Journal: Disease models & mechanisms

Article Title: The ALK inhibitor PF-06463922 is effective as a single agent in neuroblastoma driven by expression of ALK and MYCN.

doi: 10.1242/dmm.024448

Figure Lengend Snippet: Fig. 1. Inhibition of neuroblastoma cell lines employing crizotinib and PF-06463922. Treatment of neuroblastoma cells with the ALK TKIs crizotinib and PF-06463922. (A,B) Proliferation was assessed over 5 days using the resazurin cell proliferation assay in the following neuroblastoma cell lines: CLB-BAR, CLB-GE, CLB-PE, SK-N-AS and IMR32. Neuroblastoma cell line characteristics: CLB-GE – MYCN amplified, 1p deletion, 17q gain, amplified ALK amplicon containing an ALKF1174V mutation; CLB-BAR – amplified MYCN/ALK, Δexon 4-11, 1p deletion, 17q gain; IMR32 – MYCN, wt sequence but exons 2-4 are amplified, 1p deletion, 17q gain; CLB-PE – 1p gain, amplified MYCN, 17q gain; SK-N-AS – IGF-1 overexpressing, 1p deletion, 1q gain, 17q gain, 17 deletion. Cells lines were treated with increasing concentrations of either PF-06463922 (A) or crizotinib (B) as indicated. Data are mean±s.d. of the fold-relative fluorescence from treated cells relative to untreated cells from three independent experiments. (C) CLB-BAR, CLB-GE neuroblastoma cells were treated for 6 h with either crizotinib or PF-04643922 as indicated. Cellswere harvested andpre-clearedcelllysates were analyzed onSDS PAGE followedbywesternblotting for ALK, phospho-ALK-Y1278,phospho-ERK5, pan-ERK5 phospho-ERK1/2 and pan-ERK. Actin was employed as a loading control. Protein band intensities were quantified by Image Studio Lite 3.1 and normalized to untreated samples.

Article Snippet: Primary antibodies were: phospho-ALK (Y1604 (1:2000; 3341) and Y1278 (1:2000; 12127), phospho-ERK1/2, actin (1:2000; 4967), phospho-Akt (1:2000; 92711), MYCN (1:2000; 9405), Ki67 (1:100; 9129), GADPH and phospho-ERK5 (1:1000; 3371) from Cell Signaling Technology (Danvers, MA).

Techniques: Inhibition, Proliferation Assay, Amplification, Mutagenesis, Sequencing, Fluorescence, Control

Journal: Disease models & mechanisms

Article Title: The ALK inhibitor PF-06463922 is effective as a single agent in neuroblastoma driven by expression of ALK and MYCN.

doi: 10.1242/dmm.024448

Figure Lengend Snippet: Fig. 3. Comparison of inhibition effects of crizotinib and PF-06463922 on WT and neuroblastoma gain-of-function mutant TKDs by in vitro kinase assay. (A,B) Different ALK TKD proteins were incubated with either PF-06463922 (A) or crizotinib (B) in the presence of ATP (0.1 mM) and substrate peptides (0.2 mM). The incorporation of labelled γ-32P was detected under different conditions. Background counts from no-enzyme controls were subtracted, and the data were normalized to the 0 nM inhibitor reactions. (C) IC50 values from A,B were calculated by fitting data to a log (inhibitor) versus normalized response (variable slope) equation in GraphPad Prism 6.0. All data are shown as mean±s.d. from at least two independent experiments. (D) Crystal structures of ALK kinase domain in complex with PF-06463922 (top) or crizotinib (bottom). Compounds indicated in black. Gain-of-function ALK mutations F1174, R1192P, F1245, G1269 and Y1278 are shown as red spheres. The ribbon diagram displays αC helix (1157-1173; orange), catalytic loop (1246-125; magenta), activation loop (1271-1288; cyan) with DFG motif marked in blue. Figures were generated with PyMol using published coordinates (Protein data bank code: 4CLI and 2XP2).

