Journal: Aging Cell

Article Title: Liver osteopontin is required to prevent the progression of age‐related nonalcoholic fatty liver disease

doi: 10.1111/acel.13183

Figure Lengend Snippet: Liver OPN is p53 regulated. (a) Protein levels of p53 were evaluated by immunohistochemistry in liver sections of 3‐, 10‐, and 20‐month‐old (m) wild‐type (WT) mice ( n = 3–5). (b) OPN protein levels from liver homogenates were measured in WT and p53‐KO male and female mice fed a chow diet (CD) and a high‐fat diet (HFD) by immunoblotting using glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) as loading control ( n = 4–6). (c) Liver p53 and OPN protein levels were evaluated in WT mice fed a HFD injected with p53‐dominant positive adenovirus (adp53) and GFP (adGFP), using transferrin or GAPDH as a loading control ( n = 4–6). (d) Protein levels of p53 and OPN in siCtrl and sip53 HepG2 cells were measured by immunoblotting using GAPDH as loading control. Extracellular OPN was measured using an ELISA ( n = 4–5). (e) OPN protein levels from HepG2 cells silenced for p53 and Hep3B cells treated with palbociclib (Palbo) were measured by immunoblotting using GAPDH as loading control. OPN media levels from Hep3B cells treated either with vehicle (Veh) or with palbociclib (Palbo) were measured by ELISA ( n = 4–8). Values are means ± SEM . Significant differences are denoted by * p < 0.05, ** p < 0.01, and *** p < 0.001 (Student's t test)

Article Snippet: Data obtained from the Cell Line Encyclopedia (© 2019 The Broad Institute of MIT & Harvard) showed that the cell line with the least p53 expression, the Hep3B, also had the lowest OPN expression.

Techniques: Immunohistochemistry, Western Blot, Control, Injection, Enzyme-linked Immunosorbent Assay

Journal: Molecular Cancer

Article Title: Overexpression of wild type RRAS2 , without oncogenic mutations, drives chronic lymphocytic leukemia

doi: 10.1186/s12943-022-01496-x

Figure Lengend Snippet: Overexpression of the unmutated RRAS2 leads to a B cell leukemia in mice. a & b Genomic alterations in the KRAS and RRAS2 genes according to cBioportal for cancer genomics ( http://www.cbioportal.org ). Data represent a combined study of 78,278 patients/81072 samples. The X-axis represents alteration frequency of the gene (colors represent the alteration type). The Y-axis represents different cancer types. c Relative mRNA expression of RRAS2 in different cancers. Data are represented in log2 scale and were obtained from the Pan-Cancer Analysis of Whole Genomes (PCAWG). d Representative images of the relative sizes of spleens from 12 month-old control and Sox2-Cre + mice. e Quantification of the total number of B cells per spleen of 6 month-old control and Sox2-Cre + mice. Data shown correspond to triplicate measurements of one control and three Sox2-Cre mice. Unpaired t-test with Welch’s correction. f Hematoxylin and eosin images of the spleen structure in an 8-month old control mouse and in 4-, 8- and 12-month-old Sox2-Cre + mice. g Two-parameter flow cytometry of the expression of CD21 and CD23, and IgD and IgM, respectively, in B cells in spleens of 8 month-old control and Sox2Cre + mice h Left, two-parameter flow cytometry of the expression of CD5 and IgM in B cells of the blood of 8 month-old control and Sox2-Cre + mice. i, quantification of the number of CD5 + IgM+ B cells in the blood of 40 wk-old control and Sox2-Cre + mice. Data shown correspond to duplicate measurements of four control and six Sox2-Cre mice. Unpaired t-test with Welch’s correction. In all figures, control mice refer to Rosa26- RRAS2 fl/fl without Cre recombinase

Article Snippet: Ten-week Rag2 −/− γc −/− male mice were subcutaneously injected in their left flank with 10 × 10 6 MEC-1 control or MEC-1 RRAS2 knockdown cells as described in [ ], in a mixture with PBS/Matrigel (1:1) (Becton Dickinson).

Techniques: Over Expression, Expressing, Control, Flow Cytometry

Journal: Molecular Cancer

Article Title: Overexpression of wild type RRAS2 , without oncogenic mutations, drives chronic lymphocytic leukemia

