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Cusabio igf2 levels

Figure 4. <t>IGF2</t> was experimentally demonstrated as a direct target of miR-615-5p in pancreatic cancer cells. (a) Putative miR-615-5p-binding sequences in IGF2 3′-UTR and a schematic diagram of the reporter constructs showing the IGF2 3′-UTR sequence (IGF2_WT) and the mutated IGF2 3′-UTR sequence (IGF2_MUT; the mutant nucleotides of the miR-615-5p-binding site are underlined). (b) The IGF2_WT reporter or the IGF2_MUT reporter was co-transfected with the Renilla luciferase reporter into BXPC-3 and SW1990 cells containing either the miR-615-5p mimic or the negative control. Luciferase activity was assayed 48 h after transfection. (c) The expression of IGF2 mRNA was measured by real-time PCR in BXPC-3 and SW1990 cells 48 h after transfection. Beta-actin was used as a housekeeping gene control. (d) IGF2 protein levels in BXPC-3 and SW1990 cell supernatants were detected by ELISA 48 h after transfection. (e) IGF2 protein in BXPC-3 and SW1990 cells was detected by western blot 48 h post transfection. GAPDH was used as a housekeeping protein control. A grayscale analysis of the western blot data is shown.

Igf2 Levels, supplied by Cusabio, used in various techniques. Bioz Stars score: 93/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "miR-615-5p is epigenetically inactivated and functions as a tumor suppressor in pancreatic ductal adenocarcinoma."

Article Title: miR-615-5p is epigenetically inactivated and functions as a tumor suppressor in pancreatic ductal adenocarcinoma.

Journal: Oncogene

doi: 10.1038/onc.2014.101

Figure 4. IGF2 was experimentally demonstrated as a direct target of miR-615-5p in pancreatic cancer cells. (a) Putative miR-615-5p-binding sequences in IGF2 3′-UTR and a schematic diagram of the reporter constructs showing the IGF2 3′-UTR sequence (IGF2_WT) and the mutated IGF2 3′-UTR sequence (IGF2_MUT; the mutant nucleotides of the miR-615-5p-binding site are underlined). (b) The IGF2_WT reporter or the IGF2_MUT reporter was co-transfected with the Renilla luciferase reporter into BXPC-3 and SW1990 cells containing either the miR-615-5p mimic or the negative control. Luciferase activity was assayed 48 h after transfection. (c) The expression of IGF2 mRNA was measured by real-time PCR in BXPC-3 and SW1990 cells 48 h after transfection. Beta-actin was used as a housekeeping gene control. (d) IGF2 protein levels in BXPC-3 and SW1990 cell supernatants were detected by ELISA 48 h after transfection. (e) IGF2 protein in BXPC-3 and SW1990 cells was detected by western blot 48 h post transfection. GAPDH was used as a housekeeping protein control. A grayscale analysis of the western blot data is shown.

Figure Legend Snippet: Figure 4. IGF2 was experimentally demonstrated as a direct target of miR-615-5p in pancreatic cancer cells. (a) Putative miR-615-5p-binding sequences in IGF2 3′-UTR and a schematic diagram of the reporter constructs showing the IGF2 3′-UTR sequence (IGF2_WT) and the mutated IGF2 3′-UTR sequence (IGF2_MUT; the mutant nucleotides of the miR-615-5p-binding site are underlined). (b) The IGF2_WT reporter or the IGF2_MUT reporter was co-transfected with the Renilla luciferase reporter into BXPC-3 and SW1990 cells containing either the miR-615-5p mimic or the negative control. Luciferase activity was assayed 48 h after transfection. (c) The expression of IGF2 mRNA was measured by real-time PCR in BXPC-3 and SW1990 cells 48 h after transfection. Beta-actin was used as a housekeeping gene control. (d) IGF2 protein levels in BXPC-3 and SW1990 cell supernatants were detected by ELISA 48 h after transfection. (e) IGF2 protein in BXPC-3 and SW1990 cells was detected by western blot 48 h post transfection. GAPDH was used as a housekeeping protein control. A grayscale analysis of the western blot data is shown.

