Journal: PLoS ONE

Article Title: Increased Learning and Brain Long-Term Potentiation in Aged Mice Lacking DNA Polymerase μ

doi: 10.1371/journal.pone.0053243

Figure Lengend Snippet: ( A ) Hierarchical clustering of the differential mRNA expression between wild-type and Polµ −/− old (18 months) mice. ( B ) Schematic representation of the expression network detected using the software Ingenuity. ( C ) qRT-PCR analysis of genes belonging to the previously described network ( B ) and others found in DNA expression arrays carried out with brain of acutely treated animals . Black bars denote values for wild-type animals and white bars correspond to Polµ −/− animals. ( D ) Circulating levels of IGF1 and GH (ng/mL) were monitored by ELISA in old (21–24 months) animals. ( E, F ) qRT-PCR expression ( E ) and immunoconfocal analysis ( F ) of Igf1 and Igf1R in the hippocampal CA1area from wild-type and Polμ −/− young adult (4 months) mice. Igf1 immunoreactivity-labeled non-pyramidal cells (short arrows) of stratum oriens (so), stratum pyramidale (sp), and stratum radiatum (sr; not shown); no differences in labeling frequency or labeling intensity were found between Polμ −/− and wild-type mice. Igf1R-immunostaining delineated cell processes in the stratum radiatum (sr) of Polμ −/− but not of wild-type mice. As compared with the case of aged mice, unspecific punctate labeling (long arrows) was infrequent in these tissue sections. Slight differences were found, however, in the occurrence of Igf1-immunoreactive cells in the CA1 area of aged mice, and in the expression of Igf1R between young wild-type and Polμ −/− mice. Scale bar, 25 µm.

Article Snippet: Primary antibodies used were mouse monoclonal antibody M23 to IGF1 (Abcam, Cambridge, UK; 1∶300), mouse monoclonal antibody AP-20 to microtubule-associated protein 2 (map2, 2a+2b; Sigma; 1∶250), rabbit polyclonal antibody sc-713 to IGF1Rβ (Santa Cruz Biotechnology, CA, USA; 1∶150) and rabbit polyclonal antibody to the Zn 2+ vesicular transporter ZnT3 (Synaptic Systems, Göttingen, Germany; 1∶500).

Techniques: Expressing, Software, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Labeling, Immunostaining

Journal: Cell Reports Medicine

Article Title: Disrupting AGR2/IGF1 paracrine and reciprocal signaling for pancreatic cancer therapy

doi: 10.1016/j.xcrm.2024.101927

Figure Lengend Snippet: IGF1 promotes the secretion of AGR2, which in turn enhances the presentation of the IGF1R on the cell surface (A) The Venn diagram of the upper panel illustrates the count of genes down-regulated in Capan2 and Panc1 cells post AGR2 knockout. The Gene Ontology (GO) analysis of the lower panel identifies enriched biological processes, notably “regulation of IGF receptor signaling pathway” at transcriptional levels, after AGR2 knockout in these cell lines. (B) Western blot and quantitative reverse-transcription PCR (qRT-PCR) analyses assess IGF1R and AGR2 expression in Capan2 and Panc1 cells following AGR2 knockout via the CRISPR-Cas9 system (performed in triplicate). (C) Flow cytometry (FACS) quantifies cell membrane surface expression of IGF1R in Capan2 and Panc1 cells after AGR2 knockout (performed in triplicate). (D) Co-immunoprecipitation assays reveal AGR2’s interaction with pro-IGF1R in Capan2 and Panc1 cells (performed in triplicate). (E) Immunofluorescence imaging displays AGR2 and IGF1R distribution and ER labeling in Panc1 cells (scale bars: 50 μm). (F) Western blot analysis of IGF1R and AGR2 in Panc1 and Capan2 cells with controls (original cell lines), AGR2-knockout (AGR2 KO ) post-expression of AGR2 WT , AGR2 ΔNLS , AGR2 ΔSP , and AGR2 C81A mutation (performed in triplicate). (G) Western blot analysis of IGF1R expression in AGR2 in Panc1 and Capan2 cells with AGR2 knockout (KO) treated with 3-methyladenine (3-MA) (15 mM), bafilomycin A1 (30 nM), chloroquine (20 mM), MLN4929 (1 mM), or MG132 (5 mM) for 12 h (performed in triplicate). (H) Western blot analysis of IGF1R, phosphorylated IGF1R, c-JUN, phosphorylated c-JUN, and AGR2 following 12 h of serum starvation and subsequent IGF1 stimulation (50 ng/mL, performed in triplicate). (I) ELISA measures AGR2 secretion after serum starvation and treatment with PPP (1 μM) and IGF1 (50 ng/mL) over time (performed in triplicate). (J) Identification of potential c-JUN-binding sites within the AGR2 promoter region. (K) Western blot analysis of c-JUN, phosphorylated c-JUN, and AGR2 expression following c-JUN knockdown and IGF1 stimulation over time (performed in triplicate). (L) Western blot shows c-JUN, phosphorylated c-JUN, and AGR2 expression post anisomycin treatment over time (performed in triplicate). (M) Chromatin immunoprecipitation followed by quantitative PCR (ChIP-qPCR) demonstrates c-JUN enrichment at AGR2’s transcription start sites (TSSs) before and after IGF1 treatment (performed in triplicate). (N) Integrative Genomics Viewer (IGV) tracks display c-JUN peaks in AGR2’s promoter region post IGF1 treatment. (O) Dual-luciferase reporter assays in Capan2 and Panc1 cells evaluate AGR2 promoter activity under various lengths and site-specific mutations after IGF1 treatment (performed in triplicate). Statistical analyses: (B) and (C) used a one-way ANOVA with multiple comparisons. (I), (M), (N), and (O) were analyzed using two-tailed, unpaired Student’s t tests. Data are presented as mean ± SD, with significance marked as ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and “ns” indicates no significance.

Article Snippet: Mouse monoclonal anti-IGF1 , Santa Cruz Biotechnology , Cat# sc-518040;.

Techniques: Knock-Out, Western Blot, Reverse Transcription, Quantitative RT-PCR, Expressing, CRISPR, Flow Cytometry, Membrane, Immunoprecipitation, Immunofluorescence, Imaging, Labeling, Mutagenesis, Enzyme-linked Immunosorbent Assay, Binding Assay, Knockdown, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Luciferase, Activity Assay, Two Tailed Test

Journal: Cell Reports Medicine

Article Title: Disrupting AGR2/IGF1 paracrine and reciprocal signaling for pancreatic cancer therapy

doi: 10.1016/j.xcrm.2024.101927

Figure Lengend Snippet: Secreted AGR2 promotes IGF1 production from CAFs via the Wnt/β-catenin pathway (A) Western blot analysis evaluates AGR2 and IGF1 levels in three human PDAC-derived CAFs and three PDAC cell lines (Capan2, HPAC, and Panc1) across three independent experiments. (B) qRT-PCR analysis of IGF1 expression and supernatant ELISA analyses of IGF1 secretion in human PDAC-derived CAFs co-cultured with two human PDAC organoids and with or without treatment with Agr2-neutralizing antibody (5 μg/mL) for 48 h ( n = 3 independent experiments). (C) qRT-PCR assesses IGF1 expression in PDAC-derived CAFs co-cultured with AGR2-knockout Capan2 and Panc1 cells, following re-expression of AGR2 WT , AGR2 ΔNLS , and AGR2 ΔSP for 48 h (upper); Western blot analysis investigates IGF1R, phosphorylated IGF1R, c-JUN, phosphorylated c-JUN, and AGR2 levels in AGR2-knockout Capan2 and Panc1 cells after co-culture with PDAC-derived CAFs (lower, n = 3 independent experiments). (D) qRT-PCR explores IGF1 expression in two PDAC-derived CAFs after treatment with rAGR2 (500 ng/mL), rTGF-β1 (4 μg/mL), and rIL-1α (200 ng/mL) for 24 h ( n = 3 independent experiments). (E) Supernatant analysis quantifies collagen levels in two PDAC-derived CAFs following rAGR2 treatment (500 ng/mL) for 24 h ( n = 3 independent experiments). (F) Transwell assays examine cell migration in two PDAC-derived CAFs following rAGR2 treatment (500 ng/mL) for 24 h ( n = 3 independent experiments). (G) Left: scRNA-seq identifies iCAFs and myCAFs within 16 PDAC tissues (GEO: GSE155698), showing iCAFs with elevated IGF1 expression (>mean value). Right: volcano plot displays genes differentially expressed between IGF1 high and IGF1 low CAFs (FDR < 0.01; log 2 FC > 0.5), accompanied by KEGG pathway analysis of the IGF1-CAF signature. (H) Principal component analysis (PCA) of transcriptomic data from CAFs treated with rAGR2, rTGF-β1, and rIL-1α ( n = 3 per group). (I) A heatmap shows genes significantly upregulated in CAFs after treatment with rAGR2, rTGF-β1, and rIL-1α (FDR < 0.01; log2FC > 0.5; left). Bioplant pathway analysis elucidates upregulated gene pathways post rAGR2 treatment in CAFs (right). (J) Identification of potential lymphoid enhancer binding factor 1 (LEF1)-binding sites within the IGF1 promoter region. (K) Western blot analysis shows β-catenin expression in both nuclear and cytoplasmic fractions of PDAC-derived CAFs after AGR2 stimulation (500 ng/mL) for 0.5, 1, 3, and 6 h ( n = 3 independent experiments). (L) Western blot and qRT-PCR analyses evaluate β-catenin and IGF1 levels in PDAC-derived CAFs post β-catenin knockdown or following treatment with ICG-001 (Wnt pathway inhibitor) and rAGR2 (500 ng/mL) for 24 h (M) Luciferase reporter assays in three PDAC-derived CAFs transfected with wild-type and site-specific mutagenized IGF1 promoter sequences based on (J) predictions, post rAGR2 treatment (500 ng/mL) for 24 h ( n = 3 independent experiments). Statistical analysis: one-way ANOVA with multiple comparisons test was used for (B), (C), (D), and (L); two-tailed, unpaired Student’s t tests were employed for (E), (F), and (M). Data are presented as mean ± SD, with ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 indicating levels of statistical significance.

