Journal: Autophagy

Article Title: Direct regulation of FNIP1 and FNIP2 by MEF2 sustains MTORC1 activation and tumor progression in pancreatic cancer.

doi: 10.1080/15548627.2023.2259735

Figure Lengend Snippet: Figure 1. Transcription factors MEF2A and MEF2D regulate MTORC1 activity upon amino acid stimulation. (A) Gene set enrichment analysis of the MTOR pathway gene list in HeLa cells with overexpression of MEF2-VP16 versus controls by using RNA-seq data. (B) HeLa cells transfected with either dnMEF2 or vector as control were treated with amino acid (aa) starvation and amino acid restimulation with or without troin1 (250 nM). Western blot analysis of phosphorylation of RPS6KB1, EIF4EBP1 and AKT is shown. Right plots show phosphorylated p-RPS6KB1:RPS6KB1 (top), p-EIF4EBP1:ACTB (middle) and p-AKT:AKT (bottom) ratios. (C) HeLa cells transfected with MEF2-VP16 or vector were treated and then analyzed by immunoblotting similar to (B). (D) HeLa cells transfected for 48 h with indicated siRNA were starved of amino acids and restimulated before immunoblotting analysis. Right plots show the p-RPS6KB1:RPS6KB1 (top), p-EIF4EBP1:ACTB (bottom) ratios. (E) HeLa cells that stably knocked-down both MEF2A and MEF2D (shMEF2A/D) or control (NC) were starved for amino acids and restimulated with increasing levels (expressed as % of the concentration in DMEM medium) of leucine. Lysates were analyzed for MTORC1 activity as in (D). Bottom plots show the p-RPS6KB1:RPS6KB1 (right), p-EIF4EBP1:ACTB (left) ratios. (F) MEF2A and MEF2D double-knockdown HeLa cells transfected with MEF2A or/and MEF2D and then subjected to amino acid administration. MTORC1 activity was confirmed by immunoblotting. Plots on the right show p-RPS6KB1:RPS6KB1 (top) and p-EIF4EBP1:ACTB (bottom) ratios. Data are presented as the mean ± S.E.M. (n = 3 independent experiments, two-sided Student’s t-test for B, C and E, one-way analysis of variance [ANOVA] for D, two-way ANOVA post hoc test for F, *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant compared with indicated group).

Article Snippet: The antibodies and other reagents used in this study were from the following sources: phospho-RPS6KB1/p-S6K1 (9234; 1:1,000 WB), RPS6KB1/S6K1 (9202; 1:1,000 WB), phosphoEIF4EBP1/p-4E-BP1 (9451; 1:3,000 WB), EIF4EBP1/4E-BP1 (9644; 1:1,000 WB), phospho-AKT (4060; 1:6,000 WB), AKT (4691; 1:6,000 WB), MTOR (2972; 1:1,000 WB; 1:200 immunofluorescence [IF]), RPTOR/raptor (2280; 1:1,000 WB), RRAGC (3360; 1:1,000 WB), LC3B (3868; 1:1,000 WB), FNIP2 (57612; 1:1,000 WB; 1:300 immunohistochemistry [IHC]), FLCN (3697; 1:1,000 WB), MEF2D (77986; 1:1,000 WB; 1:400 IHC; 1:50 ChIP), HA (3724; 1:2,000 WB), His (2365; 1:2,000 WB), VDAC (4661; 1:1,000 WB), GOLGA2/ GM130 (12480; 1:1,000 WB), Flag (8146; 1:1,000 WB), CALR/calreticulin (12238; 1:1,000 WB), phospho-tyrosine /p-Tyr (9411; 1:3,000 WB), SRC (2109; 1:1,000 WB), phosphoSRC (59548; 1:1,000 WB) and ACTB/β-actin (3700; 1:10,000) were purchased from Cell Signaling Technology/CST.

