recombinant human igf1  (PeproTech)

Journal: bioRxiv

Article Title: Spatially Organized IGF1-mTOR Signaling Controls Human Forebrain Progenitor Fate Through Coordinated Transcriptional and Translational Programs

doi: 10.1101/2025.05.08.652851

Figure Lengend Snippet: ( A-B ) Schematic illustrating derivation of FOXG1⁺ neural progenitor cells (NPCs) from human pluripotent stem cells using a 2D culture system. Immunostaining confirms co-expression of FOXG1 (red) and Nestin (green), with DAPI (blue) marking nuclei. ( C ) Gene Ontology (GO) enrichment analysis of FOXG1⁺ NPCs highlights processes including forebrain development, neuronal differentiation, and cell fate commitment. Dot size reflects the number of associated genes; color indicates false discovery rate (FDR). ( D ) STRING network analysis identifies enriched clusters involved in immune signaling, cell cycle regulation, and metabolic networks. ( E ) Pathway enrichment analysis shows strong activation of Insulin/IGF, mTOR, Notch, Neurotrophin, and MAPK signaling pathways. ( F ) Expression analysis reveals high levels of IGF1R and IGF2R, absent IGF1 ligand, and selective expression of Relaxin receptors, suggesting reliance on paracrine IGF1 signaling. ( G ) Expression of IGFBP2, IGFBPL1, IRS1–3, and regulators of IGF bioavailability and insulin signaling is detected, favoring IGF axis competence. ( H ) Schematic of rosette-derived neural aggregate (RONA) culture system showing 3D tissue organization supported by Matrigel and nutrient exchange. ( I-L ) Immunostaining of RONAs reveals central IGF1 expression (green), peripheral CK8⁺ epithelial cells (red), and FOXG1⁺ NPCs co-expressing IGF1R at the plasma membrane. ( M ) Quantitative co-localization analysis shows substantial overlap between FOXG1 and IGF1R (Manders’ coefficients ∼0.6). ( N ) Working model proposing that IGF1⁺ niche cells secrete IGF1 to regulate neighboring FOXG1⁺ progenitors in RONAs.

Article Snippet: Recombinant human IGF1 (PeproTech, Cat# 100-11) at 100 ng/mL, IGF1-neutralizing antibody (R&D Systems, Cat# AF-291-NA), or IGF1R antibody (Abcam, Cat# ab9572) were added to designated cultures to assess ligand dependency.

Techniques: Immunostaining, Expressing, Activation Assay, Protein-Protein interactions, Derivative Assay, Clinical Proteomics, Membrane

Journal: bioRxiv

Article Title: Spatially Organized IGF1-mTOR Signaling Controls Human Forebrain Progenitor Fate Through Coordinated Transcriptional and Translational Programs

doi: 10.1101/2025.05.08.652851

Figure Lengend Snippet: (A-B) Brightfield imaging of RONAs shows regionalized progenitor (S1) and niche (S2) zones under control, IGF1 supplementation, IGF1 neutralization, and IGF1R blockade conditions. IGF1 maintains or enhances the S1/S2 ratio, while IGF1 or IGF1R blockade significantly reduces it (*p < 0.05). (C-D) Top and side-view images reveal that IGF1 promotes vertical tissue expansion, whereas IGF1 or IGF1R neutralization leads to thinner aggregates. (E) Quantification of aggregate thickness shows IGF1 significantly increases vertical growth compared to control, while neutralization treatments reduce it. (F-G) Neurosphere assays demonstrate that IGF1 enhances the size but not the total number of neurospheres, supporting its role in promoting clonal expansion. (H-I) BrdU incorporation assays show that IGF1 increases proliferation of FOXG1⁺ NPCs, while IGF1 or IGF1R blockade decreases BrdU⁺ cell fractions (*p < 0.05). (J-K) Developmental analysis reveals high IGF1R expression during early human fetal brain stages, correlating strongly with FOXG1 expression (r = 0.81). (L-O) Western blot and densitometry analyses demonstrate that IGF1 supplementation sustains FOXG1, Pax6, and Hes1 expression over time, supporting progenitor maintenance and survival.