Article Snippet: Mouse monoclonal anti-IGF1 , Santa Cruz Biotechnology , Cat# sc-518040;.

Techniques: Western Blot, Derivative Assay, Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay, Cell Culture, Knock-Out, Co-Culture Assay, Migration, Binding Assay, Knockdown, Luciferase, Transfection, Two Tailed Test

Journal: Cell Reports Medicine

Article Title: Disrupting AGR2/IGF1 paracrine and reciprocal signaling for pancreatic cancer therapy

doi: 10.1016/j.xcrm.2024.101927

Figure Lengend Snippet: High serum levels of AGR2 and IGF1 are associated with enhanced desmoplastic reactions and immunosuppression in PDAC (A) ELISA analysis of Igf1 in serum from 8-week-old KC mice, KC; Agr2 −/− mice, and KC; Agr2 OE mice reveals a significant difference (left, n = 12 mice per group). Comparison between KC mice and KC; Agr2 −/− mice with PDAC also shows marked differences (right, n = 5 mice per group). (B) Serum levels of AGR2 and IGF1 exhibit a correlation in 145 human patients with PDAC, analyzed using Pearson’s correlation coefficient. (C) IHC images display α-SMA, podoplanin (PDPN), collagen, and IL-6 positivity in tumor areas, comparing AGR2 high ; IGF1 high samples with AGR2 low ; IGF1 low samples, demonstrating a difference in desmoplastic reaction. (D) IHC images illustrate the differential presence of CD3, CD8, CD4, FOXP3, CD68, CD206, and CD20-positive cells in tumors between AGR2 high ; IGF1 high samples and AGR2 low ; IGF1 low samples, indicating variations in immune cell infiltration (scale bars: 50 μm). (E) IHC imaging further reveals the distribution of Cd3, Cd8, Cd4, Foxp3, B220, F4/80, and Cd206-positive cells in tumors from KC mice versus KC; Agr2 −/− mice, emphasizing differences in immunological responses (scale bars: 50 μm). p values in left of (A) was calculated using a one-way ANOVA with a multiple comparisons test, p values in right of (A), (C), (D), and (E) were calculated using two-tailed, unpaired Student’s t tests, and correlation coefficient in (B) was calculated using Pearson’s correlation coefficient. Data are presented as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. ns, no significance.

Article Snippet: Mouse monoclonal anti-IGF1 , Santa Cruz Biotechnology , Cat# sc-518040;.

Techniques: Enzyme-linked Immunosorbent Assay, Comparison, Imaging, Two Tailed Test

Journal: Cell Reports Medicine

Article Title: Disrupting AGR2/IGF1 paracrine and reciprocal signaling for pancreatic cancer therapy

doi: 10.1016/j.xcrm.2024.101927

Figure Lengend Snippet: Combined targeting attenuates desmoplastic reaction and normalizes immunosuppressive microenvironment (A) Western blot analysis reveals Agr2 and Igf1 levels in PSCs isolated from wild-type mice and three mouse PDAC cell lines, highlighting the differential expression patterns. (B) Schematic diagram shows the therapeutic strategy of combining IGF1R inhibitor and AGR2-neutralizing antibody. (C) ELISA and qRT-PCR analyses demonstrate Igf1 levels in PSCs co-cultured with KPC PDAC-derived organoids. The impact of treatments with the IGF1R inhibitor (PPP; 1 μM), Agr2-neutralizing antibody (5 μg/mL) alone, or their combination for 48 h is shown ( n = 3 independent experiments). (D) Western blot results display the expression levels of p-Igf1r, Igf1r, c-Jun, p-c-Jun, Akt, p-Akt, Erk, p-Erk, and Agr2 in mouse PDAC-derived organoids after co-culture with PSC cells and subsequent treatments as mentioned in (C) ( n = 3 independent experiments). (E) Representative images and quantitative analyses show the growth dynamics of PDAC organoids co-cultured with PSC cells under various treatment conditions over 0, 24, 48, and 96 h (scale bars: 50 μm, n = 3 independent experiments). (F) Tumor volume comparisons in KPC mice post caerulein-induced acute pancreatitis and subsequent treatments with Agr2 antibody (4 mg/kg; intraperitoneally [i.p.], three times per week for 2 weeks), PPP (20 mg/kg; i.p., three times per week for 2 weeks), or their combination ( n = 5 for control group, n = 3 for single treatment groups, and n = 5 for combined treatment group). (G) ELISA quantification of Agr2, Igf1, Il-1α, Lif, GM-CSF, and Il-6 in serum samples from the four groups of KPC mice underscores the systemic effects of the treatment modalities on cytokine levels ( n = 5 for control group, n = 3 for single treatment groups, and n = 5 for combined treatment group). (H and I) Representative stained sections and quantitative statistics of H&E, Pdpn, α-SMA, collagen, Cd3, Cd4, Foxp3, B220, and Cd206-positive cells within PDAC tumors (scale bars: 50 μm, n = 5 mice per group). (J) Representative IHC highlights CD8-positive cells in lymph nodes adjacent to the tumors (scale bars: 50 μm). p values in (C), (F), and (G) were calculated using a one-way ANOVA with a multiple comparisons test, and p values in (H) and (I) were calculated using two-tailed, unpaired Student’s t tests. Data are presented as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: Mouse monoclonal anti-IGF1 , Santa Cruz Biotechnology , Cat# sc-518040;.

Techniques: Western Blot, Isolation, Expressing, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Cell Culture, Derivative Assay, Co-Culture Assay, Control, Staining, Two Tailed Test

Journal: Cell Reports Medicine

Article Title: Disrupting AGR2/IGF1 paracrine and reciprocal signaling for pancreatic cancer therapy

doi: 10.1016/j.xcrm.2024.101927

Figure Lengend Snippet:

Article Snippet: Mouse monoclonal anti-IGF1 , Santa Cruz Biotechnology , Cat# sc-518040;.

