyses for DEGs (APOE−/− vs. Con) in total excitatory neuron clusters (a, b), excitatory neuron cluster 3 (c, d), radial glia clusters (e, f), and astrocyte clusters (g, h) are shown. Genes significant at the P value ≤ 0.05 and fold change ≥ 1.2 are denoted in purple. Selective DEGs are labeled in the volcano plots. i Phosphorylation levels of eIF2α in the control and APOE−/− cerebral organoids at Day 90 were quantified by Western blotting. Three cerebral organoids were pooled and analyzed as one sample (n = 3 samples/genotype). j APOE−/− cerebral organoids at Day 60 were treated with DMSO or ISRIB (100 nM) for 30 days. Phosphorylation level of eIF2α in the cerebral organoids was quantified by Western blotting. Three cerebral organoids were pooled and analyzed as one sample (n = 6 samples/genotype). k The mRNA levels of cerebral layer markers (DCX, SLC17A7, TBR1 and CTIP2) and astrocytic markers (S100B and GFAP) were quantified by RT-qPCR. Three cerebral organoids were pooled and analyzed as one sample. All data are expressed as mean ± SEM (n = 4–5 samples/genotype). l Representative microscopy images of cerebral organoids stained with neuronal layer V marker CTIP2 and DAPI. The immunoreactivity of CTIP2 was quantified and normalized by DAPI fluorescent intensity (n = 6 organoids/genotype). Scale bar: 50 μm. Experiments were repeated in two independently differentiated batches (i–l). All data are expressed as mean ± SEM. Student’s t tests were performed to determine statistical significance. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. For raw data see Additional file 1: Figs. S7 and S8
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Image Search Results


Figure 2. Dependence of induced formation of SG-like G3BP1-positive granules by TC41 on eIF2α phosphorylation. (A) U2OS cells were exposed to increasing concentrations of TC41 (1,5,10 µM) for 24 h. Cell viability was determined using MTT assay (mean ± SEM; n = 3, * p < 0.05; *** p < 0.001 in Student’s t-test). (B) U2OS cells treated with 5 µM of TC41 for 2 h. Immunoblotting of the whole cell lysate was used to assess the phosphorylation statuses of eIF2a and S6K, using specific antibodies. Actin was a loading control for the assay. (C) Relative intensity of p-S6K, p-eIf2α in each condition (mean ± SEM; n = 3, * p < 0.05; ** p < 0.01, ns, non-significant in Student’s t-test). (D) U2OS cells exposed to either Tg (1 µM) or TC41 (5 µM) either alone or in combination with cycloheximide (CHX, at 2 µg/mL) or ISRIB (a p-eIF2α inhibitor, at 200 nM) for 2 h. G3BP1-positive granules were visualized through immunofluorescence staining (Scale bars: 10 µm). (E) The proportion of U2OS cells forming granules was calculated based on G3BP1 immunofluorescence (mean ± SEM; n ≥3, * p < 0.05; ** p < 0.01; *** p < 0.001, ns, non-significant, Student’s t-test). (F) G3BP1 positive granules in U2OS cells were quantified using the Granule index (granule area/cell area), derived from G3BP1 immunofluorescence. Results are presented in arbitrary units (a.u.) (mean ± SEM; n ≥3, ** p < 0.01; in Student’s t-test).

Journal: International journal of molecular sciences

Article Title: Enhancement of Stress Granule Formation by a Chiral Compound Targeting G3BP1 via eIF2α Phosphorylation.

doi: 10.3390/ijms251910571

Figure Lengend Snippet: Figure 2. Dependence of induced formation of SG-like G3BP1-positive granules by TC41 on eIF2α phosphorylation. (A) U2OS cells were exposed to increasing concentrations of TC41 (1,5,10 µM) for 24 h. Cell viability was determined using MTT assay (mean ± SEM; n = 3, * p < 0.05; *** p < 0.001 in Student’s t-test). (B) U2OS cells treated with 5 µM of TC41 for 2 h. Immunoblotting of the whole cell lysate was used to assess the phosphorylation statuses of eIF2a and S6K, using specific antibodies. Actin was a loading control for the assay. (C) Relative intensity of p-S6K, p-eIf2α in each condition (mean ± SEM; n = 3, * p < 0.05; ** p < 0.01, ns, non-significant in Student’s t-test). (D) U2OS cells exposed to either Tg (1 µM) or TC41 (5 µM) either alone or in combination with cycloheximide (CHX, at 2 µg/mL) or ISRIB (a p-eIF2α inhibitor, at 200 nM) for 2 h. G3BP1-positive granules were visualized through immunofluorescence staining (Scale bars: 10 µm). (E) The proportion of U2OS cells forming granules was calculated based on G3BP1 immunofluorescence (mean ± SEM; n ≥3, * p < 0.05; ** p < 0.01; *** p < 0.001, ns, non-significant, Student’s t-test). (F) G3BP1 positive granules in U2OS cells were quantified using the Granule index (granule area/cell area), derived from G3BP1 immunofluorescence. Results are presented in arbitrary units (a.u.) (mean ± SEM; n ≥3, ** p < 0.01; in Student’s t-test).

