Journal: Oncotarget

Article Title: IMP1 suppresses breast tumor growth and metastasis through the regulation of its target mRNAs

doi: 10.18632/oncotarget.7464

Figure Lengend Snippet: 11 of 14 mice injected with either MDA231/GFP or MDA231/GFP-IMP1 cells gave rise palpable tumors. The volumes of individual tumors derived from the two cell lines were indicated in ( A ) and ( B ). ( C ) Average tumor volumes in two xenograft groups were calculated in cm 3 . Bars indicate standard error of mean, P < 0.01. Tumors derived from MDA231/GFP-IMP1 cells displayed smaller primary tumor masses. ( D ) Immunoblots showing the expression of GFP-IMP1 in MDA231/GFP-IMP1 and MDA231/GFP cells. ( E ) Western blots were performed using antibody against IMP1 (Sigma). GFP-IMP1 was expressed in MDA231/GFP-IMP1 cell-derived xenografts (Lanes 1–6), but not in the xenograft derived from MDA231/GFP (lane C), β-actin antibody was used as a loading control.

Article Snippet: Flag-tagged IMP1 truncate (IMP1m, aa 1–404) that lacks the KH34 domain was PCR amplified and subcloned into a lentiviral vector downstream of the GFP gene. shRNA vectors for RGS4 and PTGS2 mRNAs were purchased from OriGene Technologies (TR30007 and TG310074).

Techniques: Injection, Derivative Assay, Western Blot, Expressing

Journal: Oncotarget

Article Title: IMP1 suppresses breast tumor growth and metastasis through the regulation of its target mRNAs

doi: 10.18632/oncotarget.7464

Figure Lengend Snippet: ( A ) and ( B ) lung metastases were measured in individual mice with primary breast tumors derived from MDA231/GFP or MDA231/GFP-IMP1 cells. Numbers of lung metastasis in each individual mouse were measured. Rates of lung metastases were 73% in MDA231/GFP cell-derived tumors and 45% in MDA231/GFP-IMP1 cell-derived tumors ( P = 0.0136). ( C ) Representative low-power and high-power H & E-stained, 5-um lung sections from mice injected with the indicated cells were shown. Visible metastatic nodules in the lungs of MDA231/GFP mice were markedly higher than those in the lungs of MDA231/GFP-IMP1 mice.

Article Snippet: Flag-tagged IMP1 truncate (IMP1m, aa 1–404) that lacks the KH34 domain was PCR amplified and subcloned into a lentiviral vector downstream of the GFP gene. shRNA vectors for RGS4 and PTGS2 mRNAs were purchased from OriGene Technologies (TR30007 and TG310074).

Techniques: Derivative Assay, Staining, Injection

Journal: Oncotarget

Article Title: IMP1 suppresses breast tumor growth and metastasis through the regulation of its target mRNAs

doi: 10.18632/oncotarget.7464

Figure Lengend Snippet: Total RNA was extracted from the xenograft tumors ( A ) ( n = 5, from each group) and from the cell lines used for generating xenografts ( B ). Real-time RT-qPCR was performed to detect the mRNA levels of eight selected genes identified in microarray assays. Relative expression levels of the mRNAs are statistically analyzed and the data are presented as means ± SEM from three independent experiments. * P < 0.05. ( C ) and ( D ) Immunoprecipitations were performed in the extracts prepared from MDA231 cell lines and from the xenograft tumors using anti-flag antibodies for flag-tagged GFP-IMP1. RNAs in the precipitates were extracted and used for RT-PCR assays. (C) Upper panels: GDF15, IGF-2, RGS4 and PTGS2 mRNAs were co-precipitated with IMP1 in the extracts of breast carcinoma cells, while AMIGO2 mRNA appeared to be non-specifically precipitated. β-actin mRNA was used as a positive control for IMP1 binding. G: MDA231/GFP cells; I: MDA231/GFP-IMP1 cells. Lower panels: RT-PCR indicated the presence of selected mRNAs in the extracts of carcinoma cells. (D) Upper panels: in addition to AMIGO2 mRNA that was non-specifically precipitated, GDF15, TFPI2, IGF-2 and PTGS2 mRNAs were also co-precipitated with IMP1 in the extracts of MDA231/GFP-IMP1 cell-derived xenograft tumors. Lower panels indicated the presence of selected mRNAs in the extracts of xenografts. G: MDA231/GFP cell-derived breast tumors; I: MDA231/GFP-IMP1 cell-derived breast tumors.

