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Proteintech insulin like growth factor 2 igf2

Expression of miR-125b-5p and insulin-like growth factor 2 in vitro and in vivo . A: Quantitative real-time polymerase chain reaction (RT-qPCR) was used to detect the expression of miR-125b-5p in AR42J cell lines in activated and inactive state; B: RT-qPCR was used to detect the expression of insulin-like growth factor 2 <t>(IGF2)</t> in AR42J cell lines under activated and inactive state; C: Western blot was used to detect the expression level of IGF2, and β-actin was used as the internal reference; D: RT-qPCR was used to detect the expression of miR-125b-5p in normal pancreatic tissues and pancreatitis tissues; E: RT-qPCR was used to detect the expression of IGF2 in normal pancreatic tissues and pancreatitis tissues; F: The mRNA and protein expression of miR-125b-5p and IGF2 in 4 cases of pancreatitis tissues were detected by RT-qPCR and Western blot. Among them, sample 2 was used as the standard to calculate the fold change of miR-125b-5p expression in other samples by comparing the miR-125b-5p/U6 ratio in sample 2. Western blot was used to detect the expression level of IGF2, and β-actin was used as the internal reference. The experiment was repeated three times and is expressed as mean ± SD; G: miR-125b-5p was negatively correlated with IGF2 protein expression level, a P < 0.05, b P < 0.005. AP: Acute pancreatitis; IGF2: Insulin-like growth factor 2; N: Normal.

Insulin Like Growth Factor 2 Igf2, supplied by Proteintech, used in various techniques. Bioz Stars score: 93/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Acinous cell AR42J-derived exosome miR125b-5p promotes acute pancreatitis exacerbation by inhibiting M2 macrophage polarization via PI3K/AKT signaling pathway"

Article Title: Acinous cell AR42J-derived exosome miR125b-5p promotes acute pancreatitis exacerbation by inhibiting M2 macrophage polarization via PI3K/AKT signaling pathway

Journal: World Journal of Gastrointestinal Surgery

doi: 10.4240/wjgs.v15.i4.600

Figure Legend Snippet: Expression of miR-125b-5p and insulin-like growth factor 2 in vitro and in vivo . A: Quantitative real-time polymerase chain reaction (RT-qPCR) was used to detect the expression of miR-125b-5p in AR42J cell lines in activated and inactive state; B: RT-qPCR was used to detect the expression of insulin-like growth factor 2 (IGF2) in AR42J cell lines under activated and inactive state; C: Western blot was used to detect the expression level of IGF2, and β-actin was used as the internal reference; D: RT-qPCR was used to detect the expression of miR-125b-5p in normal pancreatic tissues and pancreatitis tissues; E: RT-qPCR was used to detect the expression of IGF2 in normal pancreatic tissues and pancreatitis tissues; F: The mRNA and protein expression of miR-125b-5p and IGF2 in 4 cases of pancreatitis tissues were detected by RT-qPCR and Western blot. Among them, sample 2 was used as the standard to calculate the fold change of miR-125b-5p expression in other samples by comparing the miR-125b-5p/U6 ratio in sample 2. Western blot was used to detect the expression level of IGF2, and β-actin was used as the internal reference. The experiment was repeated three times and is expressed as mean ± SD; G: miR-125b-5p was negatively correlated with IGF2 protein expression level, a P < 0.05, b P < 0.005. AP: Acute pancreatitis; IGF2: Insulin-like growth factor 2; N: Normal.

Techniques Used: Expressing, In Vitro, In Vivo, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot

miR-125b-5p overexpression could promote necrosis and apoptosis of AR42J cells in the activated state. A: miR-125b-5p overexpression promoted the necrosis of AR42J cells in the activated state; B: Flow cytometry analysis confirmed that the percentage of cells in G0/G1 phase increased in AR42J cells in the activated state treated with the miR-125b-5p overexpression group; C: The percentage of apoptotic cells increased as confirmed by flow cytometry; D and E: The expression of insulin-like growth factor 2, BAX, Bcl-2 and HMGB-1 was confirmed by Western blot, a P < 0.05, c P < 0.0005. AP: Acute pancreatitis; IGF2: Insulin-like growth factor 2; N: Normal; NC: Negative control.

Figure Legend Snippet: miR-125b-5p overexpression could promote necrosis and apoptosis of AR42J cells in the activated state. A: miR-125b-5p overexpression promoted the necrosis of AR42J cells in the activated state; B: Flow cytometry analysis confirmed that the percentage of cells in G0/G1 phase increased in AR42J cells in the activated state treated with the miR-125b-5p overexpression group; C: The percentage of apoptotic cells increased as confirmed by flow cytometry; D and E: The expression of insulin-like growth factor 2, BAX, Bcl-2 and HMGB-1 was confirmed by Western blot, a P < 0.05, c P < 0.0005. AP: Acute pancreatitis; IGF2: Insulin-like growth factor 2; N: Normal; NC: Negative control.

Techniques Used: Over Expression, Flow Cytometry, Expressing, Western Blot, Negative Control

miR-125b-5p promotes acute pancreatitis exacerbation by inhibiting insulin-like growth factor 2 protein expression in PI3K/AKT signaling pathway. A: The potential binding sites of miR-125b-5p in the 3¢-untranslated regions (UTR) of wild-type insulin-like growth factor 2 (IGF2) (red part). The blue section is the mutation site in mutant IGF2 3¢-UTR sequence; B: Luciferase activity in IGF2 3¢-UTR of wild-type and mutant was detected after transfection of miR-125b-5p mimics and negative control miRNAs. The normalized luciferase activity of the transfected control miRNA was set to a relative luciferase activity of 1; C: The protein expression of PI3K/AKT signaling pathway was confirmed by western blot. a P < 0.05, b P < 0.005. AP: Acute pancreatitis; IGF2: Insulin-like growth factor 2; N: Normal; NC: Negative control; WT: Wild-type; MUT: Mutant; UTR: Untranslated regions; CDS: Coding sequence.