Techniques: Virus, Recombinant, Control, Enzyme-linked Immunosorbent Assay, Isolation, Membrane, Protein Extraction, Chromatin Immunoprecipitation, Bicinchoninic Acid Protein Assay, Sircol Collagen Assay, Luciferase, RNA Sequencing Assay, Sequencing, Expressing, Real-time Polymerase Chain Reaction, shRNA, Plasmid Preparation, Software, Flow Cytometry

Journal: Cellular signalling

Article Title: Palmitate-induced Endoplasmic Reticulum stress and subsequent C/EBPα Homologous Protein activation attenuates leptin and Insulin-like Growth Factor 1 expression in the brain

doi: 10.1016/j.cellsig.2016.08.012

Figure Lengend Snippet: List of monoclonal and polyclonal antibodies used in the study

Article Snippet: 2.7 Quantitative real time RT-PCR analysis Total RNA was isolated and extracted from treated cells using the 5 prime “PerfectPure RNA tissue kit” (5 Prime, Inc., Gaithersburg, MD). cDNA was obtained by reverse transcribing 1μg of extracted RNA using an iScript cDNA synthesis kit” (BioRad, Hercules, CA). cDNA was obtained by reverse transcribing 1 μg of extracted RNA using an iScript cDNA synthesis kit" (BioRad, Hercules, CA).The quantitative Real-time RT-PCR was performed using TaqMan chemistry using “Assays-on-Demand” probes (ABI, Foster City, CA) for human Leptin ( LEP ) (Hs00174877_m1), mouse leptin ( Lep ) (Mm00434759_m1), human IGF1 ( IGF1 ) (Hs01547656_m1), and mouse IGF1 ( Igf1 ) (Mm00439560_m1).

Techniques:

Journal: Cellular signalling

Article Title: Palmitate-induced Endoplasmic Reticulum stress and subsequent C/EBPα Homologous Protein activation attenuates leptin and Insulin-like Growth Factor 1 expression in the brain

doi: 10.1016/j.cellsig.2016.08.012

Figure Lengend Snippet: List of primers used for ChIP analysis

Article Snippet: 2.7 Quantitative real time RT-PCR analysis Total RNA was isolated and extracted from treated cells using the 5 prime “PerfectPure RNA tissue kit” (5 Prime, Inc., Gaithersburg, MD). cDNA was obtained by reverse transcribing 1μg of extracted RNA using an iScript cDNA synthesis kit” (BioRad, Hercules, CA). cDNA was obtained by reverse transcribing 1 μg of extracted RNA using an iScript cDNA synthesis kit" (BioRad, Hercules, CA).The quantitative Real-time RT-PCR was performed using TaqMan chemistry using “Assays-on-Demand” probes (ABI, Foster City, CA) for human Leptin ( LEP ) (Hs00174877_m1), mouse leptin ( Lep ) (Mm00434759_m1), human IGF1 ( IGF1 ) (Hs01547656_m1), and mouse IGF1 ( Igf1 ) (Mm00439560_m1).

Techniques: Binding Assay

Journal: Cellular signalling

Article Title: Palmitate-induced Endoplasmic Reticulum stress and subsequent C/EBPα Homologous Protein activation attenuates leptin and Insulin-like Growth Factor 1 expression in the brain

doi: 10.1016/j.cellsig.2016.08.012

Figure Lengend Snippet: Representative western blots (A, B), ELISA immunoassay (C, D), and Real-time RT-PCR analysis (E, F) show that palmitate at a concentration of 100 μM and above, but not below 100 μM, significantly decreases leptin and IGF1 expression in SH-SY5Y cells (A, C, E,) and N2a cells (B, D, F). Data is expressed as Mean ± S.D and includes determination made in four (n=4) separate cell culture experiments. ***p < 0.001 versus BSA-treated control cells.

Article Snippet: 2.7 Quantitative real time RT-PCR analysis Total RNA was isolated and extracted from treated cells using the 5 prime “PerfectPure RNA tissue kit” (5 Prime, Inc., Gaithersburg, MD). cDNA was obtained by reverse transcribing 1μg of extracted RNA using an iScript cDNA synthesis kit” (BioRad, Hercules, CA). cDNA was obtained by reverse transcribing 1 μg of extracted RNA using an iScript cDNA synthesis kit" (BioRad, Hercules, CA).The quantitative Real-time RT-PCR was performed using TaqMan chemistry using “Assays-on-Demand” probes (ABI, Foster City, CA) for human Leptin ( LEP ) (Hs00174877_m1), mouse leptin ( Lep ) (Mm00434759_m1), human IGF1 ( IGF1 ) (Hs01547656_m1), and mouse IGF1 ( Igf1 ) (Mm00439560_m1).

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Concentration Assay, Expressing, Cell Culture, Control

Journal: Cellular signalling

Article Title: Palmitate-induced Endoplasmic Reticulum stress and subsequent C/EBPα Homologous Protein activation attenuates leptin and Insulin-like Growth Factor 1 expression in the brain

doi: 10.1016/j.cellsig.2016.08.012

Figure Lengend Snippet: Representative western blots (A, B), ELISA immunoassay (C, D), and Real-time RT-PCR analysis (E, F) show that palmitate treatment (100 μM for 24 hours) significantly decreases leptin and IGF1 expression in SH-SY5Y cells (A, C, E,) and N2a cells (B, D, F). Pretreatment (for 2 hours) with the molecular chaperone, 4-PBA, significantly precludes the palmitate-induced attenuation in leptin and IGF1 expression. Data is expressed as Mean ± S.D and includes determination made in four (n=4) separate cell culture experiments. *p < 0.05, **p < 0.01, ***p < 0.001 versus BSA-treated control cells; †p < 0.05, ††p < 0.01 versus palmitate-treated cells or Tunicamycin-treated cells.

Article Snippet: 2.7 Quantitative real time RT-PCR analysis Total RNA was isolated and extracted from treated cells using the 5 prime “PerfectPure RNA tissue kit” (5 Prime, Inc., Gaithersburg, MD). cDNA was obtained by reverse transcribing 1μg of extracted RNA using an iScript cDNA synthesis kit” (BioRad, Hercules, CA). cDNA was obtained by reverse transcribing 1 μg of extracted RNA using an iScript cDNA synthesis kit" (BioRad, Hercules, CA).The quantitative Real-time RT-PCR was performed using TaqMan chemistry using “Assays-on-Demand” probes (ABI, Foster City, CA) for human Leptin ( LEP ) (Hs00174877_m1), mouse leptin ( Lep ) (Mm00434759_m1), human IGF1 ( IGF1 ) (Hs01547656_m1), and mouse IGF1 ( Igf1 ) (Mm00439560_m1).

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Expressing, Cell Culture, Control

Journal: Cellular signalling

Article Title: Palmitate-induced Endoplasmic Reticulum stress and subsequent C/EBPα Homologous Protein activation attenuates leptin and Insulin-like Growth Factor 1 expression in the brain

doi: 10.1016/j.cellsig.2016.08.012

Figure Lengend Snippet: Representative western blots (A, B), ELISA immunoassay (C, D), and Real-time RT-PCR analysis (E, F) show that knocking-down CHOP using a RNAi approach significantly rescues the palmitate-induced mitigation in leptin and IGF1 expression in palmitate-treated SH-SY5Y cells (A, C, E) and palmitate-treated N2a cells (B, D, F). Data is expressed as Mean ± S.D and includes determination made in three (n=3) separate cell culture experiments. *p < 0.05, **p < 0.01, ***p < 0.001 versus GFP-shRNA knocked-down or scrambled siRNA transfected concomitant with BSA-treated control cells; †p < 0.05, ††p < 0.01, †††p<0.001 versus GFP-shRNA knocked-down or scrambled siRNA transfected concomitant with palmitate-treated cells.

Article Snippet: 2.7 Quantitative real time RT-PCR analysis Total RNA was isolated and extracted from treated cells using the 5 prime “PerfectPure RNA tissue kit” (5 Prime, Inc., Gaithersburg, MD). cDNA was obtained by reverse transcribing 1μg of extracted RNA using an iScript cDNA synthesis kit” (BioRad, Hercules, CA). cDNA was obtained by reverse transcribing 1 μg of extracted RNA using an iScript cDNA synthesis kit" (BioRad, Hercules, CA).The quantitative Real-time RT-PCR was performed using TaqMan chemistry using “Assays-on-Demand” probes (ABI, Foster City, CA) for human Leptin ( LEP ) (Hs00174877_m1), mouse leptin ( Lep ) (Mm00434759_m1), human IGF1 ( IGF1 ) (Hs01547656_m1), and mouse IGF1 ( Igf1 ) (Mm00439560_m1).

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Expressing, Cell Culture, shRNA, Transfection, Control

Journal: Cellular signalling

Article Title: Palmitate-induced Endoplasmic Reticulum stress and subsequent C/EBPα Homologous Protein activation attenuates leptin and Insulin-like Growth Factor 1 expression in the brain

doi: 10.1016/j.cellsig.2016.08.012

Figure Lengend Snippet: Representative western blots (A, B) ELISA immunoassay (C, D), and Real-time RT-PCR analysis (E, F) show that the Chop−/− mice fed a palmitate-enriched diet do not exhibit the reduction in leptin and IGF1 expression to the same degree in the cortex (A, C, E) and the hippocampus (B, D, F), compared to the C57BL/6J wild-type mice fed a palmitate-enriched diet. Data is expressed as Mean ± S.D and includes determination made in six (n=6) different animals from each group. *p < 0.05, **p < 0.01, ***p < 0.001 versus C57BL/6J wild-type mice fed a control chow diet; †p < 0.05, ††p < 0.01, †††p<0.001 versus C57BL/6J wild-type mice fed a palmitate-enriched diet.

