Journal: Cell Cycle

Article Title: Functional crosstalk between AKT/mTOR and Ras/MAPK pathways in hepatocarcinogenesis

doi: 10.4161/cc.25099

Figure Lengend Snippet: Figure 4. Rapamycin treatment induces the compensatory activation of the ERK/eIF4e pathway in the residual tumor cells from AKT/Ras mice. ( A and B ) Strong ERK activity, as assessed by positive immunolabeling for phosphorylated/activated (p-)ERK1 and 2 proteins in some (arrows) but not all (asterisks) the residual preneoplastic hepatocytes ( A ) and in a regressing tumor ( B ) following 3 wk of Rapamycin treatment. ( C and D ) Co-localization of phosphorylated/activated ERK1/2 and eIF4E immunoreactivity in one AKT/Ras preneoplastic lesion following Rapamycin treatment. ( E and F ) Proliferative activity in one preneoplastic lesion ( E ) and a small tumor ( F ), as assessed by Ki67 immunolabeling. The inset in ( F ) shows the different proliferative activity between the tumor (left part of the picture) and the surrounding non-tumorous liver (right). ( G and H ) Apoptotic bodies, indicated by arrows, in one preneoplastic lesion ( G ) and a small tumor ( H ), as assessed by TUNEL immunolabeling. Original magnifications: 100× in ( A and F ); 200× in ( B–D ); 400× in ( E, G and H ).

Article Snippet: Immunohistochemical staining was performed using the following antibodies: mouse monoclonal anti-HA-Tag (Cell Signaling Technology; 1:2,000), rabbit monoclonal anti-phosphorylated AKT, anti-phosphorylated ERK1/2, anti-phosphorylated eIF4E, anti-phosphorylated mTOR (Cell Signaling Technology; 1:100) and rabbit polyclonal anti-Ki67 (Bethyl Laboratories; 1:2,000), as previously described., TUNEL staining was performed using the ApopTag ® Peroxidase In Situ Apoptosis Detection Kit (Millipore), following the manufacturer’s instructions.

Techniques: Activation Assay, Activity Assay, Immunolabeling, TUNEL Assay

Journal: Cell Cycle

Article Title: Functional crosstalk between AKT/mTOR and Ras/MAPK pathways in hepatocarcinogenesis

doi: 10.4161/cc.25099

Figure Lengend Snippet: Figure 7. Concomitant suppression of the mTORC1 and Ras/MAPK pathways is highly detrimental for AKT/Ras-driven growth in vitro. ( A and B ) Inhibition of mTORC1 and MAPK via Rapamycin and U0126, respectively, induces a remarkable restraint over the growth of the AKT/Ras cell line, as determined by the decrease in cell proliferation and induction of massive apoptosis when compared with administration of either Rapamycin or U0126 alone. Each bar represents mean ± SD. Tukey-Kramer test: p < 0.001 a, vs. control (untreated cells); b, vs. vehicle (DMSO); c , vs. Rapamycin- (50 nM) treated cells; d , vs. Rapamycin- (100 nM) treated cells; e , vs. U0126-treated cells. ( C and D ) Representative immunoblot analysis showing that Rapamycin induces downregulation of mTORC1 and AKT activity in a concentration-independent manner but is ineffective on 4EBP1 protein and induces the compensatory activation of ERK and eIF4E proteins. On the other hand, treatment with U0126 alone suppresses phosphorylated/inactivated (p-)4EBP1 but induces the compensatory activation of phosphorylated/activated (p-)AKT and RPS6. The compensatory feed-back loops are abolished by the combination of Rapamycin and U0126 treatment. Note that combined treatment downregulates both the total and phosphorylated/activated levels of mTOR and eIF4E. The abbreviations p-AKT (1) and p-AKT (2) indicate phosphorylated/activated levels of AKT protein at threonine 308 and serine 473 residue, respectively.

Article Snippet: Immunohistochemical staining was performed using the following antibodies: mouse monoclonal anti-HA-Tag (Cell Signaling Technology; 1:2,000), rabbit monoclonal anti-phosphorylated AKT, anti-phosphorylated ERK1/2, anti-phosphorylated eIF4E, anti-phosphorylated mTOR (Cell Signaling Technology; 1:100) and rabbit polyclonal anti-Ki67 (Bethyl Laboratories; 1:2,000), as previously described., TUNEL staining was performed using the ApopTag ® Peroxidase In Situ Apoptosis Detection Kit (Millipore), following the manufacturer’s instructions.