Techniques: Activity Assay, Over Expression, RNA Sequencing, Transfection, Plasmid Preparation, Control, Western Blot, Phospho-proteomics, Stable Transfection, Concentration Assay, Knockdown

Journal: Autophagy

Article Title: Direct regulation of FNIP1 and FNIP2 by MEF2 sustains MTORC1 activation and tumor progression in pancreatic cancer.

doi: 10.1080/15548627.2023.2259735

Figure Lengend Snippet: Figure 2. MEF2A and MEF2D are required for MTORC1 mediated protein, lipid synthesis and inhibit autophagy. (A) HeLa cells depletion of both MEF2A and MEF2D by indicated siRNA were treated with amino acid administration in the presence or absence of 10 μg/ml puromycin (PURO) and cycloheximide (CHX). Cell lysates were analyzed by western blot with an antibody to puromycin (17H1). Right plots show the ratio of PURO:ACTB. (B) MEF2A and MEF2D double-knockdown HeLa cells with or without transiently re-expressing MEF2A and MEF2D after utilized TSC2-targeting (siTSC2) or control (siNC) siRNA for 48 h under normal culture condition. Oil red O staining was performed to quantify intracellular lipid levels. Scale bar: 20 μm. (C) control or shRNA-mediated depletion of MEF2A and MEF2D HeLa cells were treated with the starvation of amino acids for 1 h and then restimulated with amino acids for indicated time. Phospho-ULK1 (Ser757), ULK1, LC3B-II levels were analyzed by western blotting. Right plot shows the quantification of LC3B-II:GAPDH. Short exposure (SE), long exposure (LE). (D) MEF2A and MEF2D knockdown and control HeLa cells that stably express GFP-LC3 were starved of amino acids for 1 h and then restimulated with amino acids for indicate duration and stained with LysoTracker. Time-lapse images were taken. Scale bar: 5 μm. (E) MEF2A and MEF2D double-knockdown and control HeLa cells were transfected for 24 h with GFP-RFP- LC3 adenovirus. After that, cells were subjected to amino acid starvation for 4 h and restimulation for 30 min. Autophagy flux was tested by confocal. Plot on bottom shows the ratios of colocalization of GFP and RFP. Scale bar: 10 μm. (F) statistical analysis of the cell diameters of control HeLa cells and the cells that depletion of MEF2A and MEF2D by using a cell counter. Data are presented as the mean ± S.E.M. (n = 3 independent experiments for A, B and C, n = 3 independent fields per condition for E, n = 10 per group for F. two-sided Student’s t-test for A, C, D and E, one-way ANOVA for B and E, *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant compared with indicated group).

Article Snippet: The antibodies and other reagents used in this study were from the following sources: phospho-RPS6KB1/p-S6K1 (9234; 1:1,000 WB), RPS6KB1/S6K1 (9202; 1:1,000 WB), phosphoEIF4EBP1/p-4E-BP1 (9451; 1:3,000 WB), EIF4EBP1/4E-BP1 (9644; 1:1,000 WB), phospho-AKT (4060; 1:6,000 WB), AKT (4691; 1:6,000 WB), MTOR (2972; 1:1,000 WB; 1:200 immunofluorescence [IF]), RPTOR/raptor (2280; 1:1,000 WB), RRAGC (3360; 1:1,000 WB), LC3B (3868; 1:1,000 WB), FNIP2 (57612; 1:1,000 WB; 1:300 immunohistochemistry [IHC]), FLCN (3697; 1:1,000 WB), MEF2D (77986; 1:1,000 WB; 1:400 IHC; 1:50 ChIP), HA (3724; 1:2,000 WB), His (2365; 1:2,000 WB), VDAC (4661; 1:1,000 WB), GOLGA2/ GM130 (12480; 1:1,000 WB), Flag (8146; 1:1,000 WB), CALR/calreticulin (12238; 1:1,000 WB), phospho-tyrosine /p-Tyr (9411; 1:3,000 WB), SRC (2109; 1:1,000 WB), phosphoSRC (59548; 1:1,000 WB) and ACTB/β-actin (3700; 1:10,000) were purchased from Cell Signaling Technology/CST.

