Journal: Journal of Hematology & Oncology

Article Title: Identification of a promising PI3K inhibitor for the treatment of multiple myeloma through the structural optimization

doi: 10.1186/1756-8722-7-9

Figure Lengend Snippet: BENC-511 downregulates PI3K/AKT downstream signals. OPM2, RPMI-8226 and LP1 cells were treated with increasing concentrations of BENC-511 for 24 hours. Whole lysates were subjected to Western blot analysis. (A) p-mTOR (Ser2448), T-mTOR, Raptor; (B) p-p70S6K, p70S6K, p-4E-BP1, and 4E-BP1; (C) p-GSK-3β (Ser9). β-actin was used as an internal control.

Article Snippet: After proteins were then transferred to polyvinylidene difluoride membranes, the blots were then probed with antibodies including monoclonal PARP, Caspase-3, p-AKT(S473), p-AKT(T308), AKT, p-mTOR(S2448), Raptor, p-P70S6K, P70S6K, p-4E-BP1(S65), 4E-BP1 (all were purchased from Cell Signaling Technology, Inc.).

Techniques: Western Blot

Journal: Molecular Cancer

Article Title: The circular RNA circ-ERBIN promotes growth and metastasis of colorectal cancer by miR-125a-5p and miR-138-5p/4EBP-1 mediated cap-independent HIF-1α translation

doi: 10.1186/s12943-020-01272-9

Figure Lengend Snippet: Circ-ERBIN elevates the expression of HIF-1α via 4EBP-1 . a Venn diagram for the putative target miRNAs of circ-ERBIN. b Venn diagram for the downstream targets of circ-ERBIN targeted miRNAs. c Schematic diagram of the predicted miR-125a-5p and miR-138-5p binding sites for 4EBP-1. d Circ-ERBIN overexpression (pLCDH/circ-ERBIN) stable cells were used to perform subcutaneous tumor model and tail vein injection metastasis model, respectively. Immunohistochemical staining of 4EBP-1 were presented. Scale bars, 100 μm. e , f 4EBP-1 siRNA or 4EBP-1 plasmids were transfected into HCT116 circ-ERBIN op or knockdown cells, respectively. Western blots were performed to determine the corresponding proteins. g , h Western blot experiments were performed using HCT116 circ-ERBIN stable overexpression or knockdown cell lines, antibodies with different phosphorylation sites of 4EBP-1 were used respectively. i Circ-ERBIN op or pLCDH cells were immunopreipitated (IP) with IgG or eIF4G antibody, and the immunoprecipitates were analyzed by western blot. j Circ-ERBIN op or pLCDH cells were transiently transfected with pRnegR or pRhif-1αF plasmids. Renilla and firefly luciferase activities were determined after 24 h tranfection, and IRES activities represented as ratios of Firefly to Renilla luciferase. Data are means ± SD. *, P <0.05.

Article Snippet: Phospho-4EBP-1 (Thr37/46), non-phospho-4EBP-1 (Thr37/46), Phospho-4EBP-1 (Ser65) and Phospho-4EBP-1 (Thr70) were purchased as 4E-BP antibody sampler kit (Cell signaling technology, #9955).

Techniques: Expressing, Binding Assay, Over Expression, Injection, Immunohistochemical staining, Staining, Transfection, Western Blot, Luciferase

Journal: Molecular Cancer

Article Title: The circular RNA circ-ERBIN promotes growth and metastasis of colorectal cancer by miR-125a-5p and miR-138-5p/4EBP-1 mediated cap-independent HIF-1α translation

