Journal: Advanced Science

Article Title: Long Non‐Coding RNA IGFRIL Couples with PTBP1 to Destabilize IGFBP3 mRNA to Promote the IGF1R‐AKT‐mTOR Axis and Hepatocellular Carcinoma

doi: 10.1002/advs.202507676

Figure Lengend Snippet: IGFRIL couples with PTBP1 to enhance the activation of AKT‐mTOR signaling in HCC cells. A) Overlapping DEGs upon IGFRIL and PTBP1 knockdown in HepG2 cells. B) GO and KEGG pathway enrichment analyses of the overlapping differentially expressed genes (DEGs). C) Spearman's correlation of gene set enrichment scores between IGFRIL and PTBP1 knockdown. NES, normalized enrichment score. D) GSEA plots of “PI3K_EVENTS_IN_ERBB4_SIGNALING” in IGFRIL ‐knocked‐down and PTBP1 ‐knocked‐down HepG2 cells. E) IB assays of key cascades of the receptor tyrosine kinases (RTK) and AKT‐mTOR signaling in HepG2 cells upon IGFRIL knockdown or overexpression. F) Knockdown of IGFRIL accelerates the autophagic flux in HepG2 cells. HepG2 cells stably expressing mRFP‐GFP‐LC3 were treated with DMSO, NVP‐AEW541 (2 µ m ), or Rapamycin (10 µ m ) for 12 h upon knockdown of IGFRIL , followed by staining with DAPI. Red puncta represent the autophagosomes; and yellow puncta in the merged picture represent the autolysosomes. Scale bars, 40 µm. G,H) Rapamycin attenuates the promoting effects of IGFRIL overexpression on cell malignant phenotypes and mTOR signaling. Data are shown as mean ± SEM, * P < 0.05, ** P < 0.01, *** P < 0.001 by 1‐way ANOVA (E,G,H). n.s., not significant.

Article Snippet: When the tumor volume reached ≈100 mm 3 after implantation, the mice were randomly divided into three treatment groups for assessing the sensitivity of PDXs with different IGFRIL levels to IGF1R or mTOR inhibitors: 1) DMSO (intraperitoneal injection); 2) BMS‐754807 (25 mg kg −1 every 5 days, intraperitoneal injection; Cat. s1124, Selleck, USA); and 3) rapamycin (10 mg kg −1 every 5 days, intraperitoneal injection; Cat. s1039, Selleck, USA).

Techniques: Activation Assay, Knockdown, Over Expression, Stable Transfection, Expressing, Staining

Journal: Advanced Science

Article Title: Long Non‐Coding RNA IGFRIL Couples with PTBP1 to Destabilize IGFBP3 mRNA to Promote the IGF1R‐AKT‐mTOR Axis and Hepatocellular Carcinoma

doi: 10.1002/advs.202507676

Figure Lengend Snippet: IGFRIL activates the IGF1R‐AKT‐mTOR signaling and plays oncogenic roles dependent on IGFBP3. A) The effects of knockdown or overexpression of IGFBP3 on the activities of the IGF1R‐AKT‐mTOR axis. B) The effects of IGFBP3 overexpression on IGFRIL ‐activated IGF1R‐AKT‐mTOR axis, and the effects of IGFBP3 knockdown on IGFRIL knockdown‐repressed IGF1R‐AKT‐mTOR axis. C,D) The effects of IGFBP3 knockdown or overexpression on the abilities of plate colony formation, migration, and invasion. E) The effects of IGFBP3 overexpression on the IGFRIL ‐enhanced plate colony formation, migration, and invasion. F) The effects of IGFBP3 knockdown on the IGFRIL knockdown‐reduced plate colony formation, migration, and invasion. G) The effects of IGF1R inhibition with its inhibitor AEW541 (2 µM) or siRNAs against IGF1R on the IGFRIL‐activated IGF1R‐AKT‐mTOR axis. H,I) The effects of IGF1R inhibition on the IGFRIL ‐enhanced plate colony formation, migration, and invasion. J) j Spearman's correlations between the levels of IGFRIL and levels of IGFBP3, p‐IGF1R, p‐AKT and p‐mTOR, respectively, in HCC tumor tissues ( n = 83) from the VALI1 cohort. Data are shown as mean ± SEM, * P < 0.05, ** P < 0.01, *** P < 0.001 by 1‐way ANOVA (C,E,F,G,I) or Student's t‐ test (D).

Article Snippet: When the tumor volume reached ≈100 mm 3 after implantation, the mice were randomly divided into three treatment groups for assessing the sensitivity of PDXs with different IGFRIL levels to IGF1R or mTOR inhibitors: 1) DMSO (intraperitoneal injection); 2) BMS‐754807 (25 mg kg −1 every 5 days, intraperitoneal injection; Cat. s1124, Selleck, USA); and 3) rapamycin (10 mg kg −1 every 5 days, intraperitoneal injection; Cat. s1039, Selleck, USA).