Article Snippet: Primary antibodies were: phospho-ALK (Y1604 (1:2000; 3341) and Y1278 (1:2000; 12127), phospho-ERK1/2, actin (1:2000; 4967), phospho-Akt (1:2000; 92711), MYCN (1:2000; 9405), Ki67 (1:100; 9129), GADPH and phospho-ERK5 (1:1000; 3371) from Cell Signaling Technology (Danvers, MA).

Techniques: Comparison, Inhibition, Mutagenesis, In Vitro, Kinase Assay, Incubation, Activation Assay, Generated

Journal: Disease models & mechanisms

Article Title: The ALK inhibitor PF-06463922 is effective as a single agent in neuroblastoma driven by expression of ALK and MYCN.

doi: 10.1242/dmm.024448

Figure Lengend Snippet: Fig. 5. Efficacy of PF-06463922 in an ALKF1174 CLB-GE xenograft neuroblastoma model. 4.5×106 CLB-GE cells were injected into left shoulder subcutaneously. (A) Tumor growth curves represent the average volume of vehicle group and PF-06463922-treated group, P≤0.05 (n=5 for each group). (B) Average tumor weights in vehicle group and PF-06463922 group are displayed, P=0.02. (C) Average body weight on the day of sacrifice is shown, P>0.05. (D) Immunoblotting analysis of indicated proteins from tumors collected after 8 days of treatment with vehicle or PF-0646399 and relapsed tumors. Tumor lysates were harvested, pre-cleared and analyzed by western blotting for ALK, phospho-ALK-Y1278, MYCN, phospho-ERK1/2 and phospho-AKT. Pan-ERK and pan-Akt were employed as loading controls. (E) Immunohistochemical staining of tumors for Ki-76 as a measure of proliferation rate as indicated. Ki67-positive cells were counted manually per field of vision and quantitative results (n=15). Statistical analysis shows significant difference between vehicle and PF-06463922- treated group, P<0.002 using unpaired t-test. Data in all graphs presented as mean±s.d.

Article Snippet: Primary antibodies were: phospho-ALK (Y1604 (1:2000; 3341) and Y1278 (1:2000; 12127), phospho-ERK1/2, actin (1:2000; 4967), phospho-Akt (1:2000; 92711), MYCN (1:2000; 9405), Ki67 (1:100; 9129), GADPH and phospho-ERK5 (1:1000; 3371) from Cell Signaling Technology (Danvers, MA).

Techniques: Injection, Western Blot, Immunohistochemical staining, Staining

Journal: Disease models & mechanisms

Article Title: The ALK inhibitor PF-06463922 is effective as a single agent in neuroblastoma driven by expression of ALK and MYCN.

doi: 10.1242/dmm.024448

Figure Lengend Snippet: Fig. 6. Preclinical efficacy of PF-06463922 in a murine model of Th-ALKF1174L/MYCN-driven neuroblastoma. (A) Waterfall plots of tumoral response in Th-ALKF1174L/MYCN mice treated with vehicle, crizotinib (100 mg/kg, once daily) or PF-06463922 (10 mg/kg, twice daily). Each bar indicates percent change in the volume of an individual tumor, as assessed by T2-weighted MRI, on day 7 compared with day 0 of treatment. (B) Representative MRI of each treatment group on day 0 and day 7 after treatment of indicated vehicle or drug. Hashed white line indicates tumor border. (C) Immunoblot analysis of phosphorylated ALK (phospho-ALK-Y1278), total ALK, and MYCN of tumors treated for 2 days with vehicle or PF-06463922. GAPDH was employed as loading control. Arrow indicates pALK-Y1278. (D) Quantification of band intensity of MYCN relative to GAPDH (left) and ratio of band intensity of pALK over total ALK (right) comparing vehicle versus treated samples. (E) Meso Scale Discovery assay depicted as electrochemiluminescence signal of treated samples relative to the respective vehicle controls for total ALK, phospho-ALK-Y1568, and the ratio of pALK to ALK. (F) Representative H&E (top) and immunohistochemical images for Ki67 (bottom) of vehicle and treated samples. Quantification of overall percentage of area positive for Ki67 (right). Error bars represent s.d. between individual animals (per group: n=4 in A, n=5 in C-F). P-values equal unpaired t-test comparison between vehicle and treatment groups.