Techniques Used: Binding Assay, Construct, Sequencing, Mutagenesis, Transfection, Luciferase, Negative Control, Activity Assay, Expressing, Real-time Polymerase Chain Reaction, Control, Enzyme-linked Immunosorbent Assay, Western Blot

Figure 5. miR-615-5p suppressed pancreatic cancer cell proliferation by directly targeting IGF2. (a) Immunoﬂuorescence. BXPC-3 and SW1990 cells were ﬁxed and incubated with primary antibodies for IGF2 and then Alexa Fluor 488-conjugated anti-rabbit secondary antibodies. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). The ﬂuorescence level of IGF2 was notably decreased in miR-615-5p-transfected cells. Magniﬁcation: × 400. (b) BXPC-3 and SW1990 cell proliferation was detected by the MTT assay at 0, 24, 48 and 72 h after co-transfection with different combinations of NC or MIMIC with GFP or IGF2. For NC+GFP vs MIMIC+GFP: *Po0.05; **Po0.01. For NC+GFP vs NC+IGF2: #Po0.05; ##Po0.01. For NC+IGF2 vs MIMIC+IGF2: △△Po0.01. (c) The IGF2 protein was detected by western blot at 48 h post-transfection. GAPDH was used as a housekeeping protein control. A grayscale analysis of the western blot data is shown.

Figure Legend Snippet: Figure 5. miR-615-5p suppressed pancreatic cancer cell proliferation by directly targeting IGF2. (a) Immunoﬂuorescence. BXPC-3 and SW1990 cells were ﬁxed and incubated with primary antibodies for IGF2 and then Alexa Fluor 488-conjugated anti-rabbit secondary antibodies. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). The ﬂuorescence level of IGF2 was notably decreased in miR-615-5p-transfected cells. Magniﬁcation: × 400. (b) BXPC-3 and SW1990 cell proliferation was detected by the MTT assay at 0, 24, 48 and 72 h after co-transfection with different combinations of NC or MIMIC with GFP or IGF2. For NC+GFP vs MIMIC+GFP: *Po0.05; **Po0.01. For NC+GFP vs NC+IGF2: #Po0.05; ##Po0.01. For NC+IGF2 vs MIMIC+IGF2: △△Po0.01. (c) The IGF2 protein was detected by western blot at 48 h post-transfection. GAPDH was used as a housekeeping protein control. A grayscale analysis of the western blot data is shown.

Techniques Used: Incubation, Staining, Transfection, MTT Assay, Cotransfection, Western Blot, Control

Figure 6. Imprinting status and expression levels of IGF2. (a) Gel photo of ApaI-digested PCR products for IGF2 genotyping. The heterozygous samples had two bands, which were considered as informative. (b) Gel photo of ApaI-digested RT–PCR products for imprinting status analysis. The ratios between the two bands are listed. The samples with a ratio of o3:1 or >1:3 had LOI of IGF2. (c, e) Results of qRT–PCR analysis of IGF2 mRNA expression in the 32 pairs of tissues shown on a column chart and a scattergram. (d, f) qRT–PCR results of miR-615-5p expression levels in the 32 pairs of tissues shown on a column chart and a scattergram. (g) The inverse correlation between IGF2 and miR-615-5p expression levels was examined by Pearson’s correlation analysis (R = −0.456, P = 0.033).

Figure Legend Snippet: Figure 6. Imprinting status and expression levels of IGF2. (a) Gel photo of ApaI-digested PCR products for IGF2 genotyping. The heterozygous samples had two bands, which were considered as informative. (b) Gel photo of ApaI-digested RT–PCR products for imprinting status analysis. The ratios between the two bands are listed. The samples with a ratio of o3:1 or >1:3 had LOI of IGF2. (c, e) Results of qRT–PCR analysis of IGF2 mRNA expression in the 32 pairs of tissues shown on a column chart and a scattergram. (d, f) qRT–PCR results of miR-615-5p expression levels in the 32 pairs of tissues shown on a column chart and a scattergram. (g) The inverse correlation between IGF2 and miR-615-5p expression levels was examined by Pearson’s correlation analysis (R = −0.456, P = 0.033).