Techniques: In Vitro, Inhibition, Control, Western Blot, Activity Assay, Concentration Assay, Activation Assay, Residue

Journal: Cell Cycle

Article Title: Functional crosstalk between AKT/mTOR and Ras/MAPK pathways in hepatocarcinogenesis

doi: 10.4161/cc.25099

Figure Lengend Snippet: Figure 8. Schematic representation of the molecular mechanisms whereby AKT and Ras promote hepatocarcinogenesis in the mouse and the effect of Rapamycin treatment. ( A ) AKT induces activation (red arrows) of mTORC1, with consequent activation of RPS6, lipogenesis and glycolysis as well as inactivation (blunted, blue arrows) of 4EBP1 protein. Through inactivation of 4EBP1, mTORC1 releases the eIF4E protein, leading to protein translation. Ras activates instead the Raf/MEK/ERK/eIF4E cascade, which in turn activates mTORC1 and eIF4E. The crosstalk between activated AKT and Ras ultimately leads to unrestrained cell growth. ( B ) Rapamycin induces downregulation (thin, red arrows) of mTORC1 and AKT activity but is ineffective on 4EBP1 protein and induces the compensatory activation (thick, red arrows) of Ras/Raf/MEK/ERK and eIF4E pathways.

Article Snippet: Immunohistochemical staining was performed using the following antibodies: mouse monoclonal anti-HA-Tag (Cell Signaling Technology; 1:2,000), rabbit monoclonal anti-phosphorylated AKT, anti-phosphorylated ERK1/2, anti-phosphorylated eIF4E, anti-phosphorylated mTOR (Cell Signaling Technology; 1:100) and rabbit polyclonal anti-Ki67 (Bethyl Laboratories; 1:2,000), as previously described., TUNEL staining was performed using the ApopTag ® Peroxidase In Situ Apoptosis Detection Kit (Millipore), following the manufacturer’s instructions.

Techniques: Activation Assay, Activity Assay

Journal: The Journal of Nutrition

Article Title: Leucine and ?-Ketoisocaproic Acid, but Not Norleucine, Stimulate Skeletal Muscle Protein Synthesis in Neonatal Pigs 1–3

doi: 10.3945/jn.110.123042

Figure Lengend Snippet: Phosphorylation of 4E-BP1, percent of γ-form (A), and association of 4E-BP1 with eIF4E (B) in skeletal muscle of young pigs after 60 min of infusion with saline (Sal), leucine (Leu), KIC, or norleucine (Nleu). Values are means ± pooled SE, n = 7–8 per treatment group. Within each graph, means without a common letter differ, P < 0.04.

Article Snippet: Immunoprecipitates were subjected to protein immunoblot analysis, as described above, using a rabbit polyclonal anti-4E-BP1 antibody (Bethyl Laboratories), the aforementioned monoclonal anti-eIF4E antibody, a rabbit polyclonal anti-eIF4G antibody (Novus Biologicals), and a rabbit polyclonal antibody that recognizes site-specific phosphorylation of eIF4G at Ser-1108.

Techniques:

Journal: The Journal of Nutrition

Article Title: Leucine and ?-Ketoisocaproic Acid, but Not Norleucine, Stimulate Skeletal Muscle Protein Synthesis in Neonatal Pigs 1–3

doi: 10.3945/jn.110.123042

Figure Lengend Snippet: Association of eIF4G with eIF4E (A) and phosphorylation of eIF4G at Ser-1108 (P-Ser-1108) associated with eIF4E (B) in skeletal muscle of young pigs after 60 min of infusion with saline (Sal), leucine (Leu), KIC, or norleucine (Nleu). Values are means ± pooled SE, n = 7–8 per treatment group. Within each graph, means without a common letter differ, P < 0.05.