Techniques: Western Blot, Knockdown, Expressing, Control, Staining, shRNA, Stable Transfection, Transfection

Journal: Autophagy

Article Title: Direct regulation of FNIP1 and FNIP2 by MEF2 sustains MTORC1 activation and tumor progression in pancreatic cancer.

doi: 10.1080/15548627.2023.2259735

Figure Lengend Snippet: Figure 3. MEF2A and MEF2D positive regulate MTORC1 translocation to lysosomes. (A) MEF2A and MEF2D double-knockdown and control HeLa cells transfected with Flag-tagged TMEM192 for 24 h were subjected to amino acid starvation and restimulation. Purified lysosomes via lyso-IP assay (methods). Protein levels of MTOR and RPTOR (MTORC1 components), LAMP1 and LAMP2 (lysosome), CALR (endoplasmic reticulum), VDAC (mitochondria), GOLGA2 (Golgi) and RPS6KB1 (cytosol) were confirmed by immunoblotting. Right graph shows the quantification result of MTOR after normalization for LAMP1. (B) cells as in (A) were starved of and restimulated with amino acids for the indicated times before being analyzed by immunofluorescence and quantified to calculate the percentage of colocalization coefficient of MTOR (green) and lysosomal protein LAMP2 (red). Scale bar: 10 μm. (C) MEF2A and MEF2D double-knockdown HeLa cells were subjected to amino acid starvation and restimulation paradigm. Cell lysates were immunoprecipitated with anti-RRAGC antibody. Immunoprecipitates or total cell lysate (TCL) samples were probed for MTOR, RPTOR and RRAGC. Right graph shows the quantification result of MTOR after normalization for RRAGC. (D) MEF2A and MEF2D double-knockdown and control HeLa cells that either transfected with constitutively active RRAG GTPases (Flag-tagged RRAGAQ66L and RRAGCS75N) or vector coimmunostained for lysosomal marker LAMP2 (green), MTOR (red) and Flag-tag (violet). Cells were starved of amino acid and restimulated with amino acids before processing and imaging. The graph on the bottom shows the colocalization coefficient. Scale bar: 10 μm. (E) control (lanes 1–2) and knockdown of both MEF2A and MEF2D (lanes 3–6) HeLa cells that transfected with indicated RRAG GTPases were then treated with amino acids administration. Phosphorylation and protein levels of RPS6KB1 and EIF4EBP1 were determined by immunoblotting. Right graph shows the RPS6KB1:RPS6KB1 ratios. Data are presented as the mean ± S.E.M. (n = 3 independent experiments for A, C and E, n = 4 independent fields per condition for B, D. two-sided Student’s t-test for A, B, C and E, one-way ANOVA for D, **P < 0.01, ***P < 0.001).

Article Snippet: The antibodies and other reagents used in this study were from the following sources: phospho-RPS6KB1/p-S6K1 (9234; 1:1,000 WB), RPS6KB1/S6K1 (9202; 1:1,000 WB), phosphoEIF4EBP1/p-4E-BP1 (9451; 1:3,000 WB), EIF4EBP1/4E-BP1 (9644; 1:1,000 WB), phospho-AKT (4060; 1:6,000 WB), AKT (4691; 1:6,000 WB), MTOR (2972; 1:1,000 WB; 1:200 immunofluorescence [IF]), RPTOR/raptor (2280; 1:1,000 WB), RRAGC (3360; 1:1,000 WB), LC3B (3868; 1:1,000 WB), FNIP2 (57612; 1:1,000 WB; 1:300 immunohistochemistry [IHC]), FLCN (3697; 1:1,000 WB), MEF2D (77986; 1:1,000 WB; 1:400 IHC; 1:50 ChIP), HA (3724; 1:2,000 WB), His (2365; 1:2,000 WB), VDAC (4661; 1:1,000 WB), GOLGA2/ GM130 (12480; 1:1,000 WB), Flag (8146; 1:1,000 WB), CALR/calreticulin (12238; 1:1,000 WB), phospho-tyrosine /p-Tyr (9411; 1:3,000 WB), SRC (2109; 1:1,000 WB), phosphoSRC (59548; 1:1,000 WB) and ACTB/β-actin (3700; 1:10,000) were purchased from Cell Signaling Technology/CST.