doi: 10.1186/s12943-022-01496-x

Figure Lengend Snippet: CD5 + IgM+ B cells are oligoclonal and can be transferred to wild type mice. a Two-parameter flow cytometry of the expression of CD19 and CD5 in blood cells of 35 wk-old control and Rosa26- RRAS2 fl/fl xmb1-Cre (mb1-Cre+) mice. b Quantification of the number of CD5 + IgM+ B cells (CD19+) in the blood of 30–35 wk-old control ( n = 10) and mb1-Cre ( n = 47) mice. Unpaired t-test with Welch’s correction. c Dot plot representation of the quantification of CD5 + IgM+ B cells (CD19+) in the blood of mb1-Cre mice ( n = 14) and its evolution over time. Data points were adjusted to an exponential growth. d Kaplan-Meier survival plot of Rosa26- RRAS2 fl/fl xmb1-Cre (mb1-Cre+) mice ( n = 22) and WT control C57BL/6 J mice ( n = 40) allowed to age in the same housing conditions. Median survival for mb1-Cre = 13.57 months; median survival for WT controls = 32.39 months. P < 0.0001, long-rank Mantel-Cox test and Gehan-Breslow-Wilcoxon test. e Experimental setup for the adoptive transfer experiment. Total B cells from mouse donors bearing the hematopoietic cell marker allele CD45.2 were purified and inoculated i.v. in the tail vein of sublethaly irradiated wild type recipient of the same strain (C57BL/6) but bearing the CD45.1 allele. Donor control mice refer to Rosa26- RRAS2 fl/fl without Cre recombinase 11 wk-old and the donor problem mice corresponded to 16 wk-old Rosa26- RRAS2 fl/fl x mb1-Cre. f Quantification by flow cytometry of the expression of CD45.2 and CD5 within the CD19+ population of inoculated CD45.1+ mice ( n = 8) and bled at the indicated time points. In this figure, control mice refer to Rosa26- RRAS2 fl/fl without Cre recombinase. Two-way ANOVA test. g PCR results of using specific forward oligonucleotides for different V H families and a constant reverse oligonucleotide against the J region of the immunoglobulin heavy chain gene. The specific V H families analyzed in each case are indicated on top of each section of the agarose gel. H 2 O is used as a negative control. In each V H family, BM corresponds to bone marrow cells of a wild-type 12 wk-old mouse, T corresponds to sorted naïve thymocytes (no recombination) from a wild type 6 wk-old mouse, and each column corresponds to the spleen of an individual mb1-Cre mouse of ages between 35 and 40 weeks. h Representative flow cytometry plot showing the presence of GFP low and GFP high populations in the spleen of a 30 wk-old Rosa26- RRAS2 fl/fl xmb1-Cre mouse. i RT-qPCR for the differentially expressed RRAS2 gene in the CD19 + CD5+ GFP high and GFP low populations sorted from spleens of n = 7 30–35 wk-old Rosa26- RRAS2 fl/fl xmb1-Cre mice compared to follicular B cells of n = 6 30 wk-old WT mouse controls. One-way ANOVA test. j Dot plot representation of GFP high CD5+ leukemic B cell evolution in blood from mb1-Cre mice over time, showing each mouse individually ( n = 14). Data points were adjusted to a linear fit. k Paraffin-embedded lung tissue section stained with hematoxylin-eosin, showing representative lymphocytic infiltration in one out of three 35 wk-old Rosa26- RRAS2 fl/fl xmb1-Cre mice. l Percentage of IgM + CD5+ B cells within the lymphoid population infiltrating the lungs of n = 2 35 wk-old Rosa26- RRAS2 fl/fl xmb1-Cre mice compared to n = 2 32 wk-old Rosa26- RRAS2 fl/fl control ones. m Bar plot of the distribution of lymphoid cells between the GFP low and GFP high populations in the lungs and lymph nodes (LN) of 20 wk-old Rosa26- RRAS2 fl/fl xmb1-Cre mice ( n = 2)

Article Snippet: Ten-week Rag2 −/− γc −/− male mice were subcutaneously injected in their left flank with 10 × 10 6 MEC-1 control or MEC-1 RRAS2 knockdown cells as described in [ ], in a mixture with PBS/Matrigel (1:1) (Becton Dickinson).

Techniques: Flow Cytometry, Expressing, Control, Adoptive Transfer Assay, Marker, Purification, Irradiation, Agarose Gel Electrophoresis, Negative Control, Quantitative RT-PCR, Staining

Journal: Molecular Cancer

Article Title: Overexpression of wild type RRAS2 , without oncogenic mutations, drives chronic lymphocytic leukemia

doi: 10.1186/s12943-022-01496-x

Figure Lengend Snippet: RRAS2 -driven CLL harbors a highly conserved pattern of somatic mutations. a Pie chart representation of the somatic mutation distribution from CD19 + CD5+ leukemic cells by chromosome. Spleens from seven Rosa26- RRAS2 fl/fl xmb1-Cre mice were included in this analysis (ages in panel e ). b Ingenuity Pathway Analysis (IPA) diagram of the immunological and hematological neoplastic signature generated from the 270 mutations present in all seven different Rosa26- RRAS2 fl/fl xmb1-Cre mice. c Pie chart representation of the co-occurrence of gene mutations in murine CLL vs human CLL. d Summary of most frequently mutated genes in 1094 cases of human CLL also identified in Rosa26-RRAS2 murine CLL. The panel also shows each gene’s condition in MBL (monoclonal B lymphocytosis) and other human leukemias (BL:Burkitt Lymphoma; MCL: Mantle Cell Lymphoma; DLBCL: Diffuse Large B Cell Lymphoma; CTL:Cutaneous T cell lymphoma; FL: Follicular Lymphoma; MM: Multiple Myeloma; MZL; Marginal Zone Lymphoma; SS: Sezary Syndrome). e Heatmap of mutation rates of 107 mutated genes detected in CD19 + CD5+ leukemic cells from seven Rosa26- RRAS2 fl/fl xmb1-Cre independent mice (age of mice: Ctrl 1–3, 12 wk.; Ctrl 4–6 25 wk.; BCLL-9, 104 wk.; BCLL10–11 and BCLL16–17, 54 wk.; BCLL13–14, 32 wk). Black: homozygous; grey: heterozygous; white: unmutated. f Schematic representation of registered missense mutations in SPEN , ARID1A and AKAP13 in human CLL patients (green dots: missense mutation; black dots: truncating mutations; orange dots: splicing site mutation). Red text indicates mutations found in Rosa26- RRAS2 fl/fl xmb1-Cre mice which are also present in human genes

Article Snippet: Ten-week Rag2 −/− γc −/− male mice were subcutaneously injected in their left flank with 10 × 10 6 MEC-1 control or MEC-1 RRAS2 knockdown cells as described in [ ], in a mixture with PBS/Matrigel (1:1) (Becton Dickinson).