Figure Legend Snippet: miR-125b-5p promotes acute pancreatitis exacerbation by inhibiting insulin-like growth factor 2 protein expression in PI3K/AKT signaling pathway. A: The potential binding sites of miR-125b-5p in the 3¢-untranslated regions (UTR) of wild-type insulin-like growth factor 2 (IGF2) (red part). The blue section is the mutation site in mutant IGF2 3¢-UTR sequence; B: Luciferase activity in IGF2 3¢-UTR of wild-type and mutant was detected after transfection of miR-125b-5p mimics and negative control miRNAs. The normalized luciferase activity of the transfected control miRNA was set to a relative luciferase activity of 1; C: The protein expression of PI3K/AKT signaling pathway was confirmed by western blot. a P < 0.05, b P < 0.005. AP: Acute pancreatitis; IGF2: Insulin-like growth factor 2; N: Normal; NC: Negative control; WT: Wild-type; MUT: Mutant; UTR: Untranslated regions; CDS: Coding sequence.

Techniques Used: Expressing, Binding Assay, Mutagenesis, Sequencing, Luciferase, Activity Assay, Transfection, Negative Control, Control, Western Blot

miR125b-5p promotes the aggravation of inflammation in vivo . A: Intraperitoneal condition of rats in acute pancreatitis (AP) group and overexpression group; B: Ascites volume of rats in the three groups. The expression of miR-125b-5p in pancreatic tissues of sham group, AP group and overexpression group was detected by quantitative real-time polymerase chain reaction; C: The level of interleukin-6, tumor necrosis factor-alpha, C-reactive protein and reactive oxygen species was determined by enzyme linked immunosorbent assay; D: Histopathological image of rat pancreas in three groups; E: Pancreatic histopathology score and dry/wet ratio; F: Western blot showing the expression of BAX, Bcl-2, HMGB-1 and insulin-like growth factor 2 in the pancreatic tissues; G: Western blot showing the expression of p-PI3K, PI3K, p-AKT and AKT in the pancreatic tissues, a P < 0.05, b P < 0.005, c P < 0.0005. IGF2: Insulin-like growth factor 2; TNF-α: Tumor necrosis factor-alpha; AP: Acute pancreatitis; IL: Interleukin; TNF-α: Tumor necrosis factor-alpha; CRP: C-reactive protein; ROS: Reactive oxygen species; NC: Negative control.

Figure Legend Snippet: miR125b-5p promotes the aggravation of inflammation in vivo . A: Intraperitoneal condition of rats in acute pancreatitis (AP) group and overexpression group; B: Ascites volume of rats in the three groups. The expression of miR-125b-5p in pancreatic tissues of sham group, AP group and overexpression group was detected by quantitative real-time polymerase chain reaction; C: The level of interleukin-6, tumor necrosis factor-alpha, C-reactive protein and reactive oxygen species was determined by enzyme linked immunosorbent assay; D: Histopathological image of rat pancreas in three groups; E: Pancreatic histopathology score and dry/wet ratio; F: Western blot showing the expression of BAX, Bcl-2, HMGB-1 and insulin-like growth factor 2 in the pancreatic tissues; G: Western blot showing the expression of p-PI3K, PI3K, p-AKT and AKT in the pancreatic tissues, a P < 0.05, b P < 0.005, c P < 0.0005. IGF2: Insulin-like growth factor 2; TNF-α: Tumor necrosis factor-alpha; AP: Acute pancreatitis; IL: Interleukin; TNF-α: Tumor necrosis factor-alpha; CRP: C-reactive protein; ROS: Reactive oxygen species; NC: Negative control.

Techniques Used: In Vivo, Over Expression, Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Histopathology, Western Blot, Negative Control

Membrane:

Article Title: Acinous cell AR42J-derived exosome miR125b-5p promotes acute pancreatitis exacerbation by inhibiting M2 macrophage polarization via PI3K/AKT signaling pathway
Article Snippet: The membranes were incubated for 1 h with 5% non-fat milk solution (BD Biosciences, United States) dissolved in TBST. .. After adding primary antibodies against CD9 (Abcam, Cambridge, United Kingdom), CD81 (Abcam, Cambridge, United Kingdom), CD63 (Abcam, Cambridge, United Kingdom), TSG101 (Protein Tech Group, IL, United States), CD206 (Protein Tech Group, IL, United States), inducible nitric oxide synthase (iNOS) (Abcam, Cambridge, United Kingdom), insulin-like growth factor 2 (IGF2) (Protein Tech Group, IL, United States), Bax (Protein Tech Group, IL, United States), Bcl-2 (Protein Tech Group, IL, United States), HMGB1 (Protein Tech Group, IL, United States), PI3K (Protein Tech Group, IL, United States), p-PI3K (Protein Tech Group, IL, United States), AKT (Protein Tech Group, IL, United States), p-AKT (Protein Tech Group, IL, United States), β-tubulin (Protein Tech Group, IL, United States), β-actin (Protein Tech Group, IL, United States), and GAPDH (Abcam, ab8245, 1:1000), the membrane was incubated at 4 °C overnight. ..

Incubation:

Image Search Results

Journal: Frontiers in Immunology

Article Title: The mammosphere-derived epithelial cell secretome modulates neutrophil functions in the bovine model

doi: 10.3389/fimmu.2024.1367432

Figure Lengend Snippet: Antibodies used for immunocytochemistry assays, fluorescent bead-based multiplex assays and Western blots.

Article Snippet: Treatments consisting of either RPMI + 2% FBS (control), MDEC CM, 20 ng/ml bovine granulocyte-macrophage colony-stimulating factor (GM CSF) or 50 ng/ml bovine insulin-like growth factor 2 (IGF2) (both from Kingfisher Biotech, and diluted in RPMI + 2% FBS), were added, and cells were incubated for 1 h at 37°C.

Techniques: Immunocytochemistry, Multiplex Assay, Western Blot, Control

Primers used for reverse transcriptase (RT)-PCR.