Article Snippet: Primary antibodies were: phospho-ALK (Y1604 (1:2000; 3341) and Y1278 (1:2000; 12127), phospho-ERK1/2, actin (1:2000; 4967), phospho-Akt (1:2000; 92711), MYCN (1:2000; 9405), Ki67 (1:100; 9129), GADPH and phospho-ERK5 (1:1000; 3371) from Cell Signaling Technology (Danvers, MA).

Techniques: Western Blot, Control, Electrochemiluminescence, Immunohistochemical staining, Comparison

Journal: EMBO Reports

Article Title: Phase‐separated foci of EML4‐ALK facilitate signalling and depend upon an active kinase conformation

doi: 10.15252/embr.202153693

Figure Lengend Snippet: Antibodies used for immunofluorescence (IF) and Western blotting (WB) and dilutions.

Article Snippet: Anti‐ALK (D5F3) rabbit monoclonal , CST , 3633 , 1:100 , 1:1,000.

Techniques: Immunofluorescence, Western Blot

Journal: Cell Cycle

Article Title: Long noncoding RNA BMPR1B-AS1 facilitates endometrial cancer cell proliferation and metastasis by sponging miR-7-2-3p to modulate the DCLK1/Akt/NF-κB pathway

doi: 10.1080/15384101.2022.2060003

Figure Lengend Snippet: Sequences used in this study.

Article Snippet: Digoxigenin (DIG)-labeled BMPR1B-AS1 probes were designed and synthesized by BOSTER (Wuhan, China).

Techniques:

Journal: Cell Cycle

Article Title: Long noncoding RNA BMPR1B-AS1 facilitates endometrial cancer cell proliferation and metastasis by sponging miR-7-2-3p to modulate the DCLK1/Akt/NF-κB pathway

doi: 10.1080/15384101.2022.2060003

Figure Lengend Snippet: The expression of lncRNA BMPR1B-AS1 in endometrial cancer tissues and cell lines. A, Volcano map of dysregulated lncRNAs between EC tissues and adjacent normal tissues. The results were obtained from TCGA database. B, Expression levels of lncRNA BMPR1B-AS1 in paired EC and adjacent noncancerous tissues (n = 28). C, RT-qPCR analysis of BMPR1B-AS1 expression in human EC cell lines (Ishikawa, Hec-1a and Hec-1b cells). The data are representative of three independent experiments. Bars, ±SD. **p < 0.01, ****p < 0.0001.

Article Snippet: Digoxigenin (DIG)-labeled BMPR1B-AS1 probes were designed and synthesized by BOSTER (Wuhan, China).

Techniques: Expressing, Quantitative RT-PCR

Journal: Cell Cycle

Article Title: Long noncoding RNA BMPR1B-AS1 facilitates endometrial cancer cell proliferation and metastasis by sponging miR-7-2-3p to modulate the DCLK1/Akt/NF-κB pathway

doi: 10.1080/15384101.2022.2060003

Figure Lengend Snippet: Overexpression of BMPR1B-AS1 promotes the proliferation, migration and invasion of Hec-1b cells while inhibiting apoptosis. A, Overexpression of BMPR1B-AS1 was confirmed by RT-qPCR. B, Overexpression of BMPR1B-AS1 promoted the proliferation of Hec-1b cells. C, E, G, H, Transwell migration assay (magnification, 200×) and wound healing assay (magnification, 100×) showed that overexpression of BMPR1B-AS1 facilitated the migration of Hec-1b cells. D, F, Transwell invasion assay showed that overexpression of BMPR1B-AS1 facilitated the invasion of Hec-1b cells (magnification, 200×). I, K, Flow cytometry results showed that BMPR1B-AS1 overexpression inhibited Hec-1b cell apoptosis. J, L, Flow cytometry results showed that BMPR1B-AS1 overexpression increased the accumulation of cells in the S-phase and decreased the accumulation of cells in the G0/G1 phase. M, N, O, P, Q, Western blot and analysis showed that the protein level of E-cadherin was decreased, but the protein levels of N-cadherin, Cyclin D1 and CDK4 were increased in the BMPR1B-AS1 overexpression group. Lv, lentiviral vector. The data are representative of three independent experiments. Bars, ±SD. *P < .05, **P < .01, ***P < .001, ****P < 0.0001.