Techniques: Mutagenesis, Generated

Journal: Molecular Cancer

Article Title: Overexpression of wild type RRAS2 , without oncogenic mutations, drives chronic lymphocytic leukemia

doi: 10.1186/s12943-022-01496-x

Figure Lengend Snippet: R-RAS2 is associated with the BCR in leukemic cells and is required for human CLL cell proliferation. a Ingenuity Pathway Analysis (IPA) of differentially expressed genes in leukemic versus normal follicular B cells. Pink-filled symbols: upregulated genes. Green-filled: downregulated genes. Double circle: protein complex; horizontal ellipse: transcription regulator; vertical ellipse: transmembrane receptor, diamond: enzyme; trapezium: transporter; triangle: phosphatase; inverted triangle: kinase; circle: other. Relationship labels: A: activation; B: binding; C: causation; CO: correlation; E: expression; EC: enzyme catalysis; I: inhibition; L: molecular cleavage; LO: localization; M: biochemical modification; miT: microRNA Targeting; P: phosphorylation/dephosphorylation; PD: protein-DNA binding; PP: protein-protein binding; PR: protein-RNA binding, RB: regulation of binding; RE: reaction; T: transcription; TR: translocation; UB: ubiquitination. b Western Blot of two-dimensional (2D) gel electrophoresis under non-reducing/reducing conditions of purified CD19 + CD5+ cells from the spleen of a 45 wk-old Rosa26- RRAS2 fl/fl xmb1-Cre mouse. Left: co-immunoprecipitation of R-RAS2-interacting components using an anti-HA antibody. Right: isotype IgG2b control. Membranes were serially incubated with streptavidin-PO, anti-IgM and anti-HA. The positions of molecular weight markers are indicated to the left of each membrane. The blue line represents the mobility of proteins without inter-chain disulfide bridges. c Schematic representation of R-RAS2 interaction with the B-cell receptor and its downstream effects on canonical BCR signaling and the PI3K-Akt-mTOR pathway. d RT-qPCR analysis of RRAS2 expression in MEC-1 cell line transduced with scrambled control lentiviral particles (blue), shRNA for human RRAS2 (red), and expression of RRAS2 in healthy peripheral blood lymphocytes (grey). One-way ANOVA test. e In vitro proliferation assay of RRAS2 knockdown MEC-1 cells (red) and control (blue). Data show means ± SEM from three biological replicates. Two-way ANOVA test, row factor. f Tumor growth in immunodeficient mice. 10 × 10 6 transduced MEC-1 cells per animal were subcutaneously injected. Data show means ± SEM from a total of eight mice. Two-way ANOVA test, row factor. g & h Flow cytometry analysis of Raf-ERK pathway and proximal BCR signaling components ERK (T202/Y204), VAV (Y174), BTK (Y223) and BLNK (Y96) phosphorylation in MEC-1 cell line transduced with scrambled control lentiviral particles (blue), shRNA for human RRAS2 (red) and treated with Src-kinases inhibitor PP2 20 μM, Btk inhibitor Ibrutinib 10 μM and MEK inhibitor U0126 10 μM

Article Snippet: Ten-week Rag2 −/− γc −/− male mice were subcutaneously injected in their left flank with 10 × 10 6 MEC-1 control or MEC-1 RRAS2 knockdown cells as described in [ ], in a mixture with PBS/Matrigel (1:1) (Becton Dickinson).

Techniques: Activation Assay, Binding Assay, Expressing, Inhibition, Modification, Phospho-proteomics, De-Phosphorylation Assay, Protein Binding, RNA Binding Assay, Translocation Assay, Ubiquitin Proteomics, Western Blot, Two-Dimensional Gel Electrophoresis, Electrophoresis, Purification, Immunoprecipitation, Control, Incubation, Molecular Weight, Membrane, Quantitative RT-PCR, Transduction, shRNA, In Vitro, Proliferation Assay, Knockdown, Injection, Flow Cytometry