Article Snippet: We utilized primary antibodies for the following proteins: phospho-eIF2α (Cell Signaling, Danvers, MA, USA, 9721S), eIF2α (sc-133132), phospho-S6K (sc-8416), and S6K (sc-8418), each diluted at 1:1000.

Techniques: Phospho-proteomics, MTT Assay, Western Blot, Control, Immunofluorescence, Staining, Derivative Assay

Fig. 2 APOE deficiency alters neural differentiation in cerebral organoids by activating the EIF2 signaling pathway. a–h DEGs in cell clusters were identified through the scRNA-seq and subjected to the Ingenuity pathway analysis. Volcano plot and gene ontology analyses for DEGs (APOE−/− vs. Con) in total excitatory neuron clusters (a, b), excitatory neuron cluster 3 (c, d), radial glia clusters (e, f), and astrocyte clusters (g, h) are shown. Genes significant at the P value ≤ 0.05 and fold change ≥ 1.2 are denoted in purple. Selective DEGs are labeled in the volcano plots. i Phosphorylation levels of eIF2α in the control and APOE−/− cerebral organoids at Day 90 were quantified by Western blotting. Three cerebral organoids were pooled and analyzed as one sample (n = 3 samples/genotype). j APOE−/− cerebral organoids at Day 60 were treated with DMSO or ISRIB (100 nM) for 30 days. Phosphorylation level of eIF2α in the cerebral organoids was quantified by Western blotting. Three cerebral organoids were pooled and analyzed as one sample (n = 6 samples/genotype). k The mRNA levels of cerebral layer markers (DCX, SLC17A7, TBR1 and CTIP2) and astrocytic markers (S100B and GFAP) were quantified by RT-qPCR. Three cerebral organoids were pooled and analyzed as one sample. All data are expressed as mean ± SEM (n = 4–5 samples/genotype). l Representative microscopy images of cerebral organoids stained with neuronal layer V marker CTIP2 and DAPI. The immunoreactivity of CTIP2 was quantified and normalized by DAPI fluorescent intensity (n = 6 organoids/genotype). Scale bar: 50 μm. Experiments were repeated in two independently differentiated batches (i–l). All data are expressed as mean ± SEM. Student’s t tests were performed to determine statistical significance. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. For raw data see Additional file 1: Figs. S7 and S8

Journal: Stem cell research & therapy

Article Title: APOE deficiency impacts neural differentiation and cholesterol biosynthesis in human iPSC-derived cerebral organoids.

doi: 10.1186/s13287-023-03444-y

Figure Lengend Snippet: Fig. 2 APOE deficiency alters neural differentiation in cerebral organoids by activating the EIF2 signaling pathway. a–h DEGs in cell clusters were identified through the scRNA-seq and subjected to the Ingenuity pathway analysis. Volcano plot and gene ontology analyses for DEGs (APOE−/− vs. Con) in total excitatory neuron clusters (a, b), excitatory neuron cluster 3 (c, d), radial glia clusters (e, f), and astrocyte clusters (g, h) are shown. Genes significant at the P value ≤ 0.05 and fold change ≥ 1.2 are denoted in purple. Selective DEGs are labeled in the volcano plots. i Phosphorylation levels of eIF2α in the control and APOE−/− cerebral organoids at Day 90 were quantified by Western blotting. Three cerebral organoids were pooled and analyzed as one sample (n = 3 samples/genotype). j APOE−/− cerebral organoids at Day 60 were treated with DMSO or ISRIB (100 nM) for 30 days. Phosphorylation level of eIF2α in the cerebral organoids was quantified by Western blotting. Three cerebral organoids were pooled and analyzed as one sample (n = 6 samples/genotype). k The mRNA levels of cerebral layer markers (DCX, SLC17A7, TBR1 and CTIP2) and astrocytic markers (S100B and GFAP) were quantified by RT-qPCR. Three cerebral organoids were pooled and analyzed as one sample. All data are expressed as mean ± SEM (n = 4–5 samples/genotype). l Representative microscopy images of cerebral organoids stained with neuronal layer V marker CTIP2 and DAPI. The immunoreactivity of CTIP2 was quantified and normalized by DAPI fluorescent intensity (n = 6 organoids/genotype). Scale bar: 50 μm. Experiments were repeated in two independently differentiated batches (i–l). All data are expressed as mean ± SEM. Student’s t tests were performed to determine statistical significance. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. For raw data see Additional file 1: Figs. S7 and S8