Article Snippet: 2.7 Quantitative real time RT-PCR analysis Total RNA was isolated and extracted from treated cells using the 5 prime “PerfectPure RNA tissue kit” (5 Prime, Inc., Gaithersburg, MD). cDNA was obtained by reverse transcribing 1μg of extracted RNA using an iScript cDNA synthesis kit” (BioRad, Hercules, CA). cDNA was obtained by reverse transcribing 1 μg of extracted RNA using an iScript cDNA synthesis kit" (BioRad, Hercules, CA).The quantitative Real-time RT-PCR was performed using TaqMan chemistry using “Assays-on-Demand” probes (ABI, Foster City, CA) for human Leptin ( LEP ) (Hs00174877_m1), mouse leptin ( Lep ) (Mm00434759_m1), human IGF1 ( IGF1 ) (Hs01547656_m1), and mouse IGF1 ( Igf1 ) (Mm00439560_m1).

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Expressing, Control

Journal: Cellular signalling

Article Title: Palmitate-induced Endoplasmic Reticulum stress and subsequent C/EBPα Homologous Protein activation attenuates leptin and Insulin-like Growth Factor 1 expression in the brain

doi: 10.1016/j.cellsig.2016.08.012

Figure Lengend Snippet: Luciferase reporter assay demonstrates that knocking-down CHOP in palmitate-treated SH-SY5Y cells (A) and N2a (B) cells using an RNAi approach significantly restores the C/EBPα driven leptin and IGF1 promoter transactivation. Data is expressed as Mean ± S.D and includes determination made in three (n=3) separate cell culture experiments. *p < 0.05, **p < 0.01, ***p < 0.001 versus GFP-shRNA knocked-down or scrambled siRNA transfected concomitant with BSA-treated control cells; ††p < 0.01, †††p<0.001 versus GFP-shRNA knocked-down or scrambled siRNA transfected concomitant with palmitate-treated cells. (C, D) ChIP analysis demonstrates that Chop−/− mice fed a palmitate-enriched diet do not exhibit a reduction in C/EBPα binding to the leptin and IGF1 promoter to the same degree as the wild-type C57BL/6J mice fed a palmitate-enriched diet. Data is expressed as Mean ± S.D and includes determination made in six (n=6) different animals from each group. *p < 0.05, ***p < 0.001 versus C57BL/6J wild-type mice fed a control chow diet; ††p < 0.01 versus C57BL/6J wild-type mice fed a palmitate-enriched diet.

Article Snippet: 2.7 Quantitative real time RT-PCR analysis Total RNA was isolated and extracted from treated cells using the 5 prime “PerfectPure RNA tissue kit” (5 Prime, Inc., Gaithersburg, MD). cDNA was obtained by reverse transcribing 1μg of extracted RNA using an iScript cDNA synthesis kit” (BioRad, Hercules, CA). cDNA was obtained by reverse transcribing 1 μg of extracted RNA using an iScript cDNA synthesis kit" (BioRad, Hercules, CA).The quantitative Real-time RT-PCR was performed using TaqMan chemistry using “Assays-on-Demand” probes (ABI, Foster City, CA) for human Leptin ( LEP ) (Hs00174877_m1), mouse leptin ( Lep ) (Mm00434759_m1), human IGF1 ( IGF1 ) (Hs01547656_m1), and mouse IGF1 ( Igf1 ) (Mm00439560_m1).

Techniques: Luciferase, Reporter Assay, Cell Culture, shRNA, Transfection, Control, Binding Assay

Journal: Cellular signalling

Article Title: Palmitate-induced Endoplasmic Reticulum stress and subsequent C/EBPα Homologous Protein activation attenuates leptin and Insulin-like Growth Factor 1 expression in the brain

doi: 10.1016/j.cellsig.2016.08.012

Figure Lengend Snippet: Representative western blots (A, B), ELISA immunoassay (C, D), and Real-time RT-PCR analysis (E, F) show that the leucine zipper domain of CHOP that mediates the interaction with C/EBPα is necessary for the CHOP-induced mitigation in leptin and IGF1 expression in treated SH-SY5Y cells (A, C, E) and N2a cells (B, D, F). Luciferase reporter assay demonstrates that only the wild-type native CHOP and not the leucine zipper domain mutant of CHOP attenuates C/EBPα driven leptin and IGF1 promoter transactivation (G, H). Data is expressed as Mean ± S.D and includes determination made in three (n=3) separate cell culture experiments. ***p < 0.001 versus empty vector (EV) – transfected cells.

Article Snippet: 2.7 Quantitative real time RT-PCR analysis Total RNA was isolated and extracted from treated cells using the 5 prime “PerfectPure RNA tissue kit” (5 Prime, Inc., Gaithersburg, MD). cDNA was obtained by reverse transcribing 1μg of extracted RNA using an iScript cDNA synthesis kit” (BioRad, Hercules, CA). cDNA was obtained by reverse transcribing 1 μg of extracted RNA using an iScript cDNA synthesis kit" (BioRad, Hercules, CA).The quantitative Real-time RT-PCR was performed using TaqMan chemistry using “Assays-on-Demand” probes (ABI, Foster City, CA) for human Leptin ( LEP ) (Hs00174877_m1), mouse leptin ( Lep ) (Mm00434759_m1), human IGF1 ( IGF1 ) (Hs01547656_m1), and mouse IGF1 ( Igf1 ) (Mm00439560_m1).

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Expressing, Luciferase, Reporter Assay, Mutagenesis, Cell Culture, Plasmid Preparation, Transfection

Journal: Cellular signalling

Article Title: Palmitate-induced Endoplasmic Reticulum stress and subsequent C/EBPα Homologous Protein activation attenuates leptin and Insulin-like Growth Factor 1 expression in the brain

doi: 10.1016/j.cellsig.2016.08.012

Figure Lengend Snippet: Representative western blots (A, B), ELISA immunoassay (C, D), and Real-time RT-PCR analysis (E, F) show that ectopically overexpressing the wild-type (wt) C/EBPα, but not the bZIP deletion mutant of C/EBPα (C/EBPα bZIP--), significantly rescues the palmitate-induced mitigation in leptin and IGF1 expression in treated SH-SY5Y cells (A, C, E) and N2a cells (B, D, F). Luciferase reporter assay demonstrates that ectopically overexpressing the wild-type (wt) C/EBPα and not the bZIP deletion mutant of C/EBPα (C/EBPα bZIP--), significantly restores leptin and IGF1 promoter activation to basal levels in palmitate-treated SH-SY5Y cells and N2a cells (G, H). Data is expressed as Mean ± S.D and includes determination made in three (n=3) separate cell culture experiments. *p < 0.05, **p < 0.01, ***p < 0.001 versus empty vector (EV) – transfected concomitant with BSA-treated control cells; ††p < 0.01, †††p<0.001 versus empty vector (EV) – transfected concomitant with palmitate-treated cells.

Article Snippet: 2.7 Quantitative real time RT-PCR analysis Total RNA was isolated and extracted from treated cells using the 5 prime “PerfectPure RNA tissue kit” (5 Prime, Inc., Gaithersburg, MD). cDNA was obtained by reverse transcribing 1μg of extracted RNA using an iScript cDNA synthesis kit” (BioRad, Hercules, CA). cDNA was obtained by reverse transcribing 1 μg of extracted RNA using an iScript cDNA synthesis kit" (BioRad, Hercules, CA).The quantitative Real-time RT-PCR was performed using TaqMan chemistry using “Assays-on-Demand” probes (ABI, Foster City, CA) for human Leptin ( LEP ) (Hs00174877_m1), mouse leptin ( Lep ) (Mm00434759_m1), human IGF1 ( IGF1 ) (Hs01547656_m1), and mouse IGF1 ( Igf1 ) (Mm00439560_m1).

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Mutagenesis, Expressing, Luciferase, Reporter Assay, Activation Assay, Cell Culture, Plasmid Preparation, Transfection, Control

Journal: Science (New York, N.Y.)