Techniques: Translocation Assay, Knockdown, Control, Transfection, Purification, Western Blot, Immunofluorescence, Immunoprecipitation, Plasmid Preparation, Marker, FLAG-tag, Imaging, Phospho-proteomics

Journal: Autophagy

Article Title: Direct regulation of FNIP1 and FNIP2 by MEF2 sustains MTORC1 activation and tumor progression in pancreatic cancer.

doi: 10.1080/15548627.2023.2259735

Figure Lengend Snippet: Figure 4. MEF2A and MEF2D control MTORC1 lysosome localization and activation via transcriptional regulation of FNIP1 and FNIP2. (A) heatmap of the binding sites of MEF2s at the positions − 3.0 kb upstream to + 3.0 kb downstream relative to the transcription start site (TSS). (B) volcano plot showing DEGs in control and MEF2- VP16 overexpression HeLa cells. The significant changed upregulated genes were labeled with gene names, NR4A1 and ARC are well-characterized substrates of MEF2s. (C) venn diagrams displaying the overlap of downregulated and upregulated genes from the transcriptome with MEF2 bound genes. Bottom table shows FNIP2 and other genes which are well known as MEF2s substrates including Fos/c-fos, NR4A1, ZNF122 and ARC in the subset of overlap of upregulated genes with MEF2 bound genes. (D) left-schematic shows the predicted MEF2 binding sites by conserved MEF2 consensus at the positions upstream to FNIP1 and FNIP2 genes transcription start site (TSS). Right plots show the PCR results revealed by chromatin immunoprecipitation assay. MEF2D_1,2 indicate the ChIP-PCR results by different antibodies (1-BD biosciences, 610774; 2-CST, 77986). (E) control (lanes 1–2) and MEF2A and MEF2D double-knockdown (lanes 3–8) HeLa cells that either transfected with indicated plasmids were starved of and restimulated with amino acids. Phosphorylation and protein levels of RPS6KB1 and EIF4EBP1 were determined by immunoblotting, as indicated. Plots on the right show p-RPS6KB1:RPS6KB1 (top) and p-EIF4EBP1:ATCB (bottom) ratios. (F) MEF2A and MEF2D double-knockdown

Article Snippet: The antibodies and other reagents used in this study were from the following sources: phospho-RPS6KB1/p-S6K1 (9234; 1:1,000 WB), RPS6KB1/S6K1 (9202; 1:1,000 WB), phosphoEIF4EBP1/p-4E-BP1 (9451; 1:3,000 WB), EIF4EBP1/4E-BP1 (9644; 1:1,000 WB), phospho-AKT (4060; 1:6,000 WB), AKT (4691; 1:6,000 WB), MTOR (2972; 1:1,000 WB; 1:200 immunofluorescence [IF]), RPTOR/raptor (2280; 1:1,000 WB), RRAGC (3360; 1:1,000 WB), LC3B (3868; 1:1,000 WB), FNIP2 (57612; 1:1,000 WB; 1:300 immunohistochemistry [IHC]), FLCN (3697; 1:1,000 WB), MEF2D (77986; 1:1,000 WB; 1:400 IHC; 1:50 ChIP), HA (3724; 1:2,000 WB), His (2365; 1:2,000 WB), VDAC (4661; 1:1,000 WB), GOLGA2/ GM130 (12480; 1:1,000 WB), Flag (8146; 1:1,000 WB), CALR/calreticulin (12238; 1:1,000 WB), phospho-tyrosine /p-Tyr (9411; 1:3,000 WB), SRC (2109; 1:1,000 WB), phosphoSRC (59548; 1:1,000 WB) and ACTB/β-actin (3700; 1:10,000) were purchased from Cell Signaling Technology/CST.