Journal: Frontiers in Immunology

Article Title: The mammosphere-derived epithelial cell secretome modulates neutrophil functions in the bovine model

doi: 10.3389/fimmu.2024.1367432

Figure Lengend Snippet: Primers used for reverse transcriptase (RT)-PCR.

Techniques: Reverse Transcription

Proteins in bovine mammosphere-derived epithelial cell (MDEC) conditioned medium (CM) affecting neutrophil functions, as detected by liquid chromatography-mass spectrometry.

Journal: Frontiers in Immunology

Article Title: The mammosphere-derived epithelial cell secretome modulates neutrophil functions in the bovine model

doi: 10.3389/fimmu.2024.1367432

Figure Lengend Snippet: Proteins in bovine mammosphere-derived epithelial cell (MDEC) conditioned medium (CM) affecting neutrophil functions, as detected by liquid chromatography-mass spectrometry.

Techniques: Chromatography

Reducing Chemokine (C-X-C motif) ligand 6 (CXCL6) expression in bovine mammosphere-derived epithelial cells (MDECs) changes the effect of conditioned medium (CM) on neutrophil chemotaxis, while reducing insulin-like growth factor 2 (IGF2) expression in MDECs does not change the effect of conditioned medium (CM) on neutrophil phagocytosis. (A) . Recombinant bovine CXCL6 was added to bovine neutrophils to determine if chemotaxis changed relative to RPMI control medium (dotted line). Cells treated with MDEC CM were included as a control. (B) . CXCL6 siRNA was used to knock down CXCL6 in MDEC CM. Chemotaxis was assessed in neutrophils treated with RPMI control medium, MDEC CM, MDEC CM from cells transfected with CXCL6 siRNA, and MDEC CM from cells transfected with a non-specific (negative) siRNA. (C) . Recombinant bovine IGF2 was added to bovine neutrophils to determine if phagocytosis changed relative to the RPMI control (dotted line). Cells treated with MDEC CM were included as a control. (D) . IGF2 siRNA was used to knock down IGF2 in MDEC CM. Phagocytosis was assessed in neutrophils treated with RPMI control medium, MDEC CM, MDEC CM from cells transfected with IGF2 siRNA and MDEC CM from cells that received a non-specific (negative) siRNA. Each data point on graphs represents the results from one experiment, assessing the effects of CM from one MDEC line on 3 neutrophil preparations. Gray dotted lines on graphs indicate the control conditions expressed as 100%. n = 3 experiments.

Journal: Frontiers in Immunology

Article Title: The mammosphere-derived epithelial cell secretome modulates neutrophil functions in the bovine model

doi: 10.3389/fimmu.2024.1367432

Figure Lengend Snippet: Reducing Chemokine (C-X-C motif) ligand 6 (CXCL6) expression in bovine mammosphere-derived epithelial cells (MDECs) changes the effect of conditioned medium (CM) on neutrophil chemotaxis, while reducing insulin-like growth factor 2 (IGF2) expression in MDECs does not change the effect of conditioned medium (CM) on neutrophil phagocytosis. (A) . Recombinant bovine CXCL6 was added to bovine neutrophils to determine if chemotaxis changed relative to RPMI control medium (dotted line). Cells treated with MDEC CM were included as a control. (B) . CXCL6 siRNA was used to knock down CXCL6 in MDEC CM. Chemotaxis was assessed in neutrophils treated with RPMI control medium, MDEC CM, MDEC CM from cells transfected with CXCL6 siRNA, and MDEC CM from cells transfected with a non-specific (negative) siRNA. (C) . Recombinant bovine IGF2 was added to bovine neutrophils to determine if phagocytosis changed relative to the RPMI control (dotted line). Cells treated with MDEC CM were included as a control. (D) . IGF2 siRNA was used to knock down IGF2 in MDEC CM. Phagocytosis was assessed in neutrophils treated with RPMI control medium, MDEC CM, MDEC CM from cells transfected with IGF2 siRNA and MDEC CM from cells that received a non-specific (negative) siRNA. Each data point on graphs represents the results from one experiment, assessing the effects of CM from one MDEC line on 3 neutrophil preparations. Gray dotted lines on graphs indicate the control conditions expressed as 100%. n = 3 experiments.

Techniques: Expressing, Derivative Assay, Chemotaxis Assay, Recombinant, Control, Knockdown, Transfection

Journal: Frontiers in Immunology

Article Title: The mammosphere-derived epithelial cell secretome modulates neutrophil functions in the bovine model

doi: 10.3389/fimmu.2024.1367432

Article Snippet: Custom Silencer Select siRNA targeting bovine CXC motif chemokine ligand 6 (CXCL6) , insulin like growth factor 2 (IGF2) , superoxide dismutase 1 (SOD) , peroxiredoxin 2 (PII) and catalase (CAT) were designed by Thermo Fisher.

Techniques: Chromatography

Journal: Frontiers in Immunology

Article Title: The mammosphere-derived epithelial cell secretome modulates neutrophil functions in the bovine model

doi: 10.3389/fimmu.2024.1367432

Techniques: Expressing, Derivative Assay, Chemotaxis Assay, Recombinant, Control, Knockdown, Transfection

Journal: World Journal of Gastrointestinal Surgery

Article Title: Acinous cell AR42J-derived exosome miR125b-5p promotes acute pancreatitis exacerbation by inhibiting M2 macrophage polarization via PI3K/AKT signaling pathway

doi: 10.4240/wjgs.v15.i4.600

Figure Lengend Snippet: Expression of miR-125b-5p and insulin-like growth factor 2 in vitro and in vivo . A: Quantitative real-time polymerase chain reaction (RT-qPCR) was used to detect the expression of miR-125b-5p in AR42J cell lines in activated and inactive state; B: RT-qPCR was used to detect the expression of insulin-like growth factor 2 (IGF2) in AR42J cell lines under activated and inactive state; C: Western blot was used to detect the expression level of IGF2, and β-actin was used as the internal reference; D: RT-qPCR was used to detect the expression of miR-125b-5p in normal pancreatic tissues and pancreatitis tissues; E: RT-qPCR was used to detect the expression of IGF2 in normal pancreatic tissues and pancreatitis tissues; F: The mRNA and protein expression of miR-125b-5p and IGF2 in 4 cases of pancreatitis tissues were detected by RT-qPCR and Western blot. Among them, sample 2 was used as the standard to calculate the fold change of miR-125b-5p expression in other samples by comparing the miR-125b-5p/U6 ratio in sample 2. Western blot was used to detect the expression level of IGF2, and β-actin was used as the internal reference. The experiment was repeated three times and is expressed as mean ± SD; G: miR-125b-5p was negatively correlated with IGF2 protein expression level, a P < 0.05, b P < 0.005. AP: Acute pancreatitis; IGF2: Insulin-like growth factor 2; N: Normal.