Article Title: Stromal Gli2 activity coordinates a niche signaling program for mammary epithelial stem cells.

doi: 10.1126/science.aal3485

Figure Lengend Snippet: Fig. 6. Stromal Gli2 regulates a niche signaling program for mammary stem cell activity. (A) Fsp1Cre- labeled mammary stromal cells FACS-isolated from Gli2WT (Fsp1Cre;Rosa26mTmG/+) or Gli2ΔS (Fsp1Cre;Rosa26mTmG/+;Gli2flox/nLacZ) littermate mice were analyzed by qRT-PCR for expression of selected genes, shown as a percentage of HPRT expression, and measured in triplicate from each of three individual Gli2WT or Gli2ΔS mice. Three major classes of stromally expressed genes are down-regulated in stromal cells of Gli2ΔS mice (see text). (B) Gene expression in single, FACS-isolated Fsp1Cre-labeled stromal cells from Gli2WT and Gli2ΔS mice was analyzed by qRT-PCR. The results (fig. S4) are summarized here as the percentage of single cells analyzed that express the indicated genes. (C) Sections of Gli2WT (Fsp1Cre;Rosa26mTmG/+;Gli2nLacZ/+) and Gli2ΔS (Fsp1Cre;Rosa26mTmG/+;Gli2nLacZ/flox) mammary glands immuno-stained for collagen and β-galactosidase. Selected areas (dotted lines) are shown at higher magnification in the green channel only (β-galactosidase- expressing cells) in the right panels. The frequency of β-galactosidase-labeled cells per 100 μm length of ductal epithelium is shown in the lower left of the right panels. Scale bar, 50 μm. (D, E) Fsp1Cre-labeled mammary stromal cells FACS-isolated and cultured from Gli2WT or Gli2ΔS mice were stimulated with murine growth hormone (mGH) (D) or 17β-Estradiol (E) for 24 hours and assayed for expression of Igf1 or Hgf, respectively. Note reduced expression of Igf1 and Hgf in Gli2ΔS as compared to Gli2WT. Measurements in panels (D, E) were made in triplicate from each of three individual Gli2WT or Gli2ΔS mice.

Article Snippet: Recombinant mouse Igf1 (791-MG-050) and recombinant mouse Wnt2b (customized quantity) were purchased from R&D Systems.

Techniques: Activity Assay, Labeling, Isolation, Quantitative RT-PCR, Expressing, Gene Expression, Staining, Cell Culture

Journal: Science (New York, N.Y.)

Article Title: Stromal Gli2 activity coordinates a niche signaling program for mammary epithelial stem cells.

doi: 10.1126/science.aal3485

Figure Lengend Snippet: Fig. 7. Slow-release Igf1, Wnt2b polymer implants rescue epithelial growth defects in Gli2∆S mice. (A) Schematic of slow-release polymer-based implantation experiments to test activity of Gh, Igf1 and combined Igf1/Wnt2b. (B) Control and Gh-containing polymers (dashed lines) were implanted bilaterally into immuno-deficient Gli2ΔS (NSG: Gli2ΔS) mice and ductal morphology was examined 7 days after implantation (n=3 implantations; one representative image of three is shown). Scale bar, 1 mm. (C) Numbers of terminal structures within 2 mm of implantation site are shown as fold increase relative to control implants. (D) Induction of Igf1 induction in control and GH-containing implants. Note that GH failed to induce terminal growth or Igf1 expression in NSG;Gli2ΔS mice. (E) Control and Igf1-containing polymers (dashed lines) were implanted bilaterally into NSG;Gli2ΔS mice at pubertal (p40) or adult (p70) stages. Ductal morphology was examined after 7 days (n=3 implantations; one representative image of three is shown). Scale bar, 1 mm. (F, G) Numbers of terminal structures (F) within 2 mm of implants and Igf1-induced expression of Egr1 and M-Csf (G) was calculated and normalized to control implants. (H) Control and Igf1/Wnt2b-containing polymers (dashed lines) were implanted bilaterally into NSG;Gli2ΔS mice, followed by morphological analyses after 7 days (n=3 implantations; one representative image of three is shown). Scale bar, 1mm. (I, J) Numbers of terminal (I) structures and induction of IGF1 or WNT2b target genes (J) were quantified as fold increase relative to control implants. Note that IGF1/WNT2b implants induce more terminal end growth as compared to IGF1 alone. Data are normalized to the mean values in the control, and are presented as mean ± s.e.m.; statistical significance was calculated by an unpaired Student’s t test (*P<0.05%, **P<0.01, ***P<0.001). First release: 9 March 2017 www.sciencemag.org (Page numbers not final at time of first release) 17

Article Snippet: Recombinant mouse Igf1 (791-MG-050) and recombinant mouse Wnt2b (customized quantity) were purchased from R&D Systems.

Techniques: Polymer, Activity Assay, Control, Expressing

Journal: Annals of Translational Medicine

Article Title: Prevention of lumbar disc degeneration through co-manipulation of insulin-like growth factor 1 and vascular endothelial growth factor

doi: 10.21037/atm-21-4977

Figure Lengend Snippet: Primer sequences

Article Snippet: ELISA was performed using the IGF1 kit (MG100; R&D System, Los Angeles, CA, USA), VEGF kit (RRV00; R&D System), proteoglycan kit ( {"type":"entrez-protein","attrs":{"text":"EKC39620","term_id":"405975022","term_text":"EKC39620"}} EKC39620 ; Biomatik, Wilmington, DE, USA), and collagen II kit (LS-F23885; LSBio, Seattle, WA, USA).

Techniques: Sequencing

Journal: Annals of Translational Medicine

Article Title: Prevention of lumbar disc degeneration through co-manipulation of insulin-like growth factor 1 and vascular endothelial growth factor

doi: 10.21037/atm-21-4977

Figure Lengend Snippet: Bioinformatics analyses of IGF1 and VEGF levels in LDD patients. (A) Volcano plot for the GEO database (GSE124272) to compare the gene profiling of disc cells between LDD patients and normal controls. Data were analyzed by R language. (B) Levels of IGF1 and VEGF in disc cells in LDD patients compared to normal controls. *P<0.05. N=16. IGF1, insulin-like growth factor 1; VEGF, vascular endothelial growth factor; LDD, lumbar disc degeneration.

Article Snippet: ELISA was performed using the IGF1 kit (MG100; R&D System, Los Angeles, CA, USA), VEGF kit (RRV00; R&D System), proteoglycan kit ( {"type":"entrez-protein","attrs":{"text":"EKC39620","term_id":"405975022","term_text":"EKC39620"}} EKC39620 ; Biomatik, Wilmington, DE, USA), and collagen II kit (LS-F23885; LSBio, Seattle, WA, USA).

Techniques:

Journal: Annals of Translational Medicine

Article Title: Prevention of lumbar disc degeneration through co-manipulation of insulin-like growth factor 1 and vascular endothelial growth factor

doi: 10.21037/atm-21-4977

Figure Lengend Snippet: Preparation of AAVs overexpressing IGF1 and depleting VEGF. (A) Schematic to show the structure of AAV-IGF1/shVEGF and AAV-scramble viruses. The experimental AAV used an AAV serotype 2 carrying recombinant IGF1 and shVEGF (connected by a p2A structure) under a CMV promoter, while 2 respective scramble controls were used in the control AAV-scramble. (B-E) A disc cell line, HNPSV, was transduced with the 2 AAVs and compared to untreated cells. (B-C) IGF1 levels determined by RT-qPCR (B) and ELISA (C). (D,E) VEGF levels determined by RT-qPCR (D) and ELISA (E). *P<0.05. NS: non-significant. N=5. AAV, adeno-associated virus; IGF1, insulin-like growth factor 1; VEGF, vascular endothelial growth factor.

Article Snippet: ELISA was performed using the IGF1 kit (MG100; R&D System, Los Angeles, CA, USA), VEGF kit (RRV00; R&D System), proteoglycan kit ( {"type":"entrez-protein","attrs":{"text":"EKC39620","term_id":"405975022","term_text":"EKC39620"}} EKC39620 ; Biomatik, Wilmington, DE, USA), and collagen II kit (LS-F23885; LSBio, Seattle, WA, USA).