Article Snippet: Recombinant human IGF1 (PeproTech, Cat# 100-11) at 100 ng/mL, IGF1-neutralizing antibody (R&D Systems, Cat# AF-291-NA), or IGF1R antibody (Abcam, Cat# ab9572) were added to designated cultures to assess ligand dependency.

Techniques: Imaging, Control, Neutralization, BrdU Incorporation Assay, Expressing, Western Blot

Journal: bioRxiv

Article Title: Spatially Organized IGF1-mTOR Signaling Controls Human Forebrain Progenitor Fate Through Coordinated Transcriptional and Translational Programs

doi: 10.1101/2025.05.08.652851

Figure Lengend Snippet: (A-C) Western blot analysis shows that IGF1 rapidly and robustly induces ERK phosphorylation within 10 minutes, sustained up to 60 minutes. Total ERK levels remain stable. (D-F) IGF1 stimulation also induces Akt phosphorylation, peaking between 10–60 minutes, while total Akt levels modestly decline. (G-I) IGF1 enhances phosphorylation of S6, a downstream mTORC1 target, with peak activation at 30–60 minutes and stable total S6 levels. (J-P) BrdU incorporation assays combined with pathway-specific inhibitors demonstrate that PI3K, AKT, and mTOR pathways are essential for IGF1-driven proliferation. MEK/ERK inhibition partially reduces proliferation, indicating a secondary but supportive role.

Article Snippet: Recombinant human IGF1 (PeproTech, Cat# 100-11) at 100 ng/mL, IGF1-neutralizing antibody (R&D Systems, Cat# AF-291-NA), or IGF1R antibody (Abcam, Cat# ab9572) were added to designated cultures to assess ligand dependency.

Techniques: Western Blot, Phospho-proteomics, Activation Assay, BrdU Incorporation Assay, Inhibition

Journal: bioRxiv

Article Title: Spatially Organized IGF1-mTOR Signaling Controls Human Forebrain Progenitor Fate Through Coordinated Transcriptional and Translational Programs

doi: 10.1101/2025.05.08.652851

Figure Lengend Snippet: (A) Workflow illustrating ribosome profiling (Ribo-Seq) in cultured FOXG1⁺ neural progenitors, including cycloheximide treatment, ribosome-protected fragment (RPF) isolation, and sequencing. (B-C) Cumulative distribution plots show that IGF1 broadly enhances translation efficiency transcriptome-wide, while Torin1 attenuates this effect, particularly for known mTOR targets. (D) KEGG pathway enrichment identifies ribosome biogenesis, oxidative phosphorylation, proteasome function, and neurodegenerative pathways (e.g., Parkinson’s, Huntington’s) among IGF1-upregulated translational targets. (E-F) GO biological processes enriched among IGF1-upregulated transcripts include cytoplasmic translation, ribonucleoprotein biogenesis, and macromolecule biosynthesis. (G) IGF1-responsive translational programs prominently involve proteostasis mechanisms such as ubiquitin-dependent protein catabolism and multivesicular body transport. (H) IGF1 stimulation enhances translation of mitochondrial pathways, including the electron transport chain and oxidative phosphorylation. (I) IGF1 promotes translation of genes involved in genome stability, including DNA repair, chromatin remodeling, and cell cycle checkpoints.

Article Snippet: Recombinant human IGF1 (PeproTech, Cat# 100-11) at 100 ng/mL, IGF1-neutralizing antibody (R&D Systems, Cat# AF-291-NA), or IGF1R antibody (Abcam, Cat# ab9572) were added to designated cultures to assess ligand dependency.