Article Snippet: After adding primary antibodies against CD9 (Abcam, Cambridge, United Kingdom), CD81 (Abcam, Cambridge, United Kingdom), CD63 (Abcam, Cambridge, United Kingdom), TSG101 (Protein Tech Group, IL, United States), CD206 (Protein Tech Group, IL, United States), inducible nitric oxide synthase (iNOS) (Abcam, Cambridge, United Kingdom), insulin-like growth factor 2 (IGF2) (Protein Tech Group, IL, United States), Bax (Protein Tech Group, IL, United States), Bcl-2 (Protein Tech Group, IL, United States), HMGB1 (Protein Tech Group, IL, United States), PI3K (Protein Tech Group, IL, United States), p-PI3K (Protein Tech Group, IL, United States), AKT (Protein Tech Group, IL, United States), p-AKT (Protein Tech Group, IL, United States), β-tubulin (Protein Tech Group, IL, United States), β-actin (Protein Tech Group, IL, United States), and GAPDH (Abcam, ab8245, 1:1000), the membrane was incubated at 4 °C overnight.

Techniques: Expressing, In Vitro, In Vivo, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot

Journal: World Journal of Gastrointestinal Surgery

Article Title: Acinous cell AR42J-derived exosome miR125b-5p promotes acute pancreatitis exacerbation by inhibiting M2 macrophage polarization via PI3K/AKT signaling pathway

doi: 10.4240/wjgs.v15.i4.600

Figure Lengend Snippet: miR-125b-5p overexpression could promote necrosis and apoptosis of AR42J cells in the activated state. A: miR-125b-5p overexpression promoted the necrosis of AR42J cells in the activated state; B: Flow cytometry analysis confirmed that the percentage of cells in G0/G1 phase increased in AR42J cells in the activated state treated with the miR-125b-5p overexpression group; C: The percentage of apoptotic cells increased as confirmed by flow cytometry; D and E: The expression of insulin-like growth factor 2, BAX, Bcl-2 and HMGB-1 was confirmed by Western blot, a P < 0.05, c P < 0.0005. AP: Acute pancreatitis; IGF2: Insulin-like growth factor 2; N: Normal; NC: Negative control.

Techniques: Over Expression, Flow Cytometry, Expressing, Western Blot, Negative Control

Journal: World Journal of Gastrointestinal Surgery

Article Title: Acinous cell AR42J-derived exosome miR125b-5p promotes acute pancreatitis exacerbation by inhibiting M2 macrophage polarization via PI3K/AKT signaling pathway

doi: 10.4240/wjgs.v15.i4.600

Figure Lengend Snippet: miR-125b-5p promotes acute pancreatitis exacerbation by inhibiting insulin-like growth factor 2 protein expression in PI3K/AKT signaling pathway. A: The potential binding sites of miR-125b-5p in the 3¢-untranslated regions (UTR) of wild-type insulin-like growth factor 2 (IGF2) (red part). The blue section is the mutation site in mutant IGF2 3¢-UTR sequence; B: Luciferase activity in IGF2 3¢-UTR of wild-type and mutant was detected after transfection of miR-125b-5p mimics and negative control miRNAs. The normalized luciferase activity of the transfected control miRNA was set to a relative luciferase activity of 1; C: The protein expression of PI3K/AKT signaling pathway was confirmed by western blot. a P < 0.05, b P < 0.005. AP: Acute pancreatitis; IGF2: Insulin-like growth factor 2; N: Normal; NC: Negative control; WT: Wild-type; MUT: Mutant; UTR: Untranslated regions; CDS: Coding sequence.

Techniques: Expressing, Binding Assay, Mutagenesis, Sequencing, Luciferase, Activity Assay, Transfection, Negative Control, Control, Western Blot

Journal: World Journal of Gastrointestinal Surgery

Article Title: Acinous cell AR42J-derived exosome miR125b-5p promotes acute pancreatitis exacerbation by inhibiting M2 macrophage polarization via PI3K/AKT signaling pathway

doi: 10.4240/wjgs.v15.i4.600

Figure Lengend Snippet: miR125b-5p promotes the aggravation of inflammation in vivo . A: Intraperitoneal condition of rats in acute pancreatitis (AP) group and overexpression group; B: Ascites volume of rats in the three groups. The expression of miR-125b-5p in pancreatic tissues of sham group, AP group and overexpression group was detected by quantitative real-time polymerase chain reaction; C: The level of interleukin-6, tumor necrosis factor-alpha, C-reactive protein and reactive oxygen species was determined by enzyme linked immunosorbent assay; D: Histopathological image of rat pancreas in three groups; E: Pancreatic histopathology score and dry/wet ratio; F: Western blot showing the expression of BAX, Bcl-2, HMGB-1 and insulin-like growth factor 2 in the pancreatic tissues; G: Western blot showing the expression of p-PI3K, PI3K, p-AKT and AKT in the pancreatic tissues, a P < 0.05, b P < 0.005, c P < 0.0005. IGF2: Insulin-like growth factor 2; TNF-α: Tumor necrosis factor-alpha; AP: Acute pancreatitis; IL: Interleukin; TNF-α: Tumor necrosis factor-alpha; CRP: C-reactive protein; ROS: Reactive oxygen species; NC: Negative control.