Techniques: Recombinant, Control, Transduction, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Virus

Journal: Annals of Translational Medicine

Article Title: Prevention of lumbar disc degeneration through co-manipulation of insulin-like growth factor 1 and vascular endothelial growth factor

doi: 10.21037/atm-21-4977

Figure Lengend Snippet: AAV-IGF1/shVEGF treatment increases IGF1 in spinal tissue after LDD. We used a rat model of LDD to examine the effects of the AAV-IGF1/shVEGF gene therapy. Forty rats were randomly divided into 4 groups of 10. Group 1 was sham, group 2 was LDD and an orthotopic saline injection, group 3 was LDD and an orthotopic AAV-scr injection, and group 4 was LDD and an orthotopic AAV-IGF1/shVEGF injection. Rats were kept for 8 weeks before analysis. (A,B) The levels of IGF1 in the rat vertebral pulp and annulus fibrosus 8 weeks after treatment were examined by immunohistochemistry (A) and ELISA (B). *P<0.05. NS: non-significant. N=10. Scale bars are 100 µm. AAV, adeno-associated virus; IGF1, insulin-like growth factor 1; VEGF, vascular endothelial growth factor; LDD, lumbar disc degeneration.

Article Snippet: ELISA was performed using the IGF1 kit (MG100; R&D System, Los Angeles, CA, USA), VEGF kit (RRV00; R&D System), proteoglycan kit ( {"type":"entrez-protein","attrs":{"text":"EKC39620","term_id":"405975022","term_text":"EKC39620"}} EKC39620 ; Biomatik, Wilmington, DE, USA), and collagen II kit (LS-F23885; LSBio, Seattle, WA, USA).

Techniques: Saline, Injection, Immunohistochemistry, Enzyme-linked Immunosorbent Assay, Virus

Journal: Annals of Translational Medicine

Article Title: Prevention of lumbar disc degeneration through co-manipulation of insulin-like growth factor 1 and vascular endothelial growth factor

doi: 10.21037/atm-21-4977

Figure Lengend Snippet: AAV-IGF1/shVEGF treatment decreases VEGF in spinal tissue after LDD. (A,B) The levels of VEGF in the rat vertebral pulp and annulus fibrosus 8 weeks after treatment were examined by immunohistochemistry (A) and ELISA (B). *P<0.05. NS, non-significant. N=10. Scale bars are 100 µm. AAV, adeno-associated virus; IGF1, insulin-like growth factor 1; VEGF, vascular endothelial growth factor; LDD, lumbar disc degeneration.

Article Snippet: ELISA was performed using the IGF1 kit (MG100; R&D System, Los Angeles, CA, USA), VEGF kit (RRV00; R&D System), proteoglycan kit ( {"type":"entrez-protein","attrs":{"text":"EKC39620","term_id":"405975022","term_text":"EKC39620"}} EKC39620 ; Biomatik, Wilmington, DE, USA), and collagen II kit (LS-F23885; LSBio, Seattle, WA, USA).

Techniques: Immunohistochemistry, Enzyme-linked Immunosorbent Assay, Virus

Journal: Annals of Translational Medicine

Article Title: Prevention of lumbar disc degeneration through co-manipulation of insulin-like growth factor 1 and vascular endothelial growth factor

doi: 10.21037/atm-21-4977

Figure Lengend Snippet: AAV-IGF1/shVEGF treatment reduces cell apoptosis in spinal tissue after LDD. (A,B) Disc cell apoptosis was assessed by examining dissociated single cell suspensions from the rat vertebral pulp and annulus fibrosus 8 weeks after treatment by a flow cytometry-based apoptosis assay, shown by representative flow plots (A) and by quantification (B). *P<0.05. NS, non-significant. N=10. AAV, adeno-associated virus; IGF1, insulin-like growth factor 1; VEGF, vascular endothelial growth factor; LDD, lumbar disc degeneration.

Article Snippet: ELISA was performed using the IGF1 kit (MG100; R&D System, Los Angeles, CA, USA), VEGF kit (RRV00; R&D System), proteoglycan kit ( {"type":"entrez-protein","attrs":{"text":"EKC39620","term_id":"405975022","term_text":"EKC39620"}} EKC39620 ; Biomatik, Wilmington, DE, USA), and collagen II kit (LS-F23885; LSBio, Seattle, WA, USA).

Techniques: Flow Cytometry, Apoptosis Assay, Virus

Journal: Annals of Translational Medicine

Article Title: Prevention of lumbar disc degeneration through co-manipulation of insulin-like growth factor 1 and vascular endothelial growth factor

doi: 10.21037/atm-21-4977

Figure Lengend Snippet: AAV-IGF1/shVEGF treatment enhances spinal proteoglycan and collagen II levels after LDD. (A,B) ELISA analysis of the levels of proteoglycan (A) and collagen II (B) in the rat vertebral pulp and annulus fibrosus 8 weeks after treatment. *P<0.05. NS: non-significant. N=10. AAV, adeno-associated virus; IGF1, insulin-like growth factor 1; VEGF, vascular endothelial growth factor; LDD, lumbar disc degeneration.

Article Snippet: ELISA was performed using the IGF1 kit (MG100; R&D System, Los Angeles, CA, USA), VEGF kit (RRV00; R&D System), proteoglycan kit ( {"type":"entrez-protein","attrs":{"text":"EKC39620","term_id":"405975022","term_text":"EKC39620"}} EKC39620 ; Biomatik, Wilmington, DE, USA), and collagen II kit (LS-F23885; LSBio, Seattle, WA, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Virus

Journal: Journal of Hematology & Oncology

Article Title: CRISPR/Cas9-mediated deletion of Interleukin-30 suppresses IGF1 and CXCL5 and boosts SOCS3 reducing prostate cancer growth and mortality

doi: 10.1186/s13045-022-01357-6

Figure Lengend Snippet: IL30-dependent regulation of IGF1 production in human PC cells and IGF1-mediated autocrine growth loop. A , B Elisa assay of IGF1 release by wild-type, EV and IL30 gene-transfected DU145 ( A ) and PC3 ( B ) cells . ANOVA: p < 0.0001. * p < 0.01, Tukey HSD test compared with WT and EV-transfected cells. Results are expressed as mean ± SD. C Cytofluorimetric analyses of IGF1R expression in PC3 and DU145 cells. Red lines: isotype control. Experiments were performed in triplicate. D , E Elisa assay of IGF1 release by wild-type DU145 ( D ) and PC3 ( E ) cells, after the treatment with anti-IL30 Abs. (D) ANOVA: p < 0.05. * p < 0.05, Tukey HSD test compared with DU145 cells untreated or treated with 5 μg/mL. E ANOVA: p < 0.001. * p < 0.01, Tukey HSD test compared with untreated PC3 cells. Results are expressed as mean ± SD. F , G MTT assay of DU145 ( F ) and PC3 ( G ) cells, untreated (0.0 ng/mL) or treated with rhIGF1 (5.0, 10, 30, 50 ng/mL). ANOVA: p < 0.0001. * p < 0.01, Tukey HSD test compared with 0 ng/mL. ** p < 0.01, Tukey HSD test compared with 0 and 5 ng/mL. *** p < 0.01, Tukey HSD test compared with 0, 5 and 10 ng/mL. Results are expressed as mean ± SD. H , I MTT assay of DU145 (H) and PC3 (I) cells, untreated (0.0 μg/mL) or treated with anti-IGF1 Abs (0.1, 0.4, 0.8 μg/mL in DU145; 0.25, 0.50, 0.70 μg/mL in PC3). (H) ANOVA: p < 0.0001. * p < 0.01, Tukey HSD test compared with 0.0 μg/mL. ** p < 0.01, Tukey HSD test compared with 0.0 and 0.1 μg/mL. *** p < 0.05, Tukey HSD test compared with 0.0, 0.1 and 0.4 μg/mL. (I) ANOVA: p < 0.0001. * p < 0.01, Tukey HSD test compared with 0.00 µg/mL. ** p < 0.01, Tukey HSD test compared with 0.00 and 0.25 μg/mL. Results are expressed as mean ± SD. J , K MTT assay of wild-type and IL30 gene-transfected DU145 ( J ) and PC3 ( K ) cells, untreated (0.0 μg/mL), or treated with anti-IGF1 Abs (30 μg/mL). ANOVA: p < 0.0001. * p < 0.01, Tukey HSD test compared with wild-type cells. ** p < 0.01, Tukey HSD test compared with wild-type and IL30-transfected cells. Results are expressed as mean ± SD

Article Snippet: Quantitation of IL30, CXCL5 and IGF1, in the supernatant derived from murine or human PC cells, was carried out using the following ELISA kits, according to manufacturer’s protocols: Human CXCL5/ENA-78 Quantikine ELISA Kit (#DX000, R&D Systems, Minneapolis, MN, USA); human IGF1 ELISA Kit (#ab211651, Abcam, Cambridge, UK); and mouse Interleukin-27 subunit alpha ELISA Kit (#CSB-E08466m, Cusabio, Wuhan, China).

Techniques: Enzyme-linked Immunosorbent Assay, Transfection, Expressing, MTT Assay

Journal: Journal of Hematology & Oncology

Article Title: CRISPR/Cas9-mediated deletion of Interleukin-30 suppresses IGF1 and CXCL5 and boosts SOCS3 reducing prostate cancer growth and mortality

doi: 10.1186/s13045-022-01357-6

Figure Lengend Snippet: Tumor growth and survival of mice-bearing IL30-deficient or IL30-overexpressing PC of murine or human origin. A Mean volume of tumors developed in NSG mice, after s.c. implantation of wild type, EV- or IL30-DU145 cells. ANOVA, p < 0.0001; Tukey HSD test, p < 0.01 versus wild type or EV-transfected DU145 cells. Results are expressed as mean ± SD. B Mean volume of tumors developed in NSG mice, after s.c. implantation of wild type, NTgRNA-treated or IL30KO-DU145 cells. ANOVA, p < 0.0001; Tukey HSD test, p < 0.01 versus wild type or NTgRNA-treated DU145 cells. Results are expressed as mean ± SD. C Average number of lung metastasis spontaneously developed in NSG mice, which developed tumors after s.c. implantation of wild type, NTgRNA-treated or IL30KO-DU145 cells. ANOVA: p < 0.0001. * p < 0.01, Tukey HSD test versus DU145 or NTgRNA-treated DU145 cells. Results are expressed as mean ± SD. D Kaplan–Meier survival curves of mice-bearing tumors developed after s.c. implantation of wild type, NTgRNA-treated or IL30KO-DU145 cells. Log-rank test: p = 0.000009. E Immunopathological features of tumors developed in NSG mice, after s.c. implantation of IL30KO-DU145, wild type DU145 and IL30-DU145 cells. Expression of IGF1, proliferation (Ki67 Abs) and vascularization (CD31 Abs) were prominent in IL30-overexpressing tumors, and scanty in IL30KO tumors, compared with wild type tumors. Cytoplasmic and nuclear expression of NFKB1 was strong in IL30-overexpressing tumors and faint in IL30KO tumors. The immunopathological features of tumors developed after implantation of control, EV-transfected or NTgRNA-treated, cells were comparable to those of wild type tumors. Magnification: × 400; CD31, × 200. F Immunopathological features of tumors developed in NSG mice, after s.c. implantation of IL30KO-DU145 and wild type DU145 cells. Expression of tumor suppressor genes CDH1/E-Cadh, DKK3, PTEN, RARb and SOCS3 was stronger in IL30KO-DU145 tumors, when compared to wild type tumors, whereas the expression of PTGS2 was weaker. The immunopathological features of control tumors, developed in NSG after implantation of NTgRNA-treated cells, were comparable to those of wild type tumors. Magnification: × 400. G Automated immune cell count and microvessel density in tumors developed in NSG mice, after implantation of wild type, EV- or IL30 gene-transfected, NTgRNA-treated and IL30KO-DU145 cells, assessed by immunohistochemistry, as described in Methods. ANOVA, p < 0.0001. * p < 0.01, Tukey HSD test compared with DU145, NTgRNA-treated DU145 and EV-transfected DU145. ** p < 0.01, Tukey HSD test compared with DU145, NTgRNA-treated DU145, IL30KO-DU145 and EV-transfected DU145. Results are expressed as mean ± SD. H The immune cell contexture of tumors developed in NSG mice, after s.c. implantation of IL30KO and IL30-DU145 cells, revealed a higher content of macrophages (anti-F4/80 Abs) and granulocytes (anti-Ly-6G Abs) in IL30-overexpressing tumors, compared to wild type tumors. By contrast, these immune cell populations were scarce to absent in IL30-deficient tumors. Magnification: × 400. Scale bars: 30 μm. I Western blot analysis of IL30 protein expression in wild type, EV- and IL30 gene-transfected TRAMP-C1 cells. J Mean volume of tumors developed in C57BL/6J mice, after s.c. implantation of wild type, EV- or IL30-TRAMP-C1 cells. ANOVA, p < 0.0001. Tukey HSD test, p < 0.01 versus wild type or EV-TRAMP-C1 cells. Results are expressed as mean ± SD. K Immunopathological features of tumors developed in C57BL/6J mice, after s.c. implantation of wild type or IL30-TRAMP-C1 cells revealed that proliferation (PCNA Abs), microvascular density (CD31 Abs) and granulocyte content (Ly-6G Abs) were higher in IL30-overexpressing tumors, than in control tumors. Cancer cells that formed IL30-TRAMP-C1 tumors showed reduced cytoplasmic expression of PTEN and an increased nuclear and cytoplasmic expression of NFKB1. Magnification: × 400; CD31, × 200. L Automated immune cell count in tumors developed in C57BL/6J mice, after s.c. implantation of wild type, EV or IL30 gene-transfected TRAMP-C1 cells, assessed by immunohistochemistry, as described in Methods. ANOVA, p < 0.001. * p < 0.01, Tukey HSD test compared with wild type or EV-TRAMP-C1 cells. Results are expressed as mean ± SD. M Percentage of lung metastasis spontaneously developed in C57BL/6J mice, which developed tumors after s.c. implantation of wild type or IL30 gene-transfected TRAMP-C1 cells. *Fisher’s exact test, p = 0.03 versus EV-TRAMP-C1 or TRAMP-C1. Results from mice implanted with EV-TRAMP-C1 cells were comparable to those obtained from mice implanted with wild type cells. N Kaplan–Meier survival curves of mice-bearing tumors developed after s.c. implantation of wild type, EV- or IL30 gene-transfected TRAMP-C1 cells. Log-rank test: p = 0.000124

Article Snippet: Quantitation of IL30, CXCL5 and IGF1, in the supernatant derived from murine or human PC cells, was carried out using the following ELISA kits, according to manufacturer’s protocols: Human CXCL5/ENA-78 Quantikine ELISA Kit (#DX000, R&D Systems, Minneapolis, MN, USA); human IGF1 ELISA Kit (#ab211651, Abcam, Cambridge, UK); and mouse Interleukin-27 subunit alpha ELISA Kit (#CSB-E08466m, Cusabio, Wuhan, China).

Techniques: Transfection, Expressing, Cell Counting, Immunohistochemistry, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Anti-Müllerian Hormone, Growth Hormone, and Insulin-Like Growth Factor 1 Modulate the Migratory and Secretory Patterns of GnRH Neurons

doi: 10.3390/ijms22052445

Figure Lengend Snippet: Effect of different concentrations of insulin-like growth factor 1 (IGF1) on GN11 cell migration. Data are expressed as mean ± SD, n = 8. One experiment representative of three separate experiments is shown. * p < 0.05; **** p < 0.0001 (One-way ANOVA followed by the Dunnett post-hoc test).

Article Snippet: Human recombinant AMH and GH, and mouse recombinant IGF1, were purchased from R&D Systems, Inc. (Minneapolis, MN, USA).

Techniques: Migration

Journal: International Journal of Molecular Sciences

Article Title: Anti-Müllerian Hormone, Growth Hormone, and Insulin-Like Growth Factor 1 Modulate the Migratory and Secretory Patterns of GnRH Neurons

doi: 10.3390/ijms22052445

Figure Lengend Snippet: Insulin-like growth factor (IGF1) treatment alters GN11 cytoskeleton organization. GN11 cells treated with IGF1 (at 30 and 100 ng/mL) were immune-labeled for F-actin (red) and α-tubulin (green) to reveal microfilament and microtubule organization, respectively. Single channels are shown as black and white images. Arrowheads point at cells displaying a prominent fusiform morphology. Scale bar: 25 µm.

Article Snippet: Human recombinant AMH and GH, and mouse recombinant IGF1, were purchased from R&D Systems, Inc. (Minneapolis, MN, USA).

Techniques: Labeling

Journal: International Journal of Molecular Sciences

Article Title: Anti-Müllerian Hormone, Growth Hormone, and Insulin-Like Growth Factor 1 Modulate the Migratory and Secretory Patterns of GnRH Neurons

doi: 10.3390/ijms22052445

Figure Lengend Snippet: Quantitation (%) of GN11 cells with an elongated morphology after treatment with AMH, GH, and IGF1. AMH, Anti-Müllerian hormone; GH, growth hormone; IGF1, insulin-like growth factor 1; PBS, phosphate buffer saline; HCl, hydrochloride.

Article Snippet: Human recombinant AMH and GH, and mouse recombinant IGF1, were purchased from R&D Systems, Inc. (Minneapolis, MN, USA).

Techniques: Quantitation Assay, Saline

Journal: International Journal of Molecular Sciences

Article Title: Anti-Müllerian Hormone, Growth Hormone, and Insulin-Like Growth Factor 1 Modulate the Migratory and Secretory Patterns of GnRH Neurons

doi: 10.3390/ijms22052445

Figure Lengend Snippet: Effect of exposure to insulin-like growth factor 1 (IGF1) on gonadotropin-releasing hormone (GnRH) release by GT1-7 cells. Cells were incubated for 90 min with different concentrations of IGF1. Data are expressed as mean ± SD, n = 4. One experiment representative of three separate experiments is shown. * p < 0.05; ** p < 0.01; **** p < 0.0001 vs. Control (black bar) (One-way ANOVA followed by the Dunnett post-hoc test).

Article Snippet: Human recombinant AMH and GH, and mouse recombinant IGF1, were purchased from R&D Systems, Inc. (Minneapolis, MN, USA).

Techniques: Incubation, Control

Journal: Communications Biology

Article Title: Paradoxical regulation of IGF2 in promoting lipid metabolism in adipose tissues

doi: 10.1038/s42003-025-08458-1

Figure Lengend Snippet: A , B “U” and inverted “U” shaped relationship between IGF2 levels and the lipid species detected by LC-MS/MS assay utilizing a polynomial fourth order equation to fit the non-linear regression curve, where R² (R-squared) represents the coefficient of determination, and Sy.x represents the standard deviation of the residuals, n = 200. C , D “U” and inverted “U” shaped relationship between IGF2 levels and the lipid species detected by ELISA kit assay utilizing a polynomial fourth order equation to fit the non-linear regression curve, n = 200. E , F Pearson correlation analyses of L-IGF2 levels and H-IGF2 levels with triglyceride conducted by LC-MS/MS assay, n = 200, all p < 0.01. G , H Pearson correlation analyses of L-IGF2 levels and H-IGF2 levels with HDL-c conducted by LC-MS/MS assay, n = 200, all p < 0.05. I , J Pearson correlation analyses of L-IGF2 levels and H-IGF2 levels with triglyceride conducted by ELISA kit assay, n = 200, all p < 0.001. K , L Pearson correlation analyses of L-IGF2 levels and H-IGF2 levels with HDL-c conducted by ELISA kit assay, n = 200, all p < 0.05. M , N Multiple stepwise logistic regression analysis of MetS, HOMA-IR, and other metabolic subgroups (central obesity, hypertension, hyperglycemia, hypertriglyceridemia, and low HDL-c) connected with L-IGF2 and H-IGF2 levels conducted by LC-MS/MS assay and ELISA kit assay, respectively.

Article Snippet: The measurement of mice serum IGF1 level was conducted with an ELISA kit (E-EL-M3006, Elabscience, China).

Techniques: Liquid Chromatography with Mass Spectroscopy, Standard Deviation, Enzyme-linked Immunosorbent Assay

Journal: Communications Biology

Article Title: Paradoxical regulation of IGF2 in promoting lipid metabolism in adipose tissues

doi: 10.1038/s42003-025-08458-1

Figure Lengend Snippet: A The utilization of Nile red staining and Oil red O staining techniques revealed the presence of lipid droplets in 3T3-L1 adipocytes after IGF2 overexpression and knockdown treatment. B , C Quantification of cellular triglyceride (TG) content and the levels of free fatty acid (FFA) in the medium in 3T3-L1 adipocytes after IGF2 overexpression and knockdown treatment. * p < 0.05, ** p < 0.01. D , E The mRNA levels of genes associated with adipogenesis, lipogenesis, and lipolysis in 3T3-L1 cells transfected with Ad-IGF2 or IGF2-RNAi as well as the corresponding controls by RT-qPCR assays, using Ppia as internal controls, n = 6, Data represent the mean ± SD; * p < 0.05, ** p < 0.01, *** p < 0.001 by two-tailed, unpaired Student’s t test. F Protein expression levels of genes related to adipogenesis, lipogenesis and lipolysis in the 3T3-L1 adipocytes by western blot assays. G Mice adipose tissue has been dissected and isolated for primary adipocyte culture, and the results of 0, 4, 8 and 12 days were induced by the classic “Cocktail” induction differentiation regimen. H Primal cell supernatant was extracted during the above induction differentiation process, and IGF2 concentration was detected by ELISA kit. n = 3. I 3T3-L1 preadipocytes underwent treatment via different concentration gradients of recombinant IGF2 protein powder and induced differentiation. Oil red O staining employment demonstrated that the deposition of lipid droplets within adipocytes.

Article Snippet: The measurement of mice serum IGF1 level was conducted with an ELISA kit (E-EL-M3006, Elabscience, China).

Techniques: Staining, Over Expression, Knockdown, Transfection, Quantitative RT-PCR, Two Tailed Test, Expressing, Western Blot, Isolation, Concentration Assay, Enzyme-linked Immunosorbent Assay, Recombinant

Journal: iScience

Article Title: Contrasting consequences of podocyte insulin-like growth factor 1 receptor inhibition

doi: 10.1016/j.isci.2024.109749

Figure Lengend Snippet: In vitro differential suppression of podocyte IGF1R activity reveals that partial inhibition is beneficial but near total loss is highly detrimental (A) Representative Western blot shows >90% reduction of IGF1R protein in NC-IGF1RKD cells but no reduction of IR protein expression. Bar graphs show densitometry expressed as the mean fold change +/− SEM, t-test, ∗∗∗∗ p < 0.0001, n = 18 independent experiments. (B) Phosphorylation of AKT and p44/42MAPK in response to acute IGF1 stimulation at 10 ng and 100 ng/mL for 10 min was significantly reduced in NC-IGF1RKD podocytes. Data are expressed as the mean ± SEM, one-way ANOVA with Tukey’s multiple comparison test, ∗∗ p < 0.005, n = 3 independent experiments. (C) Western blot shows that IGF1R expression is reduced by ∼70% in wild-type podocytes exposed to 100 nM picropodophyllin for 24 h. Data are expressed as the mean ± SEM, t-test, ∗ p < 0.05, n = 3 independent experiments. No significant change in IR expression was observed. (D) Western blot shows the phosphorylation of AKT and p44/42MAPK in response to acute IGF1 stimulation at 10 ng and 100 ng/mL for 10 min in podocytes exposed to 100 nM picropodophyllin for 24 h. Data expressed as the mean ± SEM, ∗ p < 0.05, n = 3 independent experiments. (E) ∼50% of NC-IGF1RKD cells survive 7 days after gene excision. Treatment of wild-type podocytes with 100 nM picropodophyllin for 24 h has no effect on cell survival. Data are expressed as the mean ± SEM, t-test, ∗∗ p < 0.005, n = 3–4 independent experiments. See also Figures S5 and .

Article Snippet: For acute insulin and IGF1 stimulation, Conditionally immortalised wild-type mouse podocyte cell were serum starved for 4 h then 10 nM and 100 nM of insulin (Biotechne, Cat# 3435) or 10 ng/mL and 100 ng/mL IGF1 (Novus, Cat# NBP2-35081) was applied to the cells for 10 min.

Techniques: In Vitro, Activity Assay, Inhibition, Western Blot, Expressing, Phospho-proteomics, Comparison

Journal: iScience

Article Title: Contrasting consequences of podocyte insulin-like growth factor 1 receptor inhibition

doi: 10.1016/j.isci.2024.109749

Figure Lengend Snippet:

Article Snippet: For acute insulin and IGF1 stimulation, Conditionally immortalised wild-type mouse podocyte cell were serum starved for 4 h then 10 nM and 100 nM of insulin (Biotechne, Cat# 3435) or 10 ng/mL and 100 ng/mL IGF1 (Novus, Cat# NBP2-35081) was applied to the cells for 10 min.

Techniques: Recombinant, Protease Inhibitor, Enzyme-linked Immunosorbent Assay, Quantitation Assay, Staining, RNAscope, Western Blot, Cell Culture, Software, Plasmid Preparation