Techniques: Cell Culture, Isolation, Sequencing, Phospho-proteomics, Ubiquitin Proteomics

Journal: bioRxiv

Article Title: Spatially Organized IGF1-mTOR Signaling Controls Human Forebrain Progenitor Fate Through Coordinated Transcriptional and Translational Programs

doi: 10.1101/2025.05.08.652851

Figure Lengend Snippet: (A) Gene Ontology and pathway enrichment analysis identifies translational programs regulated by IGF1, including nervous system development, axon guidance, ribosome biogenesis, cytoplasmic translation, lipoprotein metabolism, and neuronal maturation pathways (e.g., SLIT– ROBO signaling). (B-C) Polysome fractionation analysis shows that IGF1 promotes GSX1 and ACTB mRNA association with heavier polysome fractions, indicating enhanced translational engagement, while Torin1 treatment shifts transcripts toward lighter fractions. (D) Bar graph quantifying polysome-to-monosome ratios reveals that IGF1 significantly enhances global translational output, while mTOR inhibition (Torin1) suppresses this effect. (E) 5’UTR reporter assays demonstrate that luciferase reporters bearing the GSX1, eEF2, or ACTB 5’UTRs exhibit reduced translation upon Torin1 treatment, with GSX1 5’UTR showing the greatest sensitivity, indicating 5’UTR-dependent mTOR control. (F-G) Immunofluorescence images and quantification show that expression of a GSX1 5’UTR– luciferase reporter reduces BrdU incorporation compared to control reporters, suggesting translational repression impairs progenitor proliferation. (H-I) Brightfield and GFP imaging reveal that GSX1 5’UTR–GFP constructs significantly reduce GFP intensity compared to control, confirming strong 5’UTR-mediated translational repression affecting protein output in neural progenitor colonies.

Article Snippet: Recombinant human IGF1 (PeproTech, Cat# 100-11) at 100 ng/mL, IGF1-neutralizing antibody (R&D Systems, Cat# AF-291-NA), or IGF1R antibody (Abcam, Cat# ab9572) were added to designated cultures to assess ligand dependency.

Techniques: Fractionation, Inhibition, Luciferase, Control, Immunofluorescence, Expressing, BrdU Incorporation Assay, Imaging, Construct

Journal: bioRxiv

Article Title: Spatially Organized IGF1-mTOR Signaling Controls Human Forebrain Progenitor Fate Through Coordinated Transcriptional and Translational Programs

doi: 10.1101/2025.05.08.652851

Figure Lengend Snippet: ( A-B ) Schematic illustrating derivation of FOXG1⁺ neural progenitor cells (NPCs) from human pluripotent stem cells using a 2D culture system. Immunostaining confirms co-expression of FOXG1 (red) and Nestin (green), with DAPI (blue) marking nuclei. ( C ) Gene Ontology (GO) enrichment analysis of FOXG1⁺ NPCs highlights processes including forebrain development, neuronal differentiation, and cell fate commitment. Dot size reflects the number of associated genes; color indicates false discovery rate (FDR). ( D ) STRING network analysis identifies enriched clusters involved in immune signaling, cell cycle regulation, and metabolic networks. ( E ) Pathway enrichment analysis shows strong activation of Insulin/IGF, mTOR, Notch, Neurotrophin, and MAPK signaling pathways. ( F ) Expression analysis reveals high levels of IGF1R and IGF2R, absent IGF1 ligand, and selective expression of Relaxin receptors, suggesting reliance on paracrine IGF1 signaling. ( G ) Expression of IGFBP2, IGFBPL1, IRS1–3, and regulators of IGF bioavailability and insulin signaling is detected, favoring IGF axis competence. ( H ) Schematic of rosette-derived neural aggregate (RONA) culture system showing 3D tissue organization supported by Matrigel and nutrient exchange. ( I-L ) Immunostaining of RONAs reveals central IGF1 expression (green), peripheral CK8⁺ epithelial cells (red), and FOXG1⁺ NPCs co-expressing IGF1R at the plasma membrane. ( M ) Quantitative co-localization analysis shows substantial overlap between FOXG1 and IGF1R (Manders’ coefficients ∼0.6). ( N ) Working model proposing that IGF1⁺ niche cells secrete IGF1 to regulate neighboring FOXG1⁺ progenitors in RONAs.