Techniques: In Vivo, Over Expression, Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Histopathology, Western Blot, Negative Control

In vitro characterization of GAA and IGF2.GAA proteins used in HSPC-LVGT (A) pRRL third-generation lentiviral vectors encoding either codon-optimized human GAA (GAAco) or IGF2-tagged GAA (the components of IGF2.GAAco are shown; SPS, signal peptide sequence of IGF2; AA, amino acid) under the spleen focus-forming virus (SFFV) promoter. (B–F) Enzyme activity in cells (B) and media (C) after uptake of IGF2.GAA, GAA, or rhGAA in Gaa −/− mouse-derived myotubes. Dashed line in (B) represents the input concentration used for the experiments in (B) and (C). (D and E) Immunoblot analysis of the experiment shown in (B) and (C) using an antibody to GAA. (D) Media before (Pre) and after (Post) uptake. (F) Gaa −/− mouse-derived myotubes after uptake stained for myosin heavy chain (MHC) in red and nuclei (using Hoechst) in blue. Fusion index is indicated at the bottom left corner. Representative images are shown. Scale bar, 100 μm. (G and H) GAA, IGF2.GAA, or rhGAA uptake in Gaa −/− mouse-derived myotubes in the presence of increasing concentrations of mannose-6-phosphate (M6P) (G) or IGF2 (H). Data are expressed as percentage of activity in the input medium. Data represent means ± SEM and are analyzed by one-way ANOVA followed by Bonferroni’s multiple testing correction. n = 3 biological replicates/condition. ∗∗∗p ≤ 0.001; ns, not significant. Comparisons are indicated by brackets.

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: IGF2-tagging of GAA promotes full correction of murine Pompe disease at a clinically relevant dosage of lentiviral gene therapy

doi: 10.1016/j.omtm.2022.09.010

Figure Lengend Snippet: In vitro characterization of GAA and IGF2.GAA proteins used in HSPC-LVGT (A) pRRL third-generation lentiviral vectors encoding either codon-optimized human GAA (GAAco) or IGF2-tagged GAA (the components of IGF2.GAAco are shown; SPS, signal peptide sequence of IGF2; AA, amino acid) under the spleen focus-forming virus (SFFV) promoter. (B–F) Enzyme activity in cells (B) and media (C) after uptake of IGF2.GAA, GAA, or rhGAA in Gaa −/− mouse-derived myotubes. Dashed line in (B) represents the input concentration used for the experiments in (B) and (C). (D and E) Immunoblot analysis of the experiment shown in (B) and (C) using an antibody to GAA. (D) Media before (Pre) and after (Post) uptake. (F) Gaa −/− mouse-derived myotubes after uptake stained for myosin heavy chain (MHC) in red and nuclei (using Hoechst) in blue. Fusion index is indicated at the bottom left corner. Representative images are shown. Scale bar, 100 μm. (G and H) GAA, IGF2.GAA, or rhGAA uptake in Gaa −/− mouse-derived myotubes in the presence of increasing concentrations of mannose-6-phosphate (M6P) (G) or IGF2 (H). Data are expressed as percentage of activity in the input medium. Data represent means ± SEM and are analyzed by one-way ANOVA followed by Bonferroni’s multiple testing correction. n = 3 biological replicates/condition. ∗∗∗p ≤ 0.001; ns, not significant. Comparisons are indicated by brackets.

Article Snippet: A codon-optimized insulin-like growth factor 2 (IGF2) cassette (GenScript, Piscataway, NJ) was subcloned into the LV- GAAco backbone after double digestion using BamHI and SgrAI.

Techniques: In Vitro, Sequencing, Activity Assay, Derivative Assay, Concentration Assay, Western Blot, Staining

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: IGF2-tagging of GAA promotes full correction of murine Pompe disease at a clinically relevant dosage of lentiviral gene therapy

doi: 10.1016/j.omtm.2022.09.010

Figure Lengend Snippet: Layout of different treatment groups

Article Snippet: A codon-optimized insulin-like growth factor 2 (IGF2) cassette (GenScript, Piscataway, NJ) was subcloned into the LV- GAAco backbone after double digestion using BamHI and SgrAI.

Techniques: Irradiation

VCN and chimerism in bone marrow after gene therapy (A) Chimerism, expressed as the percentage of reconstituted male donor cells in bone marrow of female recipients treated with gene therapy, determined by qPCR on Sry and Gapdh loci. (B) VCN measured in bone marrow by qPCR on HIV and Gapdh loci. VCN is not normalized for chimerism. MOI, multiplicity of infection; Gy, gray. Data represent means ± SEM and are analyzed by two-way ANOVA followed by Bonferroni’s multiple testing correction, using vector (LV -GAAco or LV- IGF2.GAAco ) and gene therapy dose as categorical variables. Significant results are indicated by brackets. Results of multiple comparison analysis are reported in <xref ref-type=

Table S3 . LV- GAAco and LV- IGF2.GAAco , n = 7 per group; KO, n = 5; WT, n = 5. ns, not significant; ∗p ≤ 0.05, ∗∗∗∗p ≤ 0.0001. " width="100%" height="100%">

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: IGF2-tagging of GAA promotes full correction of murine Pompe disease at a clinically relevant dosage of lentiviral gene therapy

doi: 10.1016/j.omtm.2022.09.010

Figure Lengend Snippet: VCN and chimerism in bone marrow after gene therapy (A) Chimerism, expressed as the percentage of reconstituted male donor cells in bone marrow of female recipients treated with gene therapy, determined by qPCR on Sry and Gapdh loci. (B) VCN measured in bone marrow by qPCR on HIV and Gapdh loci. VCN is not normalized for chimerism. MOI, multiplicity of infection; Gy, gray. Data represent means ± SEM and are analyzed by two-way ANOVA followed by Bonferroni’s multiple testing correction, using vector (LV -GAAco or LV- IGF2.GAAco ) and gene therapy dose as categorical variables. Significant results are indicated by brackets. Results of multiple comparison analysis are reported in Table S3 . LV- GAAco and LV- IGF2.GAAco , n = 7 per group; KO, n = 5; WT, n = 5. ns, not significant; ∗p ≤ 0.05, ∗∗∗∗p ≤ 0.0001.