Journal: Molecular Cancer

Article Title: Overexpression of wild type RRAS2 , without oncogenic mutations, drives chronic lymphocytic leukemia

doi: 10.1186/s12943-022-01496-x

Figure Lengend Snippet: RRAS2 expression is increased in CLL patients and associated to more aggressive disease. a Box and whiskers plots showing all points and median value of RT-qPCR data of RRAS2 mRNA expression in PBMCs from healthy individuals and from MBL and CLL patients. Expression data is normalized to the mean value of healthy blood donor values ( n = 17). Two-tailed unpaired t-test with Welch’s correction. b RT-qPCR analysis of RRAS2 mRNA expression compared to the lymphocyte count in the blood of CLL patients. Patients are classified according the number of total lymphocytes in blood (in ranges, in the x-axis). Values represent the mean ± SEM. All datapoints are represented. One-way ANOVA test. c RT-qPCR analysis of RRAS2 expression compared to the percentage of CD19 + CD5+ cells (in ranges in the x-axis) in the blood of CLL patients. All datapoints are represented. One-way ANOVA test. d Box and whiskers plots showing all points and median value of RT-qPCR data of RRAS2 mRNA expression in blood from MBL and CLL patients. Expression data is normalized to the mean value of healthy blood donor values. Two-tailed unpaired t-test with Welch’s correction. e Box and whiskers plots showing all points and median values of RT-qPCR data of RRAS2 mRNA expression versus age of the patient at diagnosis (in 10-year intervals) and divided according to the diagnosis as CLL or MBL. One-way ANOVA test. f Box and whiskers plot showing all points and median value of RT-qPCR data of RRAS2 mRNA expression in CLL patients classified according to the expression of a mutated or an unmutated IgH gene. ns, not significant (two-tailed unpaired t-test with Welch’s correction). g Contigency test of the distribution of mutated and unmutated IgHV gene within MBL and CLL patients in our study cohort. h Box and whiskers plot showing all points and median value of RT-qPCR data of RRAS2 mRNA expression in our study cohort classified according to MBL vs CLL diagnosis and having mutated or unmutated IgHV. i, Box and whiskers plots showing all points and median value of RT-qPCR data of RRAS2 mRNA expression in blood from all patients in our study cohort classified according to male or female sex. Two-tailed unpaired t-test with Welch’s correction. j Pie chart diagram of the relative fold variations in the expression of RRAS2 mRNA in male and female CLL patients, classified in fold intervals. 1 is the mean of expression in healthy blood donors. k Box and whiskers plot showing all points and median value of RT-qPCR RRAS2 mRNA expression in blood from healthy individuals and CLL patients, and in spleen B cells from control mice, mb1-Cre mice, and Sox2-Cre mice. One-way ANOVA test). In all panels, when there is no distinction between MBL and CLL patients, CLL refers to both groups

Article Snippet: Ten-week Rag2 −/− γc −/− male mice were subcutaneously injected in their left flank with 10 × 10 6 MEC-1 control or MEC-1 RRAS2 knockdown cells as described in [ ], in a mixture with PBS/Matrigel (1:1) (Becton Dickinson).

Techniques: Expressing, Quantitative RT-PCR, Two Tailed Test, Biomarker Discovery, Control

Journal: Molecular Cancer

Article Title: Overexpression of wild type RRAS2 , without oncogenic mutations, drives chronic lymphocytic leukemia

doi: 10.1186/s12943-022-01496-x

Figure Lengend Snippet: The C allele at SNP rs8570 is associated with several parameters of more aggressive disease in CLL patients. a Schematic representation of the rs8570 location in the 3’UTR of RRAS2 mRNA. b Representative examples of the Sanger sequencing chromatograms showing the 3 possibilities of allele dosage at the position of rs8570. The rs8570 position is highlighted by a red rectangle. c Classification of patient samples according to the genotype at SNP rs8570 detected by a dedicated RT-qPCR strategy using specific primers for each of the 2 alleles at that position. The patients sequenced as GG, GC or CC with Sanger sequencing are plotted with dark blue, purple and orange dots, respectively. d Pie chart representation of the Observed versus the Expected distribution of GG, GC and CC genotypes at position of the SNP rs8570 in our study cohort of CLL patients. e Box and whiskers plots showing all points and median value of RT-qPCR RRAS2 mRNA expression in blood from all patients in our study cohort classified according to GG, GC or CC genotype at position of the SNP rs8570. Ttwo-tailed unpaired t-test with Welch’s correction. f Box and whiskers plots showing all points and median value of RT-qPCR RRAS2 mRNA expression in blood from all patients in our study cohort harboring none (GG) or at least one (GC and CC) 124C at position of the SNP rs8570. Two-tailed unpaired t-test with Welch’s correction. g Box and whiskers plots showing all points and median value of total lymphocyte count in the blood of patients harboring two G alleles or at least one C. Two-tailed unpaired t-test with Welch’s correction. h Box and whiskers plots showing all points and median value of the percentage of CD19+ B cells in the blood of patients harboring two G alleles or at least one C. Two-tailed unpaired t-test with Welch’s correction. i Box and whiskers plots showing all points and median value of the percentage of CD19 + CD5+ leukemic B cells in the blood of patients harboring two G alleles or at least one C. Two-tailed unpaired t-test with Welch’s correction. j Box and whiskers plots showing all points and median value of the platelet count in the blood of patients harboring two G alleles or at least one C. Two-tailed unpaired t-test with Welch’s correction. k Contigency test of the distribution of patients diagnosed as having MBL or CLL according to a GG or GC + CC genotype. l Contigency test of the distribution of all samples in our study cohort according to presenting or not chromosomal alterations by FISH and a GG or GC + CC genotype. m Contigency test of the distribution of samples in our study cohort according to presenting normal caryotype or presenting deletions in chromosome 11q or in chromosome 17p (or both) and a GG or GC + CC genotype. n Contingency test of the distribution of all samples in our study cohort according to presenting or not mutated IgHV and a GG or GC genotype. o Contingency test of the distribution of all samples in our study cohort according to male or female sex and a GG or GC + CC genotype

Article Snippet: Ten-week Rag2 −/− γc −/− male mice were subcutaneously injected in their left flank with 10 × 10 6 MEC-1 control or MEC-1 RRAS2 knockdown cells as described in [ ], in a mixture with PBS/Matrigel (1:1) (Becton Dickinson).