Techniques: Control, Activation Assay, Binding Assay, Over Expression, Labeling, Chromatin Immunoprecipitation, Knockdown, Transfection, Phospho-proteomics, Western Blot

Journal: Autophagy

Article Title: Direct regulation of FNIP1 and FNIP2 by MEF2 sustains MTORC1 activation and tumor progression in pancreatic cancer.

doi: 10.1080/15548627.2023.2259735

Figure Lengend Snippet: Figure 5. SRC directly interacts with and phosphorylates MEF2D upon mitogenic stimulation. (A) lysate derived from human embryonic kidney 293T (HEK293T) cells transfected as indicated were immunoprecipitated with IgG or anti-hemagglutinin (HA) antibody. Immunoprecipitation (IP) and TCL were probed for indicated antibodies. (B) GST affinity isolation assay was performed using HEK293T cells purified Flag-tagged MEF2D protein and bacterially purified GST or GST-tagged SRC (method), followed by immunoblotting with indicated antibodies. (C) HeLa cells were cultured in a serum free medium for 4 h, then treated with or without EGF for 30 min. Cell lysates were immunoprecipitated with anti-MEF2D antibody and blotted with anti-SRC antibody (top panel). TCL was probed for indicated antibodies. (D) HEK293T purified Flag-tagged MEF2D protein was pre-treated with lambda PP and then incubated with commercial active GST-tagged SRC kinase in a kinase assay buffer, followed by immunoblotting with indicated antibodies. (E) HEK293T cells transfected as indicated were immunoprecipitated with anti-HA antibody and blotted with pan p-Tyr antibody. TCL was analyzed by immunoblotting for indicated antibodies. (F) HEK293T cells transfected HA-tagged MEF2D with either Flag- tagged SRC-WT (wild-type) or Flag-tagged SRC-KD (SRCK298M, a kinase-dead form of SRC). Cell lysates were immunoprecipitated with anti-HA antibody. IP and TCL samples were probed for indicated antibodies. (G) HeLa cells were maintained in a serum free medium for 4 h, and then treated with or without serum, EGF or serum plus EGF for 30 min. Total cell lysates and endogenous MEF2D immunoprecipitated were analyzed by immunoblotting with indicated antibodies. Actin as a loading control. (H) HeLa cells transfected with indicated siRnas against SRC (in two RNAi sequences) or control (siNC) for 48 h were subjected to serum free culture for 4 h, followed by stimulation with or without EGF for 30 min. Cells were lysed and subjected to immunoprecipitation with anti-MEF2D antibody, followed by immunoblotting with indicated antibodies. (I) HeLa cells were subjected to serum free medium for 4 h, and then either untreated or restimulation with EGF in the presence or absence of SRC kinase inhibitor Dasatinib or SU6656. Endogenous MEF2D was immunoprecipitated, followed by immunoblotting with indicated antibodies.

Article Snippet: The antibodies and other reagents used in this study were from the following sources: phospho-RPS6KB1/p-S6K1 (9234; 1:1,000 WB), RPS6KB1/S6K1 (9202; 1:1,000 WB), phosphoEIF4EBP1/p-4E-BP1 (9451; 1:3,000 WB), EIF4EBP1/4E-BP1 (9644; 1:1,000 WB), phospho-AKT (4060; 1:6,000 WB), AKT (4691; 1:6,000 WB), MTOR (2972; 1:1,000 WB; 1:200 immunofluorescence [IF]), RPTOR/raptor (2280; 1:1,000 WB), RRAGC (3360; 1:1,000 WB), LC3B (3868; 1:1,000 WB), FNIP2 (57612; 1:1,000 WB; 1:300 immunohistochemistry [IHC]), FLCN (3697; 1:1,000 WB), MEF2D (77986; 1:1,000 WB; 1:400 IHC; 1:50 ChIP), HA (3724; 1:2,000 WB), His (2365; 1:2,000 WB), VDAC (4661; 1:1,000 WB), GOLGA2/ GM130 (12480; 1:1,000 WB), Flag (8146; 1:1,000 WB), CALR/calreticulin (12238; 1:1,000 WB), phospho-tyrosine /p-Tyr (9411; 1:3,000 WB), SRC (2109; 1:1,000 WB), phosphoSRC (59548; 1:1,000 WB) and ACTB/β-actin (3700; 1:10,000) were purchased from Cell Signaling Technology/CST.