Article Snippet: Flag-tagged IMP1 truncate (IMP1m, aa 1–404) that lacks the KH34 domain was PCR amplified and subcloned into a lentiviral vector downstream of the GFP gene. shRNA vectors for RGS4 and PTGS2 mRNAs were purchased from OriGene Technologies (TR30007 and TG310074).

Techniques: Quantitative RT-PCR, Microarray, Expressing, Reverse Transcription Polymerase Chain Reaction, Positive Control, Binding Assay, Derivative Assay

Journal: Oncotarget

Article Title: IMP1 suppresses breast tumor growth and metastasis through the regulation of its target mRNAs

doi: 10.18632/oncotarget.7464

Figure Lengend Snippet: Aliquots of 32 P-labeled 3′ UTR of GDF15 mRNA were incubated with extracts prepared from MDA231/GFP and MDA231/GFP-IMP1 cells ( A ) and recombinant IMP1 ( B ). RNA-protein complexes (arrow indicated) were formed when the RNA probe incubated with IMP1-containing extracts and with recombinant IMP1. Cell extracts without IMP1 expressing did not form complex with the RNA probe. The complexes were competed by 500× excess of unlabeled 3′ UTR of GDF15 mRNA, but not the non-specific RNA (yeast tRNA). ( C ) Western blots showing the protein expression of four IMP1-bound mRNAs in MDA231/GFP and MDA231/GFP-IMP1 cells. Tubulin was used as a loading control. ( D ) Extracts of MDA231/GFP-IMP1 and MDA231/GFP cells were fractionated in 10–50% linear sucrose gradients. An OD 254 plot corresponding to the polysomal fractions was shown on the top panel. Total RNAs were isolated from the sucrose fractions. An equal amount of RNA from each fraction was subjected to Northern blots to detect the distribution of GDF15, PTGS2 and GAPDH mRNAs in the sucrose gradient.

Article Snippet: Flag-tagged IMP1 truncate (IMP1m, aa 1–404) that lacks the KH34 domain was PCR amplified and subcloned into a lentiviral vector downstream of the GFP gene. shRNA vectors for RGS4 and PTGS2 mRNAs were purchased from OriGene Technologies (TR30007 and TG310074).

Techniques: Labeling, Incubation, Recombinant, Expressing, Western Blot, Isolation, Northern Blot

Journal: Oncotarget

Article Title: IMP1 suppresses breast tumor growth and metastasis through the regulation of its target mRNAs

doi: 10.18632/oncotarget.7464

Figure Lengend Snippet: ( A ) Western blots showing the expression of IMP1 and IMP1 truncate in MDA231/GFP-IMP1 and MDA231/GFP-IMP1m lines. ( B ) Co-IP experiments were performed in the extracts prepared from MDA231 cell lines using antibodies against Flag-tag. Western blots indicated that the Flag-tagged GFP-IMP1 and GFP-IMP1m were successfully precipitated. ( C ) Co-precipitated RNAs were extracted and used for RT-PCR assays. GDF15, RGS4, PTGS2 and β-actin mRNAs were not precipitated when the KH34 domain of IMP1 was deleted. GAPDH mRNA was used as an internal control for the extracts used in the experiments. Im: MDA231/GFP-IMP1m cells. I: MDA231/GFP-IMP1 cells. ( D ) MTT assays were used to measure the rate of cell proliferation. Loss of RNA binding activity by deletion of KH34 domain abolished the ability of IMP1 in repressing proliferation of MDA231 cells ( P < 0.005). Bars indicate standard error of mean from three independent experiments, ( P < 0.01). ( E ) Average tumor volumes in MDA231/GFP and MDA231/GFP-IMP1m cell-derived xenograft animals, P > 0.5. ( F ) Western blots indicated the expression of GFP-IMP1m in MDA231/GFP-IMP1m cell-derived xenografts (Lanes 1–6). Lane C was a negative control for the xenograft derived from MDA231/GFP cells. Antibody for β-actin was used as a loading control.