Techniques: Sequencing, Quantitative RT-PCR, Expressing, Two Tailed Test

Journal: Open Life Sciences

Article Title: HK2 contributes to the proliferation, migration, and invasion of diffuse large B-cell lymphoma cells by enhancing the ERK1/2 signaling pathway

doi: 10.1515/biol-2022-0726

Figure Lengend Snippet: HK2 was overexpressed in DLBCL cell lines. (a) HK2 expression in normal (Hmy2.CIR) and DLBCL (TMD8, OCI-Ly3, U2932, OCI-Ly1, OCI-Ly7, SU-DHL-4, and SU-DHL-6) cells were measured using qRT-PCR. (b–d) Protein expression levels of HK2, p-ERK1/2, and ERK1/2 were assessed using western blotting. The University of Alabama at Birmingham Cancer (UALCAN) data analysis portal was used to analyze (e) the expression levels of HK2 at individual cancer stages and (f) overall survival of patients with DLBCL. * P < 0.05 and ** P < 0.01 versus the Hmy2.CIR group. ▲ P < 0.05 Stage 1-versus-Stage 4 ( P -value: 2.502100 × 10 −2 ). ABC-DLBCL: activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL); GCB-DLBCL: Germinal center B-cell-like (GCB) subtype of diffuse large B-cell lymphoma (DLBCL).

Article Snippet: In brief, 5 × 10 6 HK2-knockdown U2932 cells and the corresponding control cells were subcutaneously injected into the flanks of 4-week-old BALB/c nude mice (Shanghai SLAC Laboratory Animals Ltd., China).

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: Open Life Sciences

Article Title: HK2 contributes to the proliferation, migration, and invasion of diffuse large B-cell lymphoma cells by enhancing the ERK1/2 signaling pathway

doi: 10.1515/biol-2022-0726

Figure Lengend Snippet: HK2 knockdown inhibits U2932 and SU-DHL-4 cell proliferation in vitro . (a) HK2 was knocked down in U2932 and SU-DHL-4 cells using siRNAs. (b) The viability of HK2-silenced and FR180204-treated (5 μM) U2932 and SU-DHL-4 cells was assessed using the CCK-8 assays. (c) Colony formation assays were used to assess the clone numbers of HK2-silenced and FR180204-treated U2932 and SU-DHL-4 cells. * P < 0.05 and ** P < 0.01 versus the si-NC group.

Article Snippet: In brief, 5 × 10 6 HK2-knockdown U2932 cells and the corresponding control cells were subcutaneously injected into the flanks of 4-week-old BALB/c nude mice (Shanghai SLAC Laboratory Animals Ltd., China).

Techniques: In Vitro, CCK-8 Assay

Journal: Open Life Sciences

Article Title: HK2 contributes to the proliferation, migration, and invasion of diffuse large B-cell lymphoma cells by enhancing the ERK1/2 signaling pathway

doi: 10.1515/biol-2022-0726

Figure Lengend Snippet: HK2 knockdown inhibits U2932 and SU-DHL-4 cell migration and invasion in vitro . (a) Transwell assays were used to analyze the effects of HK2 knockdown on the migration and invasion of U2932 and SU-DHL-4 cells. (b) Western blotting was used to assess the expression levels of E-cadherin and N-cadherin in HK2-silenced and FR180204-treated U2932 and SU-DHL-4 cells. * P < 0.05 and ** P < 0.01 versus the si-NC group.

Article Snippet: In brief, 5 × 10 6 HK2-knockdown U2932 cells and the corresponding control cells were subcutaneously injected into the flanks of 4-week-old BALB/c nude mice (Shanghai SLAC Laboratory Animals Ltd., China).

Techniques: Migration, In Vitro, Western Blot, Expressing

Journal: Open Life Sciences

Article Title: HK2 contributes to the proliferation, migration, and invasion of diffuse large B-cell lymphoma cells by enhancing the ERK1/2 signaling pathway

doi: 10.1515/biol-2022-0726

Figure Lengend Snippet: HK2 knockdown promotes U2932 and SU-DHL-4 cell apoptosis in vitro . (a) Flow cytometry assay was employed to measure U2932 and SU-DHL-4 cell apoptosis. (b) Representative blots and statistical analysis of Bax, Bcl-2, caspase-3, ERK1/2, and p-ERK1/2 in HK2-silenced and FR180204-treated U2932 and SU-DHL-4 cells. (c) Representative blots and statistical analysis of HIF-1α and Ki67 in HK2-silenced and FR180204-treated U2932 and SU-DHL-4 cells. * P < 0.05 and ** P < 0.01 versus the si-NC group.