Techniques: Derivative Assay, Transfection, Immunoprecipitation, Isolation, Purification, Western Blot, Cell Culture, Incubation, Kinase Assay, Control

Journal: Autophagy

Article Title: Direct regulation of FNIP1 and FNIP2 by MEF2 sustains MTORC1 activation and tumor progression in pancreatic cancer.

doi: 10.1080/15548627.2023.2259735

Figure Lengend Snippet: Figure 6. SRC-mediated tyrosine phosphorylation of MEF2D is required for transcriptional activity of MEF2D and in regulation of MTORC1 activity. (A) His-tagged MEF2D-WT or MEF2D mutant constructs (mutation of Tyr33, 57, 69, 72, 117, 131, 225, 333, 337 and 478 residues) were co-transfected with or without Flag-tagged SRC in HEK293T cells for 24 h. Cells were lysed and subjected to immunoprecipitation against His-tag, followed by immunoblotting with p-tyr antibody. (B) HEK293T cells transfected His-tagged MEF2D-WT or MEF2D-3YF mutants with or without Flag-tagged SRC were lysed and subjected to immunoprecipitation against His-tag, followed by immunoblotting with indicated antibodies. (C) Flag-tagged MEF2D-WT or its 3YF mutant protein purified from HEK293T cells was incubated with commercial active GST-tagged SRC kinase in a kinase assay buffer, followed by immunoblotting with p-tyr antibody. (D) sequence alignment of the residues flanking across different species. Black arrowheads point to the tyrosine residues corresponding to the Tyr333 and tyr 337 residues in human MEF2D. (E) HeLa cells that transfected with Flag-tagged MEF2D-WT or MEF2D-3YF were maintained in a serum free medium for 4 h, followed with or without EGF treatment. Cell lysates were prepared and immunoprecipitation were analyzed by immunoblotting. (F) luciferase assay was performed in depletion of both MEF2A and MEF2D HeLa cells after co- transfection of indicated expression plasmids and wild-type (MEF2 reporter-WT) or mutated (MEF2 reporter-mt) luciferase reporter plasmids for 24 h. (G) qRT-PCR

Article Snippet: The antibodies and other reagents used in this study were from the following sources: phospho-RPS6KB1/p-S6K1 (9234; 1:1,000 WB), RPS6KB1/S6K1 (9202; 1:1,000 WB), phosphoEIF4EBP1/p-4E-BP1 (9451; 1:3,000 WB), EIF4EBP1/4E-BP1 (9644; 1:1,000 WB), phospho-AKT (4060; 1:6,000 WB), AKT (4691; 1:6,000 WB), MTOR (2972; 1:1,000 WB; 1:200 immunofluorescence [IF]), RPTOR/raptor (2280; 1:1,000 WB), RRAGC (3360; 1:1,000 WB), LC3B (3868; 1:1,000 WB), FNIP2 (57612; 1:1,000 WB; 1:300 immunohistochemistry [IHC]), FLCN (3697; 1:1,000 WB), MEF2D (77986; 1:1,000 WB; 1:400 IHC; 1:50 ChIP), HA (3724; 1:2,000 WB), His (2365; 1:2,000 WB), VDAC (4661; 1:1,000 WB), GOLGA2/ GM130 (12480; 1:1,000 WB), Flag (8146; 1:1,000 WB), CALR/calreticulin (12238; 1:1,000 WB), phospho-tyrosine /p-Tyr (9411; 1:3,000 WB), SRC (2109; 1:1,000 WB), phosphoSRC (59548; 1:1,000 WB) and ACTB/β-actin (3700; 1:10,000) were purchased from Cell Signaling Technology/CST.