Article Snippet: Immunoprecipitates were subjected to protein immunoblot analysis, as described above, using a rabbit polyclonal anti-4E-BP1 antibody (Bethyl Laboratories), the aforementioned monoclonal anti-eIF4E antibody, a rabbit polyclonal anti-eIF4G antibody (Novus Biologicals), and a rabbit polyclonal antibody that recognizes site-specific phosphorylation of eIF4G at Ser-1108.

Techniques:

Journal: eLife

Article Title: LARP1 functions as a molecular switch for mTORC1-mediated translation of an essential class of mRNAs

doi: 10.7554/eLife.25237

Figure Lengend Snippet: ( A ) Endogenous LARP1 co-IPs endogenous mTORC1 in HEK293T cells. LARP1 antibody 087A, but not 088A, co-IPs mTORC1. The LARP1 antibody, 087A or 088A recognizes amino acids 275–325 or 575–625 of LARP1, respectively. ( B ) The effect of RNAse A on the interaction of LARP1 with mTORC1 and other mRNA binding proteins (PABP1 and eIF4E). Endogenous LARP1 was IPed from the lysates treated with RNAse A. ( C ) The effect of RNase A on the interaction of LARP1 with the components of the initiation complex. Endogenous eIF4G1 was IPed from the lysates treated with RNAse A. ( D ) LARP1 interacts with mTORC1 in a manner dependent on mTORC1 activity. ( E ) LARP1 co-IPs mTORC1 in a growth factor/amino acid stimulation-dependent manner. DOI: http://dx.doi.org/10.7554/eLife.25237.007

Article Snippet: Antibodies were purchased from the following sources: Antibodies to mTOR (cat. # 2983, RRID: AB_2105622 for western blotting and immunostaining), Raptor (cat. # 2280, RRID: AB_561245 ), pT389 S6K1 (cat. # 9206, RRID: AB_2285392 ), S6K (cat. # 9202, RRID: AB_823592 ), pS473 Akt (cat. # 9270, RRID: AB_329824 ), Akt (cat # 9272, RRID: AB_329827 ), phospho-Akt Substrate (RXXS/T) (cat. # 9614, RRID: AB_2225188 ), pT37/46 4EBP1 (cat. # 2855, RRID: AB_560835 ), 4EBP1 (cat. # 9644, RRID: AB_10691384 ), S6 (cat. # 2217, RRID: AB_331355 ), pS235/236 S6 (cat. # 4856, RRID: AB_2181037 for western blotting), pS240/244 S6 (cat. # 5364, RRID: AB_10694233 for co-IP), RpL13a (cat. # 2765, RRID: AB_916223 ), PABP1 (cat # 4992, RRID: AB_2156887 ), mLST8 (cat. # 3274, RRID: AB_823685 ), PRAS40 (cat. # 2610, RRID: AB_916206 ), pULK1 (cat. # 6888, RRID: AB_10829226 ), and ULK1 (cat. # 6439, RRID: AB_11178933 ) from Cell Signaling Technology; antibodies to LARP1 (cat. # A302-087A, RRID: AB_1604274 for co-IP and western blotting, cat. # A310-088A, RRID: AB_2632225 for IP and western blotting and cat. # IHC-00559, RRID: AB_10631280 for immunostaining), Rictor (cat. # A300-458A, RRID: AB_420924 ), eIF4G (cat. # A301-776A, RRID: AB_1211011 ), eIF3D (cat. # A301-758A, RRID: AB_1210970 ), and eIF3B (cat. # A301-761A, RRID: AB_1210995 ) from Bethyl Laboratories; antibodies to eIF4E (cat. # sc-9976, RRID: AB_627502 ) and mTOR/FRAP (cat. # sc-1549, RRID: AB_631981 for co-IP) from Santa Cruz Biotechnology; antibodies to β-Actin (cat. # A1978, RRID: AB_476692 ), and Flag M2 antibodies (cat. # F1804, RRID: AB_262044 ) from Sigma-Aldrich; antibodies to HA tag (cat. # MMS-101P, RRID: AB_2314672 ), and Myc tag (cat. # MMS-150P, RRID: AB_291322 ) from Covance; antibody to LAMP2 (cat. # H4B4, RRID: AB_528129 ) from the Developmental Studies Hybridoma Bank at the University of Iowa; HRP-conjugated mouse secondary antibody (cat. # NA931-1ML, RRID: AB_772210 ) and HRP-conjugate rabbit secondary antibody (cat. # NA934-1ML, RRID: AB_772206 ) from GE Healthcare; Alexa fluor 594 goat anti-rabbit IgG (cat. # A11012, RRID: AB_141359 ) and Alexa fluor 488 goat anti-mouse IgG (cat. # A11001, RRID: AB_2534069 ) from Invitrogen.