Article Snippet: Flag-tagged IMP1 truncate (IMP1m, aa 1–404) that lacks the KH34 domain was PCR amplified and subcloned into a lentiviral vector downstream of the GFP gene. shRNA vectors for RGS4 and PTGS2 mRNAs were purchased from OriGene Technologies (TR30007 and TG310074).

Techniques: Western Blot, Expressing, Co-Immunoprecipitation Assay, FLAG-tag, Reverse Transcription Polymerase Chain Reaction, RNA Binding Assay, Activity Assay, Derivative Assay, Negative Control

Journal: Oncotarget

Article Title: IMP1 suppresses breast tumor growth and metastasis through the regulation of its target mRNAs

doi: 10.18632/oncotarget.7464

Figure Lengend Snippet: ( A ), ( B ) and ( C ) Western blots showing the effects of shRNA on silencing RGS4, PTGS2 and IMP1 expression in MDA231 stable cell lines. ( D ) and ( F ) Effects of RGS4, PTGS2 and IMP1 knockdown on proliferation of MDA231 cells. Cell numbers were determined at the indicated time points. Data shown in the figure represent the means ± standard error of data from four independent experiments. ( P < 0.01). ( E ) and ( G ) Knockdown of RGS4, PTGS2 and IMP1 expression changes the invasive potential of MDA231 cells. Cells were plated in serum-free medium into the upper chamber of 8 mm pore Matrigel-coated transwell filters. The lower chamber contained medium with 10% serum. Cells that had invaded to the underside of the filter were stained and counted in 16 hours. Relative numbers shown in the figure represent the means ± SEM. of data from three independent experiments, * P < 0.05.

Article Snippet: Flag-tagged IMP1 truncate (IMP1m, aa 1–404) that lacks the KH34 domain was PCR amplified and subcloned into a lentiviral vector downstream of the GFP gene. shRNA vectors for RGS4 and PTGS2 mRNAs were purchased from OriGene Technologies (TR30007 and TG310074).

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

Journal: Oncotarget

Article Title: IMP1 suppresses breast tumor growth and metastasis through the regulation of its target mRNAs

doi: 10.18632/oncotarget.7464

Figure Lengend Snippet: ( A ) Relative levels of IMP1 mRNA in individual human breast tumors detected by RT-qPCR (Numbers are indicated as patient ID). Total RNA was extracted from 50 mg human breast tumors ( n = 47) and breast normal tissues ( n = 4), and 1 μg of total RNA was reversely transcribed. The resulting cDNA was analyzed by ABI 7500 Fast real time PCR system. Relative expression levels of IMP1 mRNA were calculated using comparative 2 −ΔΔ Ct method, and GAPDH was used as an endogenous control. Relative expression data was normalized to normal breast tissue (labeled as N). ( P < 0.05). ( B ) Representative immunoblots indicate the expression of IMP1 protein in human breast tumors. Arrows indicate detected IMP1 and β-actin by their corresponding antibodies. ( C ) Relative levels of PTGS2, GDF15 and IGF-2 mRNAs in human breast tumors were reduced in response to increased IMP1 expression. Tumor samples were divided into three groups, one group is IMP1 negative ( N = 15), the second one has lower levels of IMP1 expression ( n = 11) and the third group expresses higher levels of IMP1 ( N = 15). Total RNA was prepared from human samples and the levels of PTGS2, GDF15 and IGF-2 mRNAs were measured by qRT-PCR. Raw Ct values for each gene were normalized to raw Ct values for GPDH mRNA that was used as an internal control. Error bars represent standard deviation from three independent experiments. (* P < 0.01).

Article Snippet: Flag-tagged IMP1 truncate (IMP1m, aa 1–404) that lacks the KH34 domain was PCR amplified and subcloned into a lentiviral vector downstream of the GFP gene. shRNA vectors for RGS4 and PTGS2 mRNAs were purchased from OriGene Technologies (TR30007 and TG310074).

Techniques: Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Expressing, Labeling, Western Blot, Standard Deviation

Journal: Clinical Epigenetics

Article Title: Comprehensive analyses of molecular features, prognostic values, and regulatory functionalities of m 6 A-modified long non-coding RNAs in lung adenocarcinoma

doi: 10.1186/s13148-023-01475-z

Figure Lengend Snippet: The expression levels of m 6 A regulators in LUAD and the correlation between m 6 A regulators. A Heatmap of the expression levels of m 