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR

Figure 7. The overexpression of miR-615-5p inhibited tumorigenicity in vivo. (a) The relative expression of miR-615-5p in the SW1990-vector group and the SW1990-mir-615 group was analyzed by qRT–PCR. (b, c) Photographs of tumors derived from the SW1990-vector group and the SW1990-mir-615 group of nude mice. (d, e) The graph is representative of tumor growth 25 days after inoculation. Tumor volume and weight were calculated, and all data are shown as mean ± s.d. (f) The expression of IGF2 was measured by immunohistochemistry in the tissues extracted from the SW1990-vector and SW1990-mir-615 nude mice. Magniﬁcation: × 40. (g) The percentage of IGF2-positive cells was calculated.

Figure Legend Snippet: Figure 7. The overexpression of miR-615-5p inhibited tumorigenicity in vivo. (a) The relative expression of miR-615-5p in the SW1990-vector group and the SW1990-mir-615 group was analyzed by qRT–PCR. (b, c) Photographs of tumors derived from the SW1990-vector group and the SW1990-mir-615 group of nude mice. (d, e) The graph is representative of tumor growth 25 days after inoculation. Tumor volume and weight were calculated, and all data are shown as mean ± s.d. (f) The expression of IGF2 was measured by immunohistochemistry in the tissues extracted from the SW1990-vector and SW1990-mir-615 nude mice. Magniﬁcation: × 40. (g) The percentage of IGF2-positive cells was calculated.

Techniques Used: Over Expression, In Vivo, Expressing, Plasmid Preparation, Quantitative RT-PCR, Derivative Assay, Immunohistochemistry

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Placentas were collected from gestation days (GD) 15 and 18 Ascl1 fl/fl and hep-Ascl1 -/- mice. (A) Frozen placental sections underwent periodic acid-Schiff (PAS) staining. Glycogen is stained red to purple. (B) Frozen placental sections were subjected to PL-I and PL-II in situ hybridization staining using RNAscope 2.5 HD Assay-BROWN kit. The PL-I and PL-II mRNAs are stained dark brown. (C) Quantification of relative intensity of Western blotting signals from . Data are presented as the mean fold changes relative to GD15 Ascl1 fl/fl mice (± SD; n = 3 for each group). *, P < 0.05, between Ascl1 fl/fl and hep-Ascl1 -/- mice. AKT, total protein kinase B; Ascl1 , achaete-scute homolog 1; ERK1/2, extracellular signal-regulated kinase 1/2; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IGF2, insulin-like growth factor; PL-II, placental lactogen II.

Journal: bioRxiv

Article Title: Activation of proneuronal transcription factor Ascl1 in maternal liver ensures a healthy pregnancy

doi: 10.1101/2021.04.27.441617

Figure Lengend Snippet: Placentas were collected from gestation days (GD) 15 and 18 Ascl1 fl/fl and hep-Ascl1 -/- mice. (A) Frozen placental sections underwent periodic acid-Schiff (PAS) staining. Glycogen is stained red to purple. (B) Frozen placental sections were subjected to PL-I and PL-II in situ hybridization staining using RNAscope 2.5 HD Assay-BROWN kit. The PL-I and PL-II mRNAs are stained dark brown. (C) Quantification of relative intensity of Western blotting signals from . Data are presented as the mean fold changes relative to GD15 Ascl1 fl/fl mice (± SD; n = 3 for each group). *, P < 0.05, between Ascl1 fl/fl and hep-Ascl1 -/- mice. AKT, total protein kinase B; Ascl1 , achaete-scute homolog 1; ERK1/2, extracellular signal-regulated kinase 1/2; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IGF2, insulin-like growth factor; PL-II, placental lactogen II.