Techniques: Knockdown, Over Expression, Migration, Inhibition

Journal: Advanced Science

Article Title: Long Non‐Coding RNA IGFRIL Couples with PTBP1 to Destabilize IGFBP3 mRNA to Promote the IGF1R‐AKT‐mTOR Axis and Hepatocellular Carcinoma

doi: 10.1002/advs.202507676

Figure Lengend Snippet: Upregulation of IGFRIL sensitizes HCC cells to IGF1R and mTOR inhibitors. A) Spearman's correlations between the levels of IGFRIL and sensitivities to inhibitors against IGF1R (BMS‐754807) and mTOR (rapamycin) in eight types of HCC cell lines. The effects of BMS‐754807 or rapamycin on HCC cell growth were determined by CCK‐8 assays. B) The effects of IGFRIL knockdown on the sensitivity to BMS‐754807 or rapamycin in HepG2 cells. C) The effects of IGFRIL overexpression on the sensitivity to BMS‐754807 or rapamycin in JHH‐2 cells. D) The effects of BMS‐754807 or rapamycin treatment on tumor growth in patient‐derived xenografts (PDX) mouse models. The mice were treated with control (DMSO), BMS‐754807 (25 mg kg −1 every 5 days, intraperitoneal injection) or rapamycin (10 mg kg −1 every 5 days, intraperitoneal injection) ( n = 5 mice/group). E–G) The effects of IGFRIL knockdown or overexpression on the cell growth, migration, and invasion (E,F), and the activities of the IGF1R‐AKT‐mTOR axis (G) in IGFRIL high and IGFRIL low patient‐derived primary tumor cells (PDCs), respectively. H) The effects of BMS‐754807 (left) or rapamycin (right) treatment on the growth of IGFRIL high or IGFRIL low PDCs. I) The effects of IGFRIL knockdown or overexpression on the sensitivities to BMS‐754807 and rapamycin in IGFRIL high and IGFRIL low PDCs, respectively. J) Schematic diagram of the function and mechanism of IGFRIL in HCC tumorigenesis and drug sensitivity. Data are shown as mean ± SEM, * P < 0.05, ** P < 0.01, *** P < 0.001 by 1‐way ANOVA (B,D,E,I) or Student's t test (C,F,H).

Article Snippet: When the tumor volume reached ≈100 mm 3 after implantation, the mice were randomly divided into three treatment groups for assessing the sensitivity of PDXs with different IGFRIL levels to IGF1R or mTOR inhibitors: 1) DMSO (intraperitoneal injection); 2) BMS‐754807 (25 mg kg −1 every 5 days, intraperitoneal injection; Cat. s1124, Selleck, USA); and 3) rapamycin (10 mg kg −1 every 5 days, intraperitoneal injection; Cat. s1039, Selleck, USA).

Techniques: CCK-8 Assay, Knockdown, Over Expression, Derivative Assay, Control, Injection, Migration

Journal: Biomolecules

Article Title: Calystegines Improve the Metabolic Activity of Human Adipose Derived Stromal Stem Cells (ASCs) under Hyperglycaemic Condition through the Reduction of Oxidative/ER Stress, Inflammation, and the Promotion of the AKT/PI3K/mTOR Pathway

doi: 10.3390/biom12030460

Figure Lengend Snippet: List of antibodies employed for protein profiling using western blot analysis.

Article Snippet: mTOR , 1:500 , Novus Biologicals, nb100-240.

Techniques: Western Blot

Journal: Journal of Biological Chemistry

Article Title: Reciprocal Regulation of AMP-activated Protein Kinase and Phospholipase D