Article Snippet: In brief, 5 × 10 6 HK2-knockdown U2932 cells and the corresponding control cells were subcutaneously injected into the flanks of 4-week-old BALB/c nude mice (Shanghai SLAC Laboratory Animals Ltd., China).

Techniques: In Vitro, Flow Cytometry

Journal: The Journal of Biological Chemistry

Article Title: The noncanonical RNA-binding protein RAN stabilizes the mRNA of intranuclear stress granule assembly factor G3BP1 in nasopharyngeal carcinoma

doi: 10.1016/j.jbc.2024.107964

Figure Lengend Snippet: Silencing RAN impairs NPC proliferation and invasion in vivo . A–D, SUNE-1 cells stably transfected with scrambled control shRNA or sh- RAN 1# were subcutaneously injected in nude mice to construct a xenograft tumor model. A, a representative image of the xenograft tumors. B, the tumor growth curves of the xenografts. Data are presented as the mean ± SD (n = 8). C, the tumor weights of the xenografts. Data are presented as the mean ± SD (n = 8). D, xenograft tumors were paraffin-embedded and sectioned. Then, the expression levels of RAN and G3BP1 were assessed by IHC staining. The scale bar represents 100 μm. E–J, SUNE-1 cells with or without RAN stable silencing were injected into the footpad of nude mice to establish an inguinal lymph node metastasis model. E, representative image of the inguinal lymph node metastasis model. F, representative images of the primary footpad tumors ( left ) and metastatic inguinal lymph nodes ( right ). G, the volume of the inguinal lymph nodes was calculated. Data are presented as the mean ± SD (n = 8). H, representative images of footpad tumor sections stained with H&E showing tumor cells invasion into muscle tissues ( left ) or lymphatic vessels ( right ). The scale bar represents 100 μm. I, representative images of IHC staining with pan-cytokeratin in metastatic ( upper ) or nonmetastatic ( lower ) inguinal lymph nodes. The scale bar represents 100 μm. J, the quantitative result of the ratios of inguinal lymph nodes metastasis. ∗ p < 0.05 and ∗∗ p < 0.01. The significant differences were assessed using two-way ANOVA ( A ) and t test ( B and G ). GEO, Gene Expression Omnibus; RT-qPCR, reverse transcription quantitative real-time PCR; IHC, immunohistochemistry; NPC, nasopharyngeal carcinoma.

Article Snippet: Cy3-labeled G3BP1 FISH probes were purchased and synthesized by RiboBio.

Techniques: In Vivo, Stable Transfection, Transfection, Control, shRNA, Injection, Construct, Expressing, Immunohistochemistry, Staining, Gene Expression, Quantitative RT-PCR, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: The noncanonical RNA-binding protein RAN stabilizes the mRNA of intranuclear stress granule assembly factor G3BP1 in nasopharyngeal carcinoma

doi: 10.1016/j.jbc.2024.107964

Figure Lengend Snippet: RAN directly binds and stabilizes G3BP1 mRNA. A, Venn diagram of genes identified by RIP-seq in HONE-1 or SUNE-1 NPC cell lines (logOddScore >1). B, pie charts showing the distribution of reads recognized by RIP-seq on gene functional elements. C, venn diagram of differentially expressed genes identified by RNA-seq after RAN silencing and overlapping genes identified by RIP-seq. D, relative levels of G3BP1 mRNA level with or without RAN silencing were detected by RT-qPCR. Data are presented as the mean ± SD (n = 3). E, G3BP1 protein level with or without RAN silencing was detected by Western blotting. The blots are the representation of three independent experiments. Data are presented as the mean ± SD (n = 3). F, nuclear/cytosol RNA fractionation assays were used to identify the suitable internal control. Data are presented as the mean ± SD (n = 3). G, relative levels of G3BP1 mRNA level in cytoplasm or nucleus, which were normalized to GAPDH or U3 , was indicated by RT-qPCR upon knockdown of RAN in HONE-1 and SUNE-1 cells. Data are presented as the mean ± SD (n = 3). H, after treatment with actinomycin D (10 μg/ml), G3BP1 mRNA level was quantified at indicated times in control and RAN-silenced cells. The half-life of G3BP1 mRNA was analyzed by plotting degradation curves. Data are presented as the mean ± 95%CI (n = 3). I, Pearson correlation analysis of RAN and G3BP1 levels in different GEO databases (GSE53819 and GSE103611). J, relative enrichment of G3BP1 mRNA immunoprecipitated by anti-RAN or anti-IgG antibody was indicated by RT-qPCR. Data are presented as the mean ± SD (n = 3). K, enrichment of RAN proteins pulled down by biotin-labeled G3BP1 probes from in vitro transcription or control antisense probes was detected by Western blotting. L, RAN protein distribution in cells was recognized by IF ( green ), G3BP1 mRNA distribution in cells was recognized by FISH ( red ), and cell nuclei were stained with DAPI ( blue ). The scale bar represents 10 μm. ∗ p < 0.05 and ∗∗ p < 0.01. The significant differences were assessed using one-way ANOVA ( D , E , and G ), two-way ANOVA ( H ), and t test ( J ). The significant differences in correlations were assessed using the Pearson correlation analysis ( I ). GEO, Gene Expression Omnibus; RT-qPCR, reverse transcription quantitative real-time PCR; NPC, nasopharyngeal carcinoma; DAPI, 4′, 6-diamino-2-phenylindole; IF, immunofluorescence; CI, confidence interval.