Article Snippet: Serum IGF2 levels were quantified using a 1/6 dilution with the Mouse IGF-2 ELISA Kit (Boster Biological Technology, EK0381) as per directions by the manufacturer and read with the SpectraMax M2e spectrophotometer using the SoftMax Pro 6 program.

Techniques: Staining, In Situ Hybridization, RNAscope, HD Assay, Western Blot

Maternal livers were collected and weighed from nonpregnant (NP) and gestation days (GD) 15 and 18 Ascl1 fl/fl and hep-Ascl1 -/- mice. (A) Hepatic Igf2 mRNA levels were measured using qRT-PCR and presented as the mean fold changes relative to NP controls ± SD (n = 4-5). (B) Western blotting was performed using liver lysates with an antibody against IGF2. (C) Igf2 in situ hybridization on liver sections. (D) IGF2 immunostaining. (E) Levels of hepatic Igf2 promoter-specific transcript variants were measured using qRT-PCR and presented as the mean fold changes relative to NP controls ± SD (n = 4-5). (F) Levels of IGF2 protein in serum were measured using ELISA and presented as the mean fold changes ± SD (n = 3-5). *, P < 0.05; **, P < 0.01; ***, P < 0.001. P0, placental-specific Igf2 promoter; P1-3, placental- and fetal liver-specific Igf2 promoter.

Journal: bioRxiv

Article Title: Activation of proneuronal transcription factor Ascl1 in maternal liver ensures a healthy pregnancy

doi: 10.1101/2021.04.27.441617

Figure Lengend Snippet: Maternal livers were collected and weighed from nonpregnant (NP) and gestation days (GD) 15 and 18 Ascl1 fl/fl and hep-Ascl1 -/- mice. (A) Hepatic Igf2 mRNA levels were measured using qRT-PCR and presented as the mean fold changes relative to NP controls ± SD (n = 4-5). (B) Western blotting was performed using liver lysates with an antibody against IGF2. (C) Igf2 in situ hybridization on liver sections. (D) IGF2 immunostaining. (E) Levels of hepatic Igf2 promoter-specific transcript variants were measured using qRT-PCR and presented as the mean fold changes relative to NP controls ± SD (n = 4-5). (F) Levels of IGF2 protein in serum were measured using ELISA and presented as the mean fold changes ± SD (n = 3-5). *, P < 0.05; **, P < 0.01; ***, P < 0.001. P0, placental-specific Igf2 promoter; P1-3, placental- and fetal liver-specific Igf2 promoter.

Techniques: Quantitative RT-PCR, Western Blot, In Situ Hybridization, Immunostaining, Enzyme-linked Immunosorbent Assay

Journal: Oncogene

Article Title: miR-615-5p is epigenetically inactivated and functions as a tumor suppressor in pancreatic ductal adenocarcinoma.

doi: 10.1038/onc.2014.101

Figure Lengend Snippet: Figure 4. IGF2 was experimentally demonstrated as a direct target of miR-615-5p in pancreatic cancer cells. (a) Putative miR-615-5p-binding sequences in IGF2 3′-UTR and a schematic diagram of the reporter constructs showing the IGF2 3′-UTR sequence (IGF2_WT) and the mutated IGF2 3′-UTR sequence (IGF2_MUT; the mutant nucleotides of the miR-615-5p-binding site are underlined). (b) The IGF2_WT reporter or the IGF2_MUT reporter was co-transfected with the Renilla luciferase reporter into BXPC-3 and SW1990 cells containing either the miR-615-5p mimic or the negative control. Luciferase activity was assayed 48 h after transfection. (c) The expression of IGF2 mRNA was measured by real-time PCR in BXPC-3 and SW1990 cells 48 h after transfection. Beta-actin was used as a housekeeping gene control. (d) IGF2 protein levels in BXPC-3 and SW1990 cell supernatants were detected by ELISA 48 h after transfection. (e) IGF2 protein in BXPC-3 and SW1990 cells was detected by western blot 48 h post transfection. GAPDH was used as a housekeeping protein control. A grayscale analysis of the western blot data is shown.