Article Snippet: Digoxigenin (DIG)-labeled BMPR1B-AS1 probes were designed and synthesized by BOSTER (Wuhan, China).

Techniques: Over Expression, Migration, Quantitative RT-PCR, Transwell Migration Assay, Wound Healing Assay, Transwell Invasion Assay, Flow Cytometry, Western Blot, Plasmid Preparation

Journal: Cell Cycle

Article Title: Long noncoding RNA BMPR1B-AS1 facilitates endometrial cancer cell proliferation and metastasis by sponging miR-7-2-3p to modulate the DCLK1/Akt/NF-κB pathway

doi: 10.1080/15384101.2022.2060003

Figure Lengend Snippet: Knockdown of BMPR1B-AS1 expression inhibits the proliferation, migration and invasion of Ishikawa cells while promoting apoptosis. A, BMPR1B-AS1 knockdown efficiency was confirmed by RT-qPCR. B, Knockdown of BMPR1B-AS1 expression inhibited the proliferation of Ishikawa cells. C, E, G, H, Transwell migration assay (magnification, 200×) and wound healing assay (magnification, 100×) showed that knockdown of BMPR1B-AS1 expression decreased the migration of Ishikawa cells. D, F, Transwell invasion assay showed that knockdown of BMPR1B-AS1 expression decreased the invasion of Ishikawa cells (magnification, 200×). I, K, Flow cytometry results showed that BMPR1B-AS1 knockdown facilitated Ishikawa cell apoptosis. J, L, Flow cytometry results showed that BMPR1B-AS1 knockdown increased the accumulation of cells in the G0/G1 phase and decreased the accumulation of cells in the S-phase. M, N, O, P, Q, Western blot and analysis showed that the protein level of E-cadherin was increased, but the protein levels of N-cadherin, Cyclin D1 and CDK4 were decreased in the BMPR1B-AS1 knockdown group. Sh, short hairpin. The data are representative of three independent experiments. Bars, ±SD. **P < .01, ***P < .001, ****P < 0.0001.

Article Snippet: Digoxigenin (DIG)-labeled BMPR1B-AS1 probes were designed and synthesized by BOSTER (Wuhan, China).

Techniques: Expressing, Migration, Quantitative RT-PCR, Transwell Migration Assay, Wound Healing Assay, Transwell Invasion Assay, Flow Cytometry, Western Blot

Journal: Cell Cycle

Article Title: Long noncoding RNA BMPR1B-AS1 facilitates endometrial cancer cell proliferation and metastasis by sponging miR-7-2-3p to modulate the DCLK1/Akt/NF-κB pathway