doi: 10.1074/jbc.m114.622571

Figure Lengend Snippet: FIGURE 3. Inhibition of PLD activates AMPK. A, MDA-MB-231 cells were plated as described for Fig. 2B and transfected with PLD1 and PLD2 siRNAs or a scrambled (scram) control siRNA as indicated. 6 h later, the cells were treated with fresh medium containing 10% serum for an additional 72 h. The cells were thenharvested,andPLDactivitywasevaluatedasdescribedunder“ExperimentalProcedures”.ValueswerenormalizedtothecontrolscrambledsiRNAs,which were given a value of 100%. For Western blot analysis, cells were harvested at 72 h, and the levels of phospho-AMPK (P-AMPK), AMPK, phospho-ACC (P-ACC), ACC, and actin were determined. B, MDA-MB-231 cells were plated as described for A and transfected with vectors expressing catalytically inactive dominant- negative (DN) mutant forms of PLD1 and PLD2. The parental vector pcDNA3.1 was used as control (Con). 48 h later, the cells were harvested, and the levels of phospho-AMPK, AMPK, phospho-ACC, and ACC were determined as described for A. Expression of PLD mutants was evaluated by probing the blots for HA tags on the PLD mutants. Blots were also probed for actin as loading controls. C, MDA-MB-231 cells were plated, the PLD1 and PLD2 inhibitors (PLDi; 10 M each) wereaddedfor1h,andtherelativePLDactivitywasthendeterminedasdescribedforFig.1C.Theconcentrationsusedwerebasedonapreviousstudyinwhich we used these inhibitors to block mTOR (24). For Western blot analysis, MDA-MB-231 cells were plated and treated with the PLD inhibitors for the indicated times. Cells were harvested, and the levels of phospho-AMPK, AMPK, phospho-ACC, ACC, and actin were determined as described for A. D, MDA-MB-231 cells were plated as described for C and treated with the PLD inhibitors and/or PA for 45 min as indicated. The cells were then harvested, and the levels of phospho-AMPK, AMPK, phospho-ACC, ACC, and actin were determined as described for A. E, Calu-1 cells were plated, and the PLD inhibitors (10 M each) were added for 1 h. The relative PLD activity was then determined as described for Fig. 1C. For Western blot analysis, Calu-1 cells were plated as described for C and treated with the PLD inhibitors and/or PA for 45 min as indicated. The cells were then harvested, and the levels of phospho-AMPK, AMPK, phospho-ACC, ACC, and actin were determined as described for A. The Western blot data are representative of experiments repeated at least two times. Error bars for PLD assays represent S.D. for at least two independent experiments. The statistical significance (p value) was determined by Student’s two-tailed paired t test. *, p 0.01 compared with the control. The relative levels of AMPK and ACC phosphorylation were normalized to total AMPK and total ACC, respectively, and quantified as described for Fig. 1A.

Article Snippet: Dialyzed fetal bovine serum (F0392) was obtained from Sigma-Aldrich. siRNAs targeting LKB1 (sc-35816), AMPK (sc-45312), Raptor (sc-44069), and mTOR (sc-35409) were obtained from Santa Cruz Biotechnology, and siRNAs targeting Rheb (M-009692-02-0005), PLD1 (M-009413-00-0010), and PLD2 (M-005064-01-0010) were obtained from Dharmacon.

Techniques: Inhibition, Transfection, Control, Western Blot, Expressing, Dominant Negative Mutation, Mutagenesis, Plasmid Preparation, Blocking Assay, Activity Assay, Two Tailed Test, Phospho-proteomics

Journal: Journal of Biological Chemistry

Article Title: Reciprocal Regulation of AMP-activated Protein Kinase and Phospholipase D

doi: 10.1074/jbc.m114.622571

Figure Lengend Snippet: FIGURE5.ModelforreciprocalregulationofAMPKandPLD.Twoscenarios are presented for the regulation of PLD by AMPK and the regulation of AMPK by PLD. A, AMPK is activated by the presence AMP and phosphorylation by LKB1. AMPK then phosphorylates TSC, which stimulates the GAP activity of TSCforRheb,resultinginthehydrolysisofboundGTPtoGDPandinactivation of Rheb. As a consequence, PLD is not activated (19), and the PA needed for stabilization of mTORC1 (21) is not generated, so mTORC1 is inactive. B, Rheb isGTP-boundandpromotestheproductionofPAbyPLDandthestabilization of mTORC1. Active mTORC1 then causes suppression of AMPK in a manner that is likely dependent on S6 kinase in that rapamycin has been reported to activate AMPK (30) at concentrations that suppress S6 kinase (38). Other sig- nals promoted by oncogenic stimuli contribute to the activation of this path- way, such as mTORC2-Akt, which suppresses TSC (16), and Ras, which leads to PLD activation (27). It is also important to note that mTOR is an integrator on nutrient and growth factor signals (54), both of which are required for cell cycle progression and proliferation. mTOR requires amino acids (55), glucose (24), and possibly lipids (51) for activity.

Article Snippet: Dialyzed fetal bovine serum (F0392) was obtained from Sigma-Aldrich. siRNAs targeting LKB1 (sc-35816), AMPK (sc-45312), Raptor (sc-44069), and mTOR (sc-35409) were obtained from Santa Cruz Biotechnology, and siRNAs targeting Rheb (M-009692-02-0005), PLD1 (M-009413-00-0010), and PLD2 (M-005064-01-0010) were obtained from Dharmacon.

Techniques: Phospho-proteomics, Activity Assay, Generated, Activation Assay