Article Snippet: IGF2 levels in the BXPC-3 and SW1990 cell Oncogene (2015) 1629 – 1640 © 2015 Macmillan Publishers Limited supernatants were detected using a human IGF2 ELISA kit (Cusabio Biotech, Newark, DE, USA) according to the manufacturer’s instructions.

Techniques: Binding Assay, Construct, Sequencing, Mutagenesis, Transfection, Luciferase, Negative Control, Activity Assay, Expressing, Real-time Polymerase Chain Reaction, Control, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Oncogene

Article Title: miR-615-5p is epigenetically inactivated and functions as a tumor suppressor in pancreatic ductal adenocarcinoma.

doi: 10.1038/onc.2014.101

Figure Lengend Snippet: Figure 5. miR-615-5p suppressed pancreatic cancer cell proliferation by directly targeting IGF2. (a) Immunoﬂuorescence. BXPC-3 and SW1990 cells were ﬁxed and incubated with primary antibodies for IGF2 and then Alexa Fluor 488-conjugated anti-rabbit secondary antibodies. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). The ﬂuorescence level of IGF2 was notably decreased in miR-615-5p-transfected cells. Magniﬁcation: × 400. (b) BXPC-3 and SW1990 cell proliferation was detected by the MTT assay at 0, 24, 48 and 72 h after co-transfection with different combinations of NC or MIMIC with GFP or IGF2. For NC+GFP vs MIMIC+GFP: *Po0.05; **Po0.01. For NC+GFP vs NC+IGF2: #Po0.05; ##Po0.01. For NC+IGF2 vs MIMIC+IGF2: △△Po0.01. (c) The IGF2 protein was detected by western blot at 48 h post-transfection. GAPDH was used as a housekeeping protein control. A grayscale analysis of the western blot data is shown.

Techniques: Incubation, Staining, Transfection, MTT Assay, Cotransfection, Western Blot, Control

Journal: Oncogene

Article Title: miR-615-5p is epigenetically inactivated and functions as a tumor suppressor in pancreatic ductal adenocarcinoma.

doi: 10.1038/onc.2014.101

Figure Lengend Snippet: Figure 6. Imprinting status and expression levels of IGF2. (a) Gel photo of ApaI-digested PCR products for IGF2 genotyping. The heterozygous samples had two bands, which were considered as informative. (b) Gel photo of ApaI-digested RT–PCR products for imprinting status analysis. The ratios between the two bands are listed. The samples with a ratio of o3:1 or >1:3 had LOI of IGF2. (c, e) Results of qRT–PCR analysis of IGF2 mRNA expression in the 32 pairs of tissues shown on a column chart and a scattergram. (d, f) qRT–PCR results of miR-615-5p expression levels in the 32 pairs of tissues shown on a column chart and a scattergram. (g) The inverse correlation between IGF2 and miR-615-5p expression levels was examined by Pearson’s correlation analysis (R = −0.456, P = 0.033).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR

Journal: Oncogene

Article Title: miR-615-5p is epigenetically inactivated and functions as a tumor suppressor in pancreatic ductal adenocarcinoma.

doi: 10.1038/onc.2014.101

Figure Lengend Snippet: Figure 7. The overexpression of miR-615-5p inhibited tumorigenicity in vivo. (a) The relative expression of miR-615-5p in the SW1990-vector group and the SW1990-mir-615 group was analyzed by qRT–PCR. (b, c) Photographs of tumors derived from the SW1990-vector group and the SW1990-mir-615 group of nude mice. (d, e) The graph is representative of tumor growth 25 days after inoculation. Tumor volume and weight were calculated, and all data are shown as mean ± s.d. (f) The expression of IGF2 was measured by immunohistochemistry in the tissues extracted from the SW1990-vector and SW1990-mir-615 nude mice. Magniﬁcation: × 40. (g) The percentage of IGF2-positive cells was calculated.

Techniques: Over Expression, In Vivo, Expressing, Plasmid Preparation, Quantitative RT-PCR, Derivative Assay, Immunohistochemistry

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