Techniques: Binding Assay, Activity Assay

Journal: eLife

Article Title: LARP1 functions as a molecular switch for mTORC1-mediated translation of an essential class of mRNAs

doi: 10.7554/eLife.25237

Figure Lengend Snippet: ( A ) LARP1 knockdown enhances 4EBP1 binding to the eIF4E precipitated with m 7 GTP sepharose beads. ( B ) Loss of LARP1 decreases the expression of eEF2 in the fractions containing active monosomes (80S) and polysomes. ( C ) RP mRNAs are accumulated in the lighter polysome fractions in LARP1 knockdown cells. ( D ) Loss of LARP1 decreases the translation of RP mRNAs. Equal amount of ribosomes were immunoprecipitated by phopho-S6 antibody from normal growing HEK293T cells in the presence or absence of LARP1 expression (right panel). Newly synthesized ribosome subunits were visualized (left panel) and quantified (middle panel). *p<0.05, mean±SEM (n = 3). ( E ) Prolonged LARP1 knockdown (96 hr) decreases the expression of RP proteins (left panels). Levels of RP proteins (middle panel) were quantified and mRNA levels of RP proteins were monitored by qPCR (right panel). Newly synthesized RP proteins were monitored by the Click-It assy *p<0.05 vs control shRNA treatment, mean±SD (n = 3). ( F ) Prolonged LARP1 knockdown (96 hr) decreases global protein synthesis. p<0.05, mean±SD (n = 3). (left panel). Equal amount of protein loading was visualized by Coomassie blue staining (middle panel). Click-It reaction was quantitated (right panel). *p<0.05 vs control shRNA treatment, mean±SD (n = 3). ( G ) The expression of LARP1 and RP proteins is enhanced in multiple cancer and transformed cell lines (left panels). Correlation between LARP1 protein vs. RpS6 protein (right upper) or RpS6 mRNA (right lower) in prostate epithelial cells. Open or close square indicates normal or benign prostate epithelial cells or metastatic prostate cancer cells, respectively. DOI: http://dx.doi.org/10.7554/eLife.25237.013

Article Snippet: Antibodies were purchased from the following sources: Antibodies to mTOR (cat. # 2983, RRID: AB_2105622 for western blotting and immunostaining), Raptor (cat. # 2280, RRID: AB_561245 ), pT389 S6K1 (cat. # 9206, RRID: AB_2285392 ), S6K (cat. # 9202, RRID: AB_823592 ), pS473 Akt (cat. # 9270, RRID: AB_329824 ), Akt (cat # 9272, RRID: AB_329827 ), phospho-Akt Substrate (RXXS/T) (cat. # 9614, RRID: AB_2225188 ), pT37/46 4EBP1 (cat. # 2855, RRID: AB_560835 ), 4EBP1 (cat. # 9644, RRID: AB_10691384 ), S6 (cat. # 2217, RRID: AB_331355 ), pS235/236 S6 (cat. # 4856, RRID: AB_2181037 for western blotting), pS240/244 S6 (cat. # 5364, RRID: AB_10694233 for co-IP), RpL13a (cat. # 2765, RRID: AB_916223 ), PABP1 (cat # 4992, RRID: AB_2156887 ), mLST8 (cat. # 3274, RRID: AB_823685 ), PRAS40 (cat. # 2610, RRID: AB_916206 ), pULK1 (cat. # 6888, RRID: AB_10829226 ), and ULK1 (cat. # 6439, RRID: AB_11178933 ) from Cell Signaling Technology; antibodies to LARP1 (cat. # A302-087A, RRID: AB_1604274 for co-IP and western blotting, cat. # A310-088A, RRID: AB_2632225 for IP and western blotting and cat. # IHC-00559, RRID: AB_10631280 for immunostaining), Rictor (cat. # A300-458A, RRID: AB_420924 ), eIF4G (cat. # A301-776A, RRID: AB_1211011 ), eIF3D (cat. # A301-758A, RRID: AB_1210970 ), and eIF3B (cat. # A301-761A, RRID: AB_1210995 ) from Bethyl Laboratories; antibodies to eIF4E (cat. # sc-9976, RRID: AB_627502 ) and mTOR/FRAP (cat. # sc-1549, RRID: AB_631981 for co-IP) from Santa Cruz Biotechnology; antibodies to β-Actin (cat. # A1978, RRID: AB_476692 ), and Flag M2 antibodies (cat. # F1804, RRID: AB_262044 ) from Sigma-Aldrich; antibodies to HA tag (cat. # MMS-101P, RRID: AB_2314672 ), and Myc tag (cat. # MMS-150P, RRID: AB_291322 ) from Covance; antibody to LAMP2 (cat. # H4B4, RRID: AB_528129 ) from the Developmental Studies Hybridoma Bank at the University of Iowa; HRP-conjugated mouse secondary antibody (cat. # NA931-1ML, RRID: AB_772210 ) and HRP-conjugate rabbit secondary antibody (cat. # NA934-1ML, RRID: AB_772206 ) from GE Healthcare; Alexa fluor 594 goat anti-rabbit IgG (cat. # A11012, RRID: AB_141359 ) and Alexa fluor 488 goat anti-mouse IgG (cat. # A11001, RRID: AB_2534069 ) from Invitrogen.