Article Snippet: Human FOXG1+ NPCs were cultured under growth factor/insulin-deprived conditions for 16 hours before treatment with 20 ng/mL recombinant human IGF1 (PeproTech, #100-11) for 30 minutes, or pre-treated with 250 nM Torin 1 (Tocris, #4247) for 30 minutes followed by 30 minutes of IGF1 in the continued presence of Torin 1.

Techniques: Immunostaining, Expressing, Activation Assay, Protein-Protein interactions, Derivative Assay, Clinical Proteomics, Membrane

Journal: bioRxiv

Article Title: Spatially Organized IGF1-mTOR Signaling Controls Human Forebrain Progenitor Fate Through Coordinated Transcriptional and Translational Programs

doi: 10.1101/2025.05.08.652851

Figure Lengend Snippet: (A-B) Brightfield imaging of RONAs shows regionalized progenitor (S1) and niche (S2) zones under control, IGF1 supplementation, IGF1 neutralization, and IGF1R blockade conditions. IGF1 maintains or enhances the S1/S2 ratio, while IGF1 or IGF1R blockade significantly reduces it (*p < 0.05). (C-D) Top and side-view images reveal that IGF1 promotes vertical tissue expansion, whereas IGF1 or IGF1R neutralization leads to thinner aggregates. (E) Quantification of aggregate thickness shows IGF1 significantly increases vertical growth compared to control, while neutralization treatments reduce it. (F-G) Neurosphere assays demonstrate that IGF1 enhances the size but not the total number of neurospheres, supporting its role in promoting clonal expansion. (H-I) BrdU incorporation assays show that IGF1 increases proliferation of FOXG1⁺ NPCs, while IGF1 or IGF1R blockade decreases BrdU⁺ cell fractions (*p < 0.05). (J-K) Developmental analysis reveals high IGF1R expression during early human fetal brain stages, correlating strongly with FOXG1 expression (r = 0.81). (L-O) Western blot and densitometry analyses demonstrate that IGF1 supplementation sustains FOXG1, Pax6, and Hes1 expression over time, supporting progenitor maintenance and survival.

Article Snippet: Human FOXG1+ NPCs were cultured under growth factor/insulin-deprived conditions for 16 hours before treatment with 20 ng/mL recombinant human IGF1 (PeproTech, #100-11) for 30 minutes, or pre-treated with 250 nM Torin 1 (Tocris, #4247) for 30 minutes followed by 30 minutes of IGF1 in the continued presence of Torin 1.

Techniques: Imaging, Control, Neutralization, BrdU Incorporation Assay, Expressing, Western Blot

Journal: bioRxiv

Article Title: Spatially Organized IGF1-mTOR Signaling Controls Human Forebrain Progenitor Fate Through Coordinated Transcriptional and Translational Programs

doi: 10.1101/2025.05.08.652851

Figure Lengend Snippet: (A-C) Western blot analysis shows that IGF1 rapidly and robustly induces ERK phosphorylation within 10 minutes, sustained up to 60 minutes. Total ERK levels remain stable. (D-F) IGF1 stimulation also induces Akt phosphorylation, peaking between 10–60 minutes, while total Akt levels modestly decline. (G-I) IGF1 enhances phosphorylation of S6, a downstream mTORC1 target, with peak activation at 30–60 minutes and stable total S6 levels. (J-P) BrdU incorporation assays combined with pathway-specific inhibitors demonstrate that PI3K, AKT, and mTOR pathways are essential for IGF1-driven proliferation. MEK/ERK inhibition partially reduces proliferation, indicating a secondary but supportive role.

Article Snippet: Human FOXG1+ NPCs were cultured under growth factor/insulin-deprived conditions for 16 hours before treatment with 20 ng/mL recombinant human IGF1 (PeproTech, #100-11) for 30 minutes, or pre-treated with 250 nM Torin 1 (Tocris, #4247) for 30 minutes followed by 30 minutes of IGF1 in the continued presence of Torin 1.