Article Snippet: Primary antibodies and their dilutions used in this study are as follows: ABCA1 (Millipore, MAB10005, 1:1000), ABCG1 (Abcam, ab52617, 1: 1000), PhosphoPlus eIF2α (Ser51) Antibody Duet (Cell Signaling Technology, 89,117, 1:1000), Soluble frizzled-related protein 1 (SFRP1) (Invitrogen, MA5-38,193, 1:1000), β-catenin (BD Biosciences, 610,154, 1:4000), Wnt7B (Abcam, ab227607, 1:1000) and β-actin (Sigma, A2228, 1:4000).

Techniques: Labeling, Phospho-proteomics, Control, Western Blot, Quantitative RT-PCR, Microscopy, Staining, Marker

Fig. 5 APOE4 induces loss-of-function phenotypes in neurogenesis and cholesterol metabolism in the iPSC-derived cerebral organoids. The iPSC-derived cerebral organoids from an AD patient carrying APOE ε4/ε4 and the APOE ε3/ε3 isogenic line were analyzed at Day 90. a The mRNA levels of cerebral layer markers (DCX, SLC17A7, TBR1, BCL11B and SATB2) and astrocytic markers (S100B and GFAP) were quantified by RT-qPCR. Three cerebral organoids were pooled and analyzed as one sample (n = 5–8 samples/genotype). b Phosphorylation levels of eIF2α in APOE3 and APOE4 cerebral organoids at Day 90 were quantified by Western blotting. Three cerebral organoids were pooled and analyzed as one sample (n = 3 samples/genotype). c, d The mRNA levels of selective cholesterol biosynthesis genes in the neurons (c) and astrocytes (d) sorted from cerebral organoids were quantified by RT-qPCR (n = 6 wells/genotype). e, f The isolated neurons (e) and astrocytes (f) were plated on coverslips and stained with Filipin III for cholesterol. Filipin III intensities were quantified in 5 fields of each coverslip and averaged (n = 5 coverslips/genotype), Scale bars: 20 μm. Experiments were repeated in two independently differentiated batches. All data are expressed as mean ± SEM Student’s t tests were performed to determine statistical significance. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. For raw data see Additional file 1: Fig. S11

Journal: Stem cell research & therapy

Article Title: APOE deficiency impacts neural differentiation and cholesterol biosynthesis in human iPSC-derived cerebral organoids.

doi: 10.1186/s13287-023-03444-y

Figure Lengend Snippet: Fig. 5 APOE4 induces loss-of-function phenotypes in neurogenesis and cholesterol metabolism in the iPSC-derived cerebral organoids. The iPSC-derived cerebral organoids from an AD patient carrying APOE ε4/ε4 and the APOE ε3/ε3 isogenic line were analyzed at Day 90. a The mRNA levels of cerebral layer markers (DCX, SLC17A7, TBR1, BCL11B and SATB2) and astrocytic markers (S100B and GFAP) were quantified by RT-qPCR. Three cerebral organoids were pooled and analyzed as one sample (n = 5–8 samples/genotype). b Phosphorylation levels of eIF2α in APOE3 and APOE4 cerebral organoids at Day 90 were quantified by Western blotting. Three cerebral organoids were pooled and analyzed as one sample (n = 3 samples/genotype). c, d The mRNA levels of selective cholesterol biosynthesis genes in the neurons (c) and astrocytes (d) sorted from cerebral organoids were quantified by RT-qPCR (n = 6 wells/genotype). e, f The isolated neurons (e) and astrocytes (f) were plated on coverslips and stained with Filipin III for cholesterol. Filipin III intensities were quantified in 5 fields of each coverslip and averaged (n = 5 coverslips/genotype), Scale bars: 20 μm. Experiments were repeated in two independently differentiated batches. All data are expressed as mean ± SEM Student’s t tests were performed to determine statistical significance. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. For raw data see Additional file 1: Fig. S11

Article Snippet: Primary antibodies and their dilutions used in this study are as follows: ABCA1 (Millipore, MAB10005, 1:1000), ABCG1 (Abcam, ab52617, 1: 1000), PhosphoPlus eIF2α (Ser51) Antibody Duet (Cell Signaling Technology, 89,117, 1:1000), Soluble frizzled-related protein 1 (SFRP1) (Invitrogen, MA5-38,193, 1:1000), β-catenin (BD Biosciences, 610,154, 1:4000), Wnt7B (Abcam, ab227607, 1:1000) and β-actin (Sigma, A2228, 1:4000).

Techniques: Derivative Assay, Quantitative RT-PCR, Phospho-proteomics, Western Blot, Isolation, Staining