Article Snippet: Cy3-labeled G3BP1 FISH probes were purchased and synthesized by RiboBio.

Techniques: Functional Assay, RNA Sequencing, Quantitative RT-PCR, Western Blot, Fractionation, Control, Knockdown, Immunoprecipitation, Labeling, In Vitro, Staining, Gene Expression, Reverse Transcription, Real-time Polymerase Chain Reaction, Immunofluorescence

Journal: The Journal of Biological Chemistry

Article Title: The noncanonical RNA-binding protein RAN stabilizes the mRNA of intranuclear stress granule assembly factor G3BP1 in nasopharyngeal carcinoma

doi: 10.1016/j.jbc.2024.107964

Figure Lengend Snippet: RAN, TDP43, and G3BP1 mRNA form a complex in the nucleus. A, diagrams of full-length G3BP1 transcript consist of CDS and 3′ UTR, as well as its deletion fragments. B and C, the in vitro –transcribed full-length G3BP1 transcript and deletion fragments with the correct sizes were indicated ( upper ). RNA pull-down assay and Western blotting showed whether these biotin-labeled G3BP1 fragments could pull down RAN in cell lysates ( lower ). D, the motif discovery algorithm DREME was used to recognize the top consensus motif based on the RIP-seq data. E, construction of mutant fragments based on predicted motifs. F, the in vitro –transcribed D2 fragments and mutant fragments with the correct sizes are indicated ( upper ). RNA pull-down assay and Western blotting indicated whether these biotin-labeled fragments could pull down RAN in cell lysates ( lower ). G, the interaction between endogenous RAN and TDP43 was evaluated by Co-IP assays using anti-RAN antibody or normal rabbit IgG in HONE-1 and SUNE-1 cell lysates. H, the interaction between endogenous TDP43 and RAN was evaluated by Co-IP assays using anti-TDP43 antibody or normal rabbit IgG in HONE-1 and SUNE-1 cell lysates. I, RAN ( red ) or TDP43 ( green ) protein distribution in cells was recognized by IF, and cell nuclei were stained with DAPI ( blue ). The scale bar represents 10 μm. J, relative enrichment of G3BP1 mRNA immunoprecipitated by anti-TDP43 or anti-IgG antibody was indicated by RT-qPCR. Data are presented as the mean ± SD (n = 3). K, enrichment of TDP43 pulled down by biotin-labeled G3BP1 probes from in vitro transcription or control antisense probes was detected by Western blotting. L, RNA pull-down assay and Western blotting showed whether biotin-labeled full-length G3BP1 transcript or deletion fragments could pull down TDP43 in cell lysates. M, TDP43 protein distribution in cells was recognized by IF ( green ), G3BP1 mRNA distribution in cells was recognized by FISH ( red ), and cell nuclei were stained with DAPI ( blue ). The scale bar represents 10 μm. N, in vitro purified RAN-GST, TDP43, eEF2-His (positive control), and GST (negative control) proteins were used to perform a cell-free RNA pull-down experiment. Coomassie brilliant blue staining showed the purification of these proteins ( left ). Enrichment of proteins pulled down by biotin-labeled G3BP1 probes from in vitro transcription was detected by Western blotting ( right ). O, in vitro purified RAN-GST, TDP43, and GST (negative control) proteins were used to perform a cell-free co-IP experiment. Coomassie brilliant blue staining showed the purification of these proteins ( left ). Enrichment of proteins immunoprecipitated by anti-GST magnetic beads was detected by Western blotting ( right ). ∗ p < 0.05 and ∗∗ p < 0.01. The significant differences were assessed using t test ( J ). RT-qPCR, reverse transcription quantitative real-time PCR; ; NPC, nasopharyngeal carcinoma; Co-IP, coimmunoprecipitation; GST, glutathione- S -transferase; IF, immunofluorescence; DAPI, 4′, 6-diamino-2-phenylindole; CDS, coding sequence.

Article Snippet: Cy3-labeled G3BP1 FISH probes were purchased and synthesized by RiboBio.

Techniques: In Vitro, Pull Down Assay, Western Blot, Labeling, Mutagenesis, Co-Immunoprecipitation Assay, Staining, Immunoprecipitation, Quantitative RT-PCR, Control, Purification, Positive Control, Negative Control, Magnetic Beads, Reverse Transcription, Real-time Polymerase Chain Reaction, Immunofluorescence, Sequencing