doi: 10.1186/s12943-020-01272-9

Figure Lengend Snippet: Circ-ERBIN elevates HIF-1 α expression by miR-125a-5p/miR-138-5p/4EBP-1 signaling. a Luciferase activity of LUC-circ-ERBIN WT or LUC-circ-ERBIN Mutant in HCT116 cells after co-transfection with miR-125a-5p or/and miR-138-5p mimics. b qRT-PCR was performed to detect related miRNAs and 4EBP-1 levels using mouse subcutaneous tumor samples. c , d qRT-PCR experiments were performed to detect miR-125a-5p and miR-138-5p mRNA levels using clinical CRC patient samples ( n = 32). The correlations between circ-ERBIN and miR-125a-5p or miR-138-5p were represented. All the qRT-PCR experiments were normalized using GAPDH as an endogenous control. Data are means ± SD. P <0.05. e Luciferase activity of 4EBP-1 3’UTR constructs harboring wide type sequence or mutant sequence were analyzed after being transfected with miR-125a-5p or miR-138-5p mimics alone or together. β-gal was transfected as control. f miR-125a-5p and miR-138-5p mimics were transfected into circ-ERBIN stable cell lines alone or together. Western blots were performed to analyze the protein of 4EBP-1 and HIF-1α. g miR-125a-5p or miR-138-5p inhibitor was transfected alone or together into HCT116 sh-circ-ERBIN stable cell lines. Western blots were performed to analyze the protein levels of 4EBP-11 and HIF-1α. h , i The correlations between 4EBP-1 and miR-125a-5p or miR-138-5p were represented. j The correlation between circ-ERBIN and 4EBP-1 was represented. All the qRT-PCR experiments were normalized using GAPDH as an endogenous control. Data are means ± SD. P <0.05.

Article Snippet: Phospho-4EBP-1 (Thr37/46), non-phospho-4EBP-1 (Thr37/46), Phospho-4EBP-1 (Ser65) and Phospho-4EBP-1 (Thr70) were purchased as 4E-BP antibody sampler kit (Cell signaling technology, #9955).

Techniques: Expressing, Luciferase, Activity Assay, Mutagenesis, Cotransfection, Quantitative RT-PCR, Construct, Sequencing, Transfection, Stable Transfection, Western Blot

Journal: Molecular Cancer

Article Title: The circular RNA circ-ERBIN promotes growth and metastasis of colorectal cancer by miR-125a-5p and miR-138-5p/4EBP-1 mediated cap-independent HIF-1α translation

doi: 10.1186/s12943-020-01272-9

Figure Lengend Snippet: Circ-ERBIN accelerates the growth and metastasis of CRC by activating HIF-1α signaling through the circ-ERBIN/miR-125a-5p/miR-138-5p/ 4EBP-1 pathway. a Transwell assays for HCT116 cells that stably overexpressed circ-ERBIN or co-transfected with miR-125a-5p or miR-138-5p mimics after 2 days. b MiR-125a-5p or miR-138-5p inhibitor were transfected alone or together into HCT116 sh-circ-ERBIN #1 stable cell lines for 2 days followed by the analysis of cell migration using transwell assays. Cells were stained by Giemsa’s staining and visualized under a phase-contrast microscope. c - e HCT116 stably expressed circ-ERBIN or pLCDH vector were injected into BALB/C nude mice ( n = 5 for each group). AgomiRs were injected into tumors as represented. The tumors were excised and photographed, and the size and weight of tumors were presented. f Representative immunohistochemical stainings of 4EBP-1 and HIF-1α in subcutaneous tumors with agomiRs injection as described. Scale bars, 100 μm. g A schematic model displaying the role of circ-ERBIN/miR-125a-5p/miR-138-5p/ 4EBP-1/HIF-1α axis in CRC metastasis.

Article Snippet: Phospho-4EBP-1 (Thr37/46), non-phospho-4EBP-1 (Thr37/46), Phospho-4EBP-1 (Ser65) and Phospho-4EBP-1 (Thr70) were purchased as 4E-BP antibody sampler kit (Cell signaling technology, #9955).

Techniques: Stable Transfection, Transfection, Migration, Staining, Microscopy, Plasmid Preparation, Injection, Immunohistochemical staining

Journal: bioRxiv

Article Title: CK1 Delta Is an mRNA Cap-Associated Protein That Drives Translation Initiation and Tumor Growth