doi: 10.1080/15384101.2022.2060003

Figure Lengend Snippet: BMPR1B-AS1 functions as an efficient miR-7-2-3p sponge in Hec-1b and Ishikawa cells. A, FISH assay showed that the intracellular localization of BMPR1B-AS1 was mainly in the cytoplasm in Ishikawa cells. 18S and U6 were used as controls. B, Putative binding sites of miR-7-2-3p and BMPR1B-AS1. C, Negative correlation was found between BMPR1B-AS1 expression and miR-7-2-3p expression among the 28 endometrial cancer tissue specimens. R = −.549, P = .002. D, E, The effect of the miR-7-2-3p mimic on the luciferase activities of 293 T cells transfected with WT or MUT BMPR1B-AS1 was detected 24 h and 48 h after transfection, respectively. F, RT-qPCR results showed that miR-7-2-3p expression was downregulated after BMPR1B-AS1 overexpression in Hec-1b cells. G, RT-qPCR results showed that miR-7-2-3p expression was upregulated after BMPR1B-AS1 knockdown in Ishikawa cells. H, miR-7-2-3p expression was upregulated after transfection with the miR-7-2-3p mimic in Hec-1b cells. I, The miR-7-2-3p mimic reversed the BMPR1B-AS1-mediated downregulation of miR-7-2-3p expression. J, miR-7-2-3p expression was downregulated after transfection of Ishikawa cells with the miR-7-2-3p inhibitor. K, The miR-7-2-3p inhibitor attenuated the BMPR1B-AS1-mediated upregulation of miR-7-2-3p expression. The data are representative of three independent experiments. Bars, ±SD. *P < .05, **P < .01, ***P < .001, ****P < 0.0001.

Article Snippet: Digoxigenin (DIG)-labeled BMPR1B-AS1 probes were designed and synthesized by BOSTER (Wuhan, China).

Techniques: Binding Assay, Expressing, Luciferase, Transfection, Quantitative RT-PCR, Over Expression

Journal: Cell Cycle

Article Title: Long noncoding RNA BMPR1B-AS1 facilitates endometrial cancer cell proliferation and metastasis by sponging miR-7-2-3p to modulate the DCLK1/Akt/NF-κB pathway

doi: 10.1080/15384101.2022.2060003

Figure Lengend Snippet: Upregulation of miR-7-2-3p expression reverses the BMPR1B-AS1 overexpression-induced progression of Hec-1b cells. A, CCK8 assay showed that cotransfection with the miR-7-2-3p mimic suppressed the BMPR1B-AS1 overexpression-induced proliferation of Hec-1b cells. B, D, F, G, Transwell migration assay (magnification, 200×) and wound healing assay (magnification, 100×) showed that cotransfection with the miR-7-2-3p mimic suppressed the BMPR1B-AS1 overexpression-induced migration of Hec-1b cells. C, E, Transwell invasion assay (magnification, 200×) showed that cotransfection with the miR-7-2-3p mimic suppressed the BMPR1B-AS1 overexpression-induced invasion of Hec-1b cells. H-K, Flow cytometry showed that cotransfection with the miR-7-2-3p mimic accelerated apoptosis and cell cycle arrest, and these effects were inhibited by BMPR1B-AS1 overexpression in Hec-1b cells. The data are representative of three independent experiments. Bars, ±SD. *P < .05, **P < .01, ***P < .001, ****P < 0.0001.

Article Snippet: Digoxigenin (DIG)-labeled BMPR1B-AS1 probes were designed and synthesized by BOSTER (Wuhan, China).

Techniques: Expressing, Over Expression, CCK-8 Assay, Cotransfection, Transwell Migration Assay, Wound Healing Assay, Migration, Transwell Invasion Assay, Flow Cytometry

Journal: Cell Cycle

Article Title: Long noncoding RNA BMPR1B-AS1 facilitates endometrial cancer cell proliferation and metastasis by sponging miR-7-2-3p to modulate the DCLK1/Akt/NF-κB pathway

doi: 10.1080/15384101.2022.2060003

Figure Lengend Snippet: Downregulation of miR-7-2-3p expression reverses the BMPR1B-AS1 knockdown-mediated inhibition of Ishikawa cells. A, CCK8 assay showed that cotransfection with the miR-7-2-3p inhibitor reversed the BMPR1B-AS1 knockdown-mediated inhibition of proliferation of Ishikawa cells. B, D, F, G, Transwell migration assay (magnification, 200×) and wound healing assay (magnification, 100×) showed that cotransfection with the miR-7-2-3p inhibitor reversed the BMPR1B-AS1 knockdown-mediated inhibition of migration of Ishikawa cells. C, E, Transwell invasion assay (magnification, 200×) showed that cotransfection with the miR-7-2-3p inhibitor reversed the BMPR1B-AS1 knockdown-mediated inhibition of invasion of Ishikawa cells. H-K, Flow cytometry showed that cotransfection with the miR-7-2-3p inhibitor inhibited apoptosis and cell cycle arrest, and these effects were enhanced by BMPR1B-AS1 knockdown in Ishikawa cells. The data are representative of three independent experiments. Bars, ±SD. **P < .01, ***P < .001, ****P < 0.0001.