Journal: The Journal of Biological Chemistry

Article Title: The noncanonical RNA-binding protein RAN stabilizes the mRNA of intranuclear stress granule assembly factor G3BP1 in nasopharyngeal carcinoma

doi: 10.1016/j.jbc.2024.107964

Figure Lengend Snippet: RAN increases TDP43 nucleus import and facilitates TDP43 binding G3BP1 mRNA. A, after treatment with actinomycin D (10 μg/ml), G3BP1 mRNA level was quantified at indicated times in control, RAN-silenced, TDP43-silenced, and both RAN and TDP43-silenced cells. The half-life of G3BP1 mRNA was analyzed by plotting degradation curves. Data are presented as the mean ± 95%CI (n = 3). B, TDP43 protein levels with knockdown of RAN were detected by Western blotting. The blots are the representation of three independent experiments. Data are presented as the mean ± SD (n = 3). C, relative enrichment of G3BP1 mRNA immunoprecipitated by anti-TDP43 or anti-IgG antibody was indicated by RT-qPCR upon knockdown of RAN. Data are presented as the mean ± SD (n = 3). D, the nucleus and cytoplasm proteins in HONE-1 and SUNE-1 cells were isolated by nuclear/cytosol fractionation assays. TDP43 distribution in the nucleus or cytoplasm was identified by Western blotting with or without RAN silencing, GAPDH as the cytoplasm marker, and Lamin B as the nucleus marker. The blots are the representation of three independent experiments. Data are presented as the mean ± SD (n = 3). E, TDP43-GFP overexpression vectors were transfected in HONE-1 and SUNE-1 cells after RAN silencing. Recognizing TDP43-GFP protein distribution ( green ) in HONE-1 and SUNE-1 cells after 24 h of TDP43-GFP overexpression vectors transfected, range indicator was performed to show clearer distribution of TDP43-GFP in the nucleus or cytoplasm, cell nuclei were stained with DAPI ( blue ). The scale bar represents 10 μm. F, the average fluorescence intensity of TDP43-GFP in the nucleus ( lower ) and cytoplasm ( upper ) of each cell was calculated. Data are presented as the mean ± SD (n = 20). ∗ p < 0.05 and ∗∗ p < 0.01. The significant differences were assessed using one-way ANOVA ( B , D , and F ), two-way ANOVA ( A ), and t test ( C ). RT-qPCR, reverse transcription quantitative real-time PCR; DAPI, 4′, 6-diamino-2-phenylindole; CI, confidence interval.

Article Snippet: Cy3-labeled G3BP1 FISH probes were purchased and synthesized by RiboBio.

Techniques: Binding Assay, Control, Knockdown, Western Blot, Immunoprecipitation, Quantitative RT-PCR, Isolation, Fractionation, Marker, Over Expression, Transfection, Staining, Fluorescence, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: The noncanonical RNA-binding protein RAN stabilizes the mRNA of intranuclear stress granule assembly factor G3BP1 in nasopharyngeal carcinoma

doi: 10.1016/j.jbc.2024.107964

Figure Lengend Snippet: RAN facilitates AKT/ERK phosphorylation via G3BP1 and further promotes NPC progression. A and B, cell proliferation ability was evaluated by CCK-8 assays in SUNE-1 and HONE-1 cells after silencing of G3BP1. Data are presented as the mean ± SD (n = 6). B, cell proliferation ability was evaluated by colony formation assays in SUNE-1 and HONE-1 cells after silencing of G3BP1. Data are presented as the mean ± SD (n = 3). C, migration and invasion capacity were analyzed by transwell assays in G3BP1-silenced SUNE-1 and HONE-1 cells. The scale bar represents 200 μm. Data are presented as the mean ± SD (n = 4). D, cell proliferation ability was evaluated by CCK-8 assays in HONE-1 cells after cotransfected with scrambled control or si-RAN, together with empty vector or HA-tagged G3BP1 overexpression vector. Data are presented as the mean ± SD (n = 6). E, migration and invasion capacity were analyzed by transwell assays in HONE-1 cells after cotransfected with scrambled control or si-RAN, together with empty vector or HA-tagged G3BP1 overexpression vector. Data are presented as the mean ± SD (n = 4). F, AKT, p-AKT, ERK, and p-ERK protein levels with knockdown of RAN or G3BP1 were detected by Western blotting in HONE-1 cells. The blots are the representation of three independent experiments. Data are presented as the mean ± SD (n = 3). ∗ p < 0.05 and ∗∗ p < 0.01. The significant differences were assessed using one-way ANOVA ( B , C , and F ) and two-way ANOVA ( A and D ). CCK-8, Cell Counting Kit-8; HA, hemagglutinin; IHC, immunohistochemistry; NPC, nasopharyngeal carcinoma.

Article Snippet: Cy3-labeled G3BP1 FISH probes were purchased and synthesized by RiboBio.

Techniques: Phospho-proteomics, CCK-8 Assay, Migration, Control, Plasmid Preparation, Over Expression, Knockdown, Western Blot, Cell Counting, Immunohistochemistry

Journal: The Journal of Biological Chemistry

Article Title: The noncanonical RNA-binding protein RAN stabilizes the mRNA of intranuclear stress granule assembly factor G3BP1 in nasopharyngeal carcinoma

doi: 10.1016/j.jbc.2024.107964

Figure Lengend Snippet: Example diagram of RAN–TDP43–G3BP1 axis modulation. RAN increases the nucleus import of TDP43 and acts as an adapter to facilitate TDP43 direct binding to G3BP1 mRNA in the nucleus, thereby increasing G3BP1 mRNA stability and leading to upregulation of G3BP1, which further enhances AKT and ERK signaling and ultimately facilitates NPC proliferation and metastasis. NPC, nasopharyngeal carcinoma.

Article Snippet: Cy3-labeled G3BP1 FISH probes were purchased and synthesized by RiboBio.

Techniques: Binding Assay