Article Snippet: A codon-optimized insulin-like growth factor 2 (IGF2) cassette (GenScript, Piscataway, NJ) was subcloned into the LV- GAAco backbone after double digestion using BamHI and SgrAI.

Techniques: Infection, Plasmid Preparation

Tissue GAA activity 6 months after gene therapy

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: IGF2-tagging of GAA promotes full correction of murine Pompe disease at a clinically relevant dosage of lentiviral gene therapy

doi: 10.1016/j.omtm.2022.09.010

Figure Lengend Snippet: Tissue GAA activity 6 months after gene therapy

Article Snippet: A codon-optimized insulin-like growth factor 2 (IGF2) cassette (GenScript, Piscataway, NJ) was subcloned into the LV- GAAco backbone after double digestion using BamHI and SgrAI.

Techniques: Activity Assay

Gene therapy with LV- IGF2.GAAco results in correction in skeletal muscles (A–D) Total glycogen content in skeletal muscles after different doses of gene therapy with LV- IGF2.GAAco or LV- GAAco . MOI, multiplicity of infection; Gy, gray. (E) Correlation between bone marrow VCN and glycogen clearance in tibialis anterior. λ, exponential decay constant. VCN is not normalized for chimerism. (F–I) Immunoblot analysis in biological triplicates of tibialis anterior of mice treated with a high (MOI 7, 9 Gy, 10 6 cells) or low dose (MOI 7, 6 Gy, 3 × 10 cells) of gene therapy using antibodies against beclin 1, p62, and LC3. Density levels of beclin 1 (G), p62 (H), or LC3 (I) are quantified from (F) (loading controls are shown in <xref ref-type=

Figure S5 A). In (G)–(I), values are relative to WT. (J and K) Latency on a rotarod after gene therapy with LV- GAAco or (J) LV- IGF2.GAAco (K). (L and M) Representative images of PAS (upper row) and AP (lower row) stainings in tibialis anterior (L) and diaphragm (M) after high-dose gene therapy. Scale bar, 50 μm. Data are presented as means ± SEM. In (A)–(D), data are analyzed by two-way ANOVA with Bonferroni’s correction, using vector (LV -GAAco or LV- IGF2.GAAco ) and gene therapy dose as categorical variables. Significant results are indicated by brackets. Results of multiple comparison analysis are reported in Table S3 . In (G)–(K), data are analyzed by one-way ANOVA followed by Bonferroni’s multiple testing. Significance is expressed as relative to WT; other significant comparisons are indicated by brackets. (A–D; J, K) LV -GAAco and LV- IGF2.GAAco , n = 7; KO, n = 5; WT, n = 5. (G–I) n = 3 for all groups. (L and M) LV -GAAco and LV- IGF2.GAAco , n = 3 per group; KO, n = 2; WT, n = 2. ns, not significant; ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0 .001, ∗∗∗∗p ≤ 0 .0001. " width="100%" height="100%">

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: IGF2-tagging of GAA promotes full correction of murine Pompe disease at a clinically relevant dosage of lentiviral gene therapy

doi: 10.1016/j.omtm.2022.09.010

Figure Lengend Snippet: Gene therapy with LV- IGF2.GAAco results in correction in skeletal muscles (A–D) Total glycogen content in skeletal muscles after different doses of gene therapy with LV- IGF2.GAAco or LV- GAAco . MOI, multiplicity of infection; Gy, gray. (E) Correlation between bone marrow VCN and glycogen clearance in tibialis anterior. λ, exponential decay constant. VCN is not normalized for chimerism. (F–I) Immunoblot analysis in biological triplicates of tibialis anterior of mice treated with a high (MOI 7, 9 Gy, 10 6 cells) or low dose (MOI 7, 6 Gy, 3 × 10 cells) of gene therapy using antibodies against beclin 1, p62, and LC3. Density levels of beclin 1 (G), p62 (H), or LC3 (I) are quantified from (F) (loading controls are shown in Figure S5 A). In (G)–(I), values are relative to WT. (J and K) Latency on a rotarod after gene therapy with LV- GAAco or (J) LV- IGF2.GAAco (K). (L and M) Representative images of PAS (upper row) and AP (lower row) stainings in tibialis anterior (L) and diaphragm (M) after high-dose gene therapy. Scale bar, 50 μm. Data are presented as means ± SEM. In (A)–(D), data are analyzed by two-way ANOVA with Bonferroni’s correction, using vector (LV -GAAco or LV- IGF2.GAAco ) and gene therapy dose as categorical variables. Significant results are indicated by brackets. Results of multiple comparison analysis are reported in Table S3 . In (G)–(K), data are analyzed by one-way ANOVA followed by Bonferroni’s multiple testing. Significance is expressed as relative to WT; other significant comparisons are indicated by brackets. (A–D; J, K) LV -GAAco and LV- IGF2.GAAco , n = 7; KO, n = 5; WT, n = 5. (G–I) n = 3 for all groups. (L and M) LV -GAAco and LV- IGF2.GAAco , n = 3 per group; KO, n = 2; WT, n = 2. ns, not significant; ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0 .001, ∗∗∗∗p ≤ 0 .0001.

Article Snippet: A codon-optimized insulin-like growth factor 2 (IGF2) cassette (GenScript, Piscataway, NJ) was subcloned into the LV- GAAco backbone after double digestion using BamHI and SgrAI.