Article Snippet: Digoxigenin (DIG)-labeled BMPR1B-AS1 probes were designed and synthesized by BOSTER (Wuhan, China).

Techniques: Expressing, Inhibition, CCK-8 Assay, Cotransfection, Transwell Migration Assay, Wound Healing Assay, Migration, Transwell Invasion Assay, Flow Cytometry

Journal: Cell Cycle

Article Title: Long noncoding RNA BMPR1B-AS1 facilitates endometrial cancer cell proliferation and metastasis by sponging miR-7-2-3p to modulate the DCLK1/Akt/NF-κB pathway

doi: 10.1080/15384101.2022.2060003

Figure Lengend Snippet: DCLK1 is a target gene of miR-7-2-3p, and BMPR1B-AS1 regulates DCLK1 expression by competitively binding to miR-7-2-3p in EC cell lines. A, Putative binding sites of miR-7-2-3p and DCLK1 mRNA. B, C, The effect of the miR-7-2-3p mimic on the luciferase activities of cells transfected with WT or MUT DCLK1 was detected after 24 h and 48 h, respectively. D, Negative correlation was found between miR-7-2-3p expression and DCLK1 mRNA expression among the 28 endometrial cancer tissue specimens. R = −.531, P = .004. E, Positive correlation was found between BMPR1B-AS1 expression and DCLK1 mRNA expression among the 28 endometrial cancer tissue specimens. R = .690, P = .000. F, J, N, RT-qPCR and western blotting results showed that DCLK1 mRNA and protein expression was upregulated after BMPR1B-AS1 overexpression. G, K, O, RT-qPCR and western blotting results showed that DCLK1 mRNA and protein expression was downregulated after BMPR1B-AS1 knockdown. H, L, P, RT-qPCR and western blotting results showed that the miR-7-2-3p mimic attenuated the BMPR1B-AS1-mediated upregulation of DCLK1 mRNA and protein expression. I, M, Q, RT-qPCR and western blotting results showed that the miR-7-2-3p inhibitor reversed the effects of BMPR1B-AS1 knockdown on DCLK1 mRNA and protein expression. The data are representative of three independent experiments. Bars, ±SD. *P < .05, **P < .01, ***P < .001, ****P < 0.0001.

Article Snippet: Digoxigenin (DIG)-labeled BMPR1B-AS1 probes were designed and synthesized by BOSTER (Wuhan, China).

Techniques: Expressing, Binding Assay, Luciferase, Transfection, Quantitative RT-PCR, Western Blot, Over Expression

Journal: Cell Cycle

Article Title: Long noncoding RNA BMPR1B-AS1 facilitates endometrial cancer cell proliferation and metastasis by sponging miR-7-2-3p to modulate the DCLK1/Akt/NF-κB pathway

doi: 10.1080/15384101.2022.2060003

Figure Lengend Snippet: BMPR1B-AS1 promotes endometrial cancer cell growth in vivo by targeting miR-7-2-3p. A, Images of tumor xenografts. B, The growth curves of tumor xenografts demonstrated that BMPR1B-AS1 significantly promote tumor growth in vivo , which was reversed by treatment with agomir-7-2-3p. **P < 0.01 vs. Lv-BMPR1B-AS1, ***P < 0.001 vs. Lv-BMPR1B-AS1, ****P < 0.0001 vs. Lv-BMPR1B-AS1, # P < 0.05 vs. Lv-BMPR1B-AS1+ agomiR-7-2-3p. C, The weights of tumor xenografts derived from five groups. D, The expression levels of BMPR1B-AS1 in five groups were examined by RT-qPCR. E, The expression levels of miR-7-2-3p in five groups were examined by RT-qPCR. F, G, The expression levels of DCLK1 in five groups were examined by RT-qPCR and IHC. H, Negative correlation was found between BMPR1B-AS1 and miR-7-2-3p expression in xenografts tissues. R = −.506, P = .000. I, Negative correlation was found between miR-7-2-3p and DCLK1 mRNA expression in xenografts tissues. R = −.452, P = .001. J. Positive correlation was found between BMPR1B-AS1 and DCLK1 mRNA expression in xenografts tissues. R = .387, P = .006. *P < .05, **P < .01, ***P < .001.