Techniques: Knockdown, Binding Assay, Expressing, Immunoprecipitation, Synthesized, Control, shRNA, Staining, Transformation Assay

Journal: Oncotarget

Article Title: The effect of the type of dietary protein on the development of ovarian cancer

doi: 10.18632/oncotarget.25253

Figure Lengend Snippet: (A, B) The rate of p-4EBP1 positivity in the tumors of the 20% animal protein group was higher than that of the 20% plant protein group (p<0.001). (C, D) The rate of positivity for Ki67, an index of proliferation, in the tumors of the 20% animal protein group was also higher than that of the 20% plant protein group (p<0.01).

Article Snippet: To examine the expression of Ki67 and 4EBP1, tissue sections were blocked and incubated overnight in primary antibodies against Ki67 (1:100; Bethyl Laboratories, Inc., TX), and rabbit monoclonal antibody against phosphorylated 4EBP1 (Thr37/46) (1:100).

Techniques:

Journal: Frontiers in Oncology

Article Title: Selinexor inhibits growth of patient derived chordomas in vivo as a single agent and in combination with abemaciclib through diverse mechanisms

doi: 10.3389/fonc.2022.808021

Figure Lengend Snippet: Effect of selinexor on indicators of cell health, chordoma markers, and XPO1 cargo proteins. Immunohistochemistry analysis of tumor samples from CF466 PDX models treated for six weeks with either vehicle control or selinexor. (A) Markers of cell survival (cleaved caspase 3) and proliferation (Ki67), as well as the selinexor target XPO1. (B) XPO1 cargo proteins APC, FOXO3A, eIF4E and SMAD4. (C) Proteins involved in sonic hedgehog signaling pathways such as SOX9, and YAP1, and regulators of chordoma cell growth, Brachyury and Survivin, as well as DNA repair enzyme PARP1.

Article Snippet: Primary antibodies against XPO1 (Bethyl Laboratories, A300-469A, 1:15k), Brachyury (Abcam, ab209665, 1:30k), APC (Abcam, ab15270, 1:3k), FOXO3A (Cell Signaling Technology, 12829, 1:1k), eIF4E (Protein Tech, 11149-1-AP, 1:500), Survivin (Abcam, ab76424, 1:1k), SOX9 (Sigma-Aldrich, HPA001758, 1:1k), YAP1 (Cell Signaling Technology, 14074, 1:500), PARP1 (Santa Cruz, sc-8007, 1:1k), Ki67 (Biocare, Prediluted), SMAD4 (Santa Cruz, sc-7966, 1:600), and cleaved Caspase 3 (Cell Signaling Technology, #9661, 1:1k) were used for IHC analysis.

Techniques: Immunohistochemistry, Control, Protein-Protein interactions