Techniques: Infection, Western Blot, Plasmid Preparation

Gene therapy results in correction in heart (A) Total glycogen content in cardiac muscle after different doses of gene therapy. MOI, multiplicity of infection; Gy, gray. (B) VCN in bone marrow versus glycogen content after gene therapy. λ, exponential decay constant. VCN is not normalized for chimerism. (C–F) Immunoblot analysis of heart lysate with antibody to beclin 1, p62, and LC3 after high-dose or low-dose gene therapy (C). Density levels of beclin 1 (D), p62 (E), and LC3 (F) were quantified from C (total protein control is shown in <xref ref-type=

Figure S3 B). In (D)–(F), quantification density values are relative to WT value (set as 1). (G) Heart wet weight after gene therapy. (H) PAS (upper row) and AP (lower row) stainings in cardiac tissue from mice treated with high-dose gene therapy. Scale bar, 50 μm. Data are presented as means ± SEM. In (A) and (G), data are analyzed by two-way ANOVA with Bonferroni’s correction, using vector (LV -GAAco or LV- IGF2.GAAco ) and gene therapy dose as categorical variables. Significant results are indicated by brackets. Results of multiple comparison analysis are reported in Table S3 . Western blot quantifications (D–F) are analyzed by one-way ANOVA followed by Bonferroni’s multiple testing. Significance is expressed as relative to WT levels; other significant comparisons are indicated by brackets. (A and G) LV- GAAco and LV- IGF2.GAAco , n = 7; KO, n = 5; and WT, n = 5. (D-F) n = 3. In (H), LV -GAAco and LV- IGF2.GAAco , n = 3 per group; KO, n = 2; and WT, n = 2. ns, not significant; ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0 .001, ∗∗∗∗p ≤ 0 .0001. " width="100%" height="100%">

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: IGF2-tagging of GAA promotes full correction of murine Pompe disease at a clinically relevant dosage of lentiviral gene therapy

doi: 10.1016/j.omtm.2022.09.010

Figure Lengend Snippet: Gene therapy results in correction in heart (A) Total glycogen content in cardiac muscle after different doses of gene therapy. MOI, multiplicity of infection; Gy, gray. (B) VCN in bone marrow versus glycogen content after gene therapy. λ, exponential decay constant. VCN is not normalized for chimerism. (C–F) Immunoblot analysis of heart lysate with antibody to beclin 1, p62, and LC3 after high-dose or low-dose gene therapy (C). Density levels of beclin 1 (D), p62 (E), and LC3 (F) were quantified from C (total protein control is shown in Figure S3 B). In (D)–(F), quantification density values are relative to WT value (set as 1). (G) Heart wet weight after gene therapy. (H) PAS (upper row) and AP (lower row) stainings in cardiac tissue from mice treated with high-dose gene therapy. Scale bar, 50 μm. Data are presented as means ± SEM. In (A) and (G), data are analyzed by two-way ANOVA with Bonferroni’s correction, using vector (LV -GAAco or LV- IGF2.GAAco ) and gene therapy dose as categorical variables. Significant results are indicated by brackets. Results of multiple comparison analysis are reported in Table S3 . Western blot quantifications (D–F) are analyzed by one-way ANOVA followed by Bonferroni’s multiple testing. Significance is expressed as relative to WT levels; other significant comparisons are indicated by brackets. (A and G) LV- GAAco and LV- IGF2.GAAco , n = 7; KO, n = 5; and WT, n = 5. (D-F) n = 3. In (H), LV -GAAco and LV- IGF2.GAAco , n = 3 per group; KO, n = 2; and WT, n = 2. ns, not significant; ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0 .001, ∗∗∗∗p ≤ 0 .0001.

Article Snippet: A codon-optimized insulin-like growth factor 2 (IGF2) cassette (GenScript, Piscataway, NJ) was subcloned into the LV- GAAco backbone after double digestion using BamHI and SgrAI.

Techniques: Infection, Western Blot, Plasmid Preparation

Gene therapy with LV- IGF2.GAAco results in correction in brain (A and B) Glycogen content in control and gene-therapy-treated mice in cerebrum and cerebellum. MOI, multiplicity of infection; Gy, gray. (C) Correlation between VCN and glycogen clearance in cerebrum. λ, exponential decay constant. VCN is not normalized for chimerism. (D–G) Immunoblot analysis for beclin 1, p62, and LC3 in cerebrum after gene therapy. Age-matched WT and Gaa −/− animals were taken as control. Density levels of beclin 1 (E), p62 (F), or LC3 (G) were quantified from (D) (total protein control is shown in <xref ref-type=

Figure S5 C). (E–G) Quantification density values are relative to WT level (set as 1). (H and I) PAS staining analysis in cerebrum and cerebellum after high-dose gene therapy. An overview of the stained area is presented in (H). Scale bar, 0.5 mm (hippocampus) or 1 mm (cerebellum). Representative images at indicated areas are shown in (I). Scale bar, 50 μm. Black arrows point to pyramidal neurons (in hippocampus CA1) and Purkinje cells (in cerebellum). Black arrowheads refer to the glial cells. DG, dentate gyrus; KO, untreated Gaa −/− mice; WT, untreated WT mice. Data are presented as means ± SEM. (A and B) Data are analyzed by two-way ANOVA with Bonferroni’s correction, using vector (LV -GAAco or LV- IGF2.GAAco ) and gene therapy dose as nominal predictor variables. Significant results are indicated by brackets. Results of multiple comparison analysis are reported in Table S3 . Western blot quantifications (E–G) are analyzed by one-way ANOVA followed by Bonferroni’s multiple testing. Significance versus WT is shown; other significant comparisons are indicated by brackets. LV- GAAco and LV- IGF2.GAAco , n = 7; KO, n = 5; and WT, n = 5. (E–G) n = 3. (H and I) LV -GAAco and LV- IGF2.GAAco , n = 3 per group; KO, n = 2; and WT, n = 2. ns, not significant; ∗p ≤ 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p ≤ 0 .0001. " width="100%" height="100%">