Techniques: Western Blot, Phospho-proteomics, Activation Assay, BrdU Incorporation Assay, Inhibition

Journal: bioRxiv

Article Title: Spatially Organized IGF1-mTOR Signaling Controls Human Forebrain Progenitor Fate Through Coordinated Transcriptional and Translational Programs

doi: 10.1101/2025.05.08.652851

Figure Lengend Snippet: (A) Workflow illustrating ribosome profiling (Ribo-Seq) in cultured FOXG1⁺ neural progenitors, including cycloheximide treatment, ribosome-protected fragment (RPF) isolation, and sequencing. (B-C) Cumulative distribution plots show that IGF1 broadly enhances translation efficiency transcriptome-wide, while Torin1 attenuates this effect, particularly for known mTOR targets. (D) KEGG pathway enrichment identifies ribosome biogenesis, oxidative phosphorylation, proteasome function, and neurodegenerative pathways (e.g., Parkinson’s, Huntington’s) among IGF1-upregulated translational targets. (E-F) GO biological processes enriched among IGF1-upregulated transcripts include cytoplasmic translation, ribonucleoprotein biogenesis, and macromolecule biosynthesis. (G) IGF1-responsive translational programs prominently involve proteostasis mechanisms such as ubiquitin-dependent protein catabolism and multivesicular body transport. (H) IGF1 stimulation enhances translation of mitochondrial pathways, including the electron transport chain and oxidative phosphorylation. (I) IGF1 promotes translation of genes involved in genome stability, including DNA repair, chromatin remodeling, and cell cycle checkpoints.

Article Snippet: Human FOXG1+ NPCs were cultured under growth factor/insulin-deprived conditions for 16 hours before treatment with 20 ng/mL recombinant human IGF1 (PeproTech, #100-11) for 30 minutes, or pre-treated with 250 nM Torin 1 (Tocris, #4247) for 30 minutes followed by 30 minutes of IGF1 in the continued presence of Torin 1.

Techniques: Cell Culture, Isolation, Sequencing, Phospho-proteomics, Ubiquitin Proteomics

Journal: bioRxiv

Article Title: Spatially Organized IGF1-mTOR Signaling Controls Human Forebrain Progenitor Fate Through Coordinated Transcriptional and Translational Programs

doi: 10.1101/2025.05.08.652851

Figure Lengend Snippet: (A) Gene Ontology and pathway enrichment analysis identifies translational programs regulated by IGF1, including nervous system development, axon guidance, ribosome biogenesis, cytoplasmic translation, lipoprotein metabolism, and neuronal maturation pathways (e.g., SLIT– ROBO signaling). (B-C) Polysome fractionation analysis shows that IGF1 promotes GSX1 and ACTB mRNA association with heavier polysome fractions, indicating enhanced translational engagement, while Torin1 treatment shifts transcripts toward lighter fractions. (D) Bar graph quantifying polysome-to-monosome ratios reveals that IGF1 significantly enhances global translational output, while mTOR inhibition (Torin1) suppresses this effect. (E) 5’UTR reporter assays demonstrate that luciferase reporters bearing the GSX1, eEF2, or ACTB 5’UTRs exhibit reduced translation upon Torin1 treatment, with GSX1 5’UTR showing the greatest sensitivity, indicating 5’UTR-dependent mTOR control. (F-G) Immunofluorescence images and quantification show that expression of a GSX1 5’UTR– luciferase reporter reduces BrdU incorporation compared to control reporters, suggesting translational repression impairs progenitor proliferation. (H-I) Brightfield and GFP imaging reveal that GSX1 5’UTR–GFP constructs significantly reduce GFP intensity compared to control, confirming strong 5’UTR-mediated translational repression affecting protein output in neural progenitor colonies.

Article Snippet: Human FOXG1+ NPCs were cultured under growth factor/insulin-deprived conditions for 16 hours before treatment with 20 ng/mL recombinant human IGF1 (PeproTech, #100-11) for 30 minutes, or pre-treated with 250 nM Torin 1 (Tocris, #4247) for 30 minutes followed by 30 minutes of IGF1 in the continued presence of Torin 1.

Techniques: Fractionation, Inhibition, Luciferase, Control, Immunofluorescence, Expressing, BrdU Incorporation Assay, Imaging, Construct