Article Snippet: Digoxigenin (DIG)-labeled BMPR1B-AS1 probes were designed and synthesized by BOSTER (Wuhan, China).

Techniques: In Vivo, Derivative Assay, Expressing, Quantitative RT-PCR

Journal: Cell Cycle

Article Title: Long noncoding RNA BMPR1B-AS1 facilitates endometrial cancer cell proliferation and metastasis by sponging miR-7-2-3p to modulate the DCLK1/Akt/NF-κB pathway

doi: 10.1080/15384101.2022.2060003

Figure Lengend Snippet: A schematic model of the mechanism by which lncRNA BMPR1B-AS1 regulates endometrial cancer cell malignant behaviors. The BMPR1B-AS1/miR-7-2-3p/DCLK1 axis promotes the progression of endometrial cancer cells via the PI3K/Akt/NF-κB signaling pathway.

Article Snippet: Digoxigenin (DIG)-labeled BMPR1B-AS1 probes were designed and synthesized by BOSTER (Wuhan, China).

Techniques:

Journal: Journal of Ginseng Research

Article Title: Ginsenoside Rb1 and compound K improve insulin signaling and inhibit ER stress-associated NLRP3 inflammasome activation in adipose tissue

doi: 10.1016/j.jgr.2015.11.002

Figure Lengend Snippet: Rb1 and CK modulate IRS-1 phosphorylation in the presence of high glucose concentrations. Epididymal adipose tissue was separated after the mice were scarified. The tissue was pretreated with Rb1, CK, or TUDCA at given concentrations, followed by stimulation with high glucose (33mM) for 24 h with or without insulin treatment for an additional 30 min. (A,B) Serine phosphorylation of IRS-1 (S307) and tyrosine phosphorylation IRS-1 (PY99), respectively, were determined by western blot. (C) The level of PI3K in the supernatant of lysed tissue was assayed with an ELISA kit. All results were derived from three independent experiments for the western blot or expressed as the mean ± SD ( n = 4) for ELISA. * p < 0.05 versus high glucose-only treatment; ** p < 0.05 versus the indicated treatment. CK, ginsenoside compound K; ELISA, enzyme-linked immunosorbent assay; SD, standard deviation; TUDCA, tauroursodeoxycholic acid.

Article Snippet: Monoclonal antibodies were procured from the cited commercial sources: anti-TXNIP (NBP1-54578) and anti-NLRP3 (NBP2-12446), Novus Biologicals (Littleton, CO, USA); anti-PERK (#3192), Cell Signaling Technology (Beverly, MA, USA); anti-phospho-PERK (Thr 981; sc-32577) and anti-phospho-IRS-1 (PY99; sc-7020), Santa Cruz Biotechnology, Inc. (Dallas, TX, USA); anti-phospho-IRE1α (S724; ab104157) and anti-IRE1α (ab37073), Abcam (Cambridge, MA, USA); anti-phospho-IRS-1 (Ser307; BS4104), anti-IRS-1 (BS1408), anti-phospho-Akt (T308; BS40080), anti-Akt (BS1810), anti-GAPDH (AP0063), and goat anti-mouse IgG (H+L) horseradish peroxidase (HRP; BS12478), Bioworld Technology (St. Paul, MN,USA).

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Derivative Assay, Standard Deviation