doi: 10.1101/2020.02.20.955229

Figure Lengend Snippet: The HEK293 cell line was targeted for CK1δ-/- knockout by the CRISPR-Cas9 system, using 2 different single guide RNA (sgRNA) sequences. Knockout clones were selected from single cells by GFP sorting followed by serial dilution, and gene editing was confirmed by sequencing. KO1 and KO2 had mono-allelic knockout of CK1δ, and KO3 and KO4 has bi-allelic knockout of CK1δ (Figure S1A). (A-B) Four clones of CK1δ knockout, KO1-4, were compared with the vector Cas9 in HEK293 for the levels of CK1δ, CK1ε, 4E-BP1, p70S6K, RPS6, and actin by immunoblotting. (C) The same CK1δ knockout clones of HEK293 and Cas9 HEK293 cells were processed for the cap binding assay using m 7 GTP agarose beads to pull down cap binding proteins, which were then analyzed by immunoblotting. The ratio of eIF4G in the pulldown relative to the input cell lysate is an index of eIF4F assembly. (D) Polysome profiling was performed the in HEK293 cells with CK1δ knockout clones KO3 and KO4, in comparison to the control HEK293 cells with non-targeting Cas9. X-axis showed the sedimentations or fractions collected from 10–50% sucrose density gradient centrifugation. Y-axis showed the OD260 UV recording of RNA abundance. Data shown were the average of three experiments. RNP, ribonuclear protein; 40S, 40S small ribosome subunit; 60S, 60S large ribosome subunit; 80S, 80S monosome. (E) The same CK1δ knockout clones of HEK293 and Cas9 HEK293 cells were grown in 1μg/ml puromycin for 30 minutes. Cell lysates were subjected to Western blotting using the anti-puromycin antibody in the SUnSET assay.

Article Snippet: Membranes were developed using the chemiluminescence detection system from Thermo Scientific.p-4E-BP1-S65, p-4E-BP1-T70, p-4E-BP1-T37/46, 4E-BP1, β-catenin, p-p70SK, P-RPS6, p70SK, RPS6, eIF4E, eIF4G, PARP, RAPTOR, Lamin B1, β-actin were purchased from Cell Signaling Technology.

Techniques: Knock-Out, CRISPR, Clone Assay, Serial Dilution, Sequencing, Plasmid Preparation, Western Blot, Binding Assay, Gradient Centrifugation

Journal: Frontiers in Pharmacology

Article Title: Modified Si–Jun–Zi–Tang Attenuates Airway Inflammation in a Murine Model of Chronic Asthma by Inhibiting Teff Cells via the mTORC1 Pathway

doi: 10.3389/fphar.2019.00161

Figure Lengend Snippet: MSJZT treatment inhibited the mTORC1 pathway, but not the mTORC2 pathway in lung homogenates. Western blot analysis was performed to investigate mTORC1 and mTORC2 activities and involved investigating the phosphorylation of their substrates, S6RP, p70S6K, 4E-BP1, Akt, and mTOR 2481, respectively. The expression of these proteins was quantified and represented as band intensity of phosphorylated proteins normalized to the relevant total proteins. The amount of loaded material was 100 μg. Data are expressed as means ± SEM ( n = 6 in each group). ∗ P < 0.05 and ∗∗ P < 0.01 versus the OVA group.

Article Snippet: Antibodies for phospho-p70S6K (Thr389), p70 S6K, phospho-S6 ribosomal protein (Ser235/236), S6 ribosomal protein, phospho-Akt (Ser473), Akt, phospho-4E-BP1 (Ser65), 4E-BP1, phospho-mTOR (Ser2481), and mTOR were obtained from Cell Signaling Technology, Danvers, MA, United States.

Techniques: Western Blot, Expressing

Journal: Oncotarget

Article Title: PAFR selectively mediates radioresistance and irradiation-induced autophagy suppression in prostate cancer cells

doi: 10.18632/oncotarget.14647

Figure Lengend Snippet: ( A ) Western blot analysis of LC3B in PC3 and PC3-shPAFR cells in indicated time postradiation. ( B ) Quantification of LC3B expression in PC3 and PC3-shPAFR cells in indicated time postradiation. The results are normalized by β-actin. Data represents at least 3 independent experiments. ( C ) Western blot analysis of LC3B in xenograft tumors harvested from Figure . ( D ) Western blot analysis of LC3B, P62, p-Beclin 1, total Beclin 1, p-4E-BP1, total 4E-BP1 (21KD-band), p-ULK1 and total ULK1 in DMSO or GB-treated treated PC3 72 hours (72 h) post sham or 6 Gy of X-ray. ( E ) Immunoprecipitation of Beclin 1 with PAFR (and the reverse direction CO-IP) in PC3 cells in conditions shown in (D).

Article Snippet: Antibodies specific for activated caspase 3, LC3B, Beclin-1, phosphorylated Beclin 1 at Ser93 ( p -Beclin 1), 4E-BP1, phosphorylated 4E-BP1 at Ser65 ( p -4E-BP1), ULK1 and phosphorylated ULK1 at ser757 ( p -ULK1) were obtained from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Western Blot, Expressing, Immunoprecipitation, Co-Immunoprecipitation Assay