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: IGF2-tagging of GAA promotes full correction of murine Pompe disease at a clinically relevant dosage of lentiviral gene therapy

doi: 10.1016/j.omtm.2022.09.010

Figure Lengend Snippet: Gene therapy with LV- IGF2.GAAco results in correction in brain (A and B) Glycogen content in control and gene-therapy-treated mice in cerebrum and cerebellum. MOI, multiplicity of infection; Gy, gray. (C) Correlation between VCN and glycogen clearance in cerebrum. λ, exponential decay constant. VCN is not normalized for chimerism. (D–G) Immunoblot analysis for beclin 1, p62, and LC3 in cerebrum after gene therapy. Age-matched WT and Gaa −/− animals were taken as control. Density levels of beclin 1 (E), p62 (F), or LC3 (G) were quantified from (D) (total protein control is shown in Figure S5 C). (E–G) Quantification density values are relative to WT level (set as 1). (H and I) PAS staining analysis in cerebrum and cerebellum after high-dose gene therapy. An overview of the stained area is presented in (H). Scale bar, 0.5 mm (hippocampus) or 1 mm (cerebellum). Representative images at indicated areas are shown in (I). Scale bar, 50 μm. Black arrows point to pyramidal neurons (in hippocampus CA1) and Purkinje cells (in cerebellum). Black arrowheads refer to the glial cells. DG, dentate gyrus; KO, untreated Gaa −/− mice; WT, untreated WT mice. Data are presented as means ± SEM. (A and B) Data are analyzed by two-way ANOVA with Bonferroni’s correction, using vector (LV -GAAco or LV- IGF2.GAAco ) and gene therapy dose as nominal predictor variables. Significant results are indicated by brackets. Results of multiple comparison analysis are reported in Table S3 . Western blot quantifications (E–G) are analyzed by one-way ANOVA followed by Bonferroni’s multiple testing. Significance versus WT is shown; other significant comparisons are indicated by brackets. LV- GAAco and LV- IGF2.GAAco , n = 7; KO, n = 5; and WT, n = 5. (E–G) n = 3. (H and I) LV -GAAco and LV- IGF2.GAAco , n = 3 per group; KO, n = 2; and WT, n = 2. ns, not significant; ∗p ≤ 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p ≤ 0 .0001.

Article Snippet: A codon-optimized insulin-like growth factor 2 (IGF2) cassette (GenScript, Piscataway, NJ) was subcloned into the LV- GAAco backbone after double digestion using BamHI and SgrAI.

Techniques: Infection, Western Blot, Staining, Plasmid Preparation

Gene therapy with LV- IGF2.GAAco relieves astrogliosis in brain (A–D) Sagittal sections of hippocampus, corpus callosum, and thalamus after high-dose gene therapy stained for GFAP (red) and LAMP1 (green). An overview of the stained area is presented in (A). Scale bar, 500 μm. Boxed areas in (A) are magnified in (B) (1, hippocampus), (C) (2, corpus callosum), and (D) (3, thalamus). Scale bar, 25 μm.

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: IGF2-tagging of GAA promotes full correction of murine Pompe disease at a clinically relevant dosage of lentiviral gene therapy

doi: 10.1016/j.omtm.2022.09.010

Figure Lengend Snippet: Gene therapy with LV- IGF2.GAAco relieves astrogliosis in brain (A–D) Sagittal sections of hippocampus, corpus callosum, and thalamus after high-dose gene therapy stained for GFAP (red) and LAMP1 (green). An overview of the stained area is presented in (A). Scale bar, 500 μm. Boxed areas in (A) are magnified in (B) (1, hippocampus), (C) (2, corpus callosum), and (D) (3, thalamus). Scale bar, 25 μm.

Article Snippet: A codon-optimized insulin-like growth factor 2 (IGF2) cassette (GenScript, Piscataway, NJ) was subcloned into the LV- GAAco backbone after double digestion using BamHI and SgrAI.

Techniques: Staining

Gene therapy with LV- IGF2.GAAco relieves microgliosis in brain (A–D) Sagittal sections of hippocampus, corpus callosum and thalamus after high-dose gene therapy stained with Iba1 (red) and LAMP1 (green). An overview of the stained area is presented in (A). Scale bar, 500 μm. Boxed areas in (A) are magnified in (B) (1, hippocampus), (C) (2, corpus callosum), and (D) (3, thalamus). Scale bar, 25 μm.

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: IGF2-tagging of GAA promotes full correction of murine Pompe disease at a clinically relevant dosage of lentiviral gene therapy

doi: 10.1016/j.omtm.2022.09.010

Figure Lengend Snippet: Gene therapy with LV- IGF2.GAAco relieves microgliosis in brain (A–D) Sagittal sections of hippocampus, corpus callosum and thalamus after high-dose gene therapy stained with Iba1 (red) and LAMP1 (green). An overview of the stained area is presented in (A). Scale bar, 500 μm. Boxed areas in (A) are magnified in (B) (1, hippocampus), (C) (2, corpus callosum), and (D) (3, thalamus). Scale bar, 25 μm.

Article Snippet: A codon-optimized insulin-like growth factor 2 (IGF2) cassette (GenScript, Piscataway, NJ) was subcloned into the LV- GAAco backbone after double digestion using BamHI and SgrAI.

Techniques: Staining

Glucose levels are not affected by gene therapy with LV- IGF2.GAAco Monthly glucose levels in plasma of mice treated as indicated. Mice were fasted for 15 h before glucose sampling. Data are presented as means ± SEM and analyzed by ANOVA with post hoc Turkey’s analysis. n = 10 per gene-therapy-treated groups; n = 6 per control groups; ns, not significant.

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: IGF2-tagging of GAA promotes full correction of murine Pompe disease at a clinically relevant dosage of lentiviral gene therapy

doi: 10.1016/j.omtm.2022.09.010

Figure Lengend Snippet: Glucose levels are not affected by gene therapy with LV- IGF2.GAAco Monthly glucose levels in plasma of mice treated as indicated. Mice were fasted for 15 h before glucose sampling. Data are presented as means ± SEM and analyzed by ANOVA with post hoc Turkey’s analysis. n = 10 per gene-therapy-treated groups; n = 6 per control groups; ns, not significant.

Article Snippet: A codon-optimized insulin-like growth factor 2 (IGF2) cassette (GenScript, Piscataway, NJ) was subcloned into the LV- GAAco backbone after double digestion using BamHI and SgrAI.

Techniques: Sampling

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