Techniques: Phospho-proteomics, Activity Assay, Mutagenesis, Construct, Transfection, Immunoprecipitation, Western Blot, Purification, Incubation, Kinase Assay, Sequencing, Luciferase, Cotransfection, Expressing, Quantitative RT-PCR

Journal: Autophagy

Article Title: Direct regulation of FNIP1 and FNIP2 by MEF2 sustains MTORC1 activation and tumor progression in pancreatic cancer.

doi: 10.1080/15548627.2023.2259735

Figure Lengend Snippet: Figure 7. MEF2D promotes pancreatic cancer cell proliferation and correlates with clinical and pathological parameters. (A) pancreatic cancer cell line AsPC-1 (top) and PANC-1 (bottom) that depletion of MEF2A and MEF2D, transfected with either Flag-tagged MEF2D-WT or MEF2D-3YF, respectively and subjected to the cell proliferation assay (CCK8) to determine viable cell number. NC and knockdown of both MEF2A and MEF2D group co-transfected with empty vector. (B) the colony formation assay was performed with MEF2A and MEF2D double-knockdown AsPC-1 cells (left) or PANC-1 cells (right). The cells were maintained in complete medium containing 10% FBS for two weeks before staining and imaging. Scale bars: 5 mm. (C) Representative images (left) and weights (right) of xenograft tumors from nude mice implanted with MEF2A and MEF2D double-knockdown AsPC-1 cells versus control cells. n = 10 mice per group. (D) volumes of tumor burden in nude mice implanted with MEF2A and MEF2D double-knockdown or control AsPC-1 cells were measured after implantation. n = 10 mice per group. (E) TCGA cohort of pancreatic cancer patients were divided into two groups according to the median level of MEF2D mRNA expression. Overall survival was compared between these two groups, as shown in Kaplan-Meier curves. Log-rank P values are indicated. (F) immunoblot analysis of MEF2D, FNIP1, FNIP2, EIF4EBP1 and p-EIF4EBP1 were

Article Snippet: The antibodies and other reagents used in this study were from the following sources: phospho-RPS6KB1/p-S6K1 (9234; 1:1,000 WB), RPS6KB1/S6K1 (9202; 1:1,000 WB), phosphoEIF4EBP1/p-4E-BP1 (9451; 1:3,000 WB), EIF4EBP1/4E-BP1 (9644; 1:1,000 WB), phospho-AKT (4060; 1:6,000 WB), AKT (4691; 1:6,000 WB), MTOR (2972; 1:1,000 WB; 1:200 immunofluorescence [IF]), RPTOR/raptor (2280; 1:1,000 WB), RRAGC (3360; 1:1,000 WB), LC3B (3868; 1:1,000 WB), FNIP2 (57612; 1:1,000 WB; 1:300 immunohistochemistry [IHC]), FLCN (3697; 1:1,000 WB), MEF2D (77986; 1:1,000 WB; 1:400 IHC; 1:50 ChIP), HA (3724; 1:2,000 WB), His (2365; 1:2,000 WB), VDAC (4661; 1:1,000 WB), GOLGA2/ GM130 (12480; 1:1,000 WB), Flag (8146; 1:1,000 WB), CALR/calreticulin (12238; 1:1,000 WB), phospho-tyrosine /p-Tyr (9411; 1:3,000 WB), SRC (2109; 1:1,000 WB), phosphoSRC (59548; 1:1,000 WB) and ACTB/β-actin (3700; 1:10,000) were purchased from Cell Signaling Technology/CST.

Techniques: Transfection, Proliferation Assay, Knockdown, Plasmid Preparation, Colony Assay, Staining, Imaging, Control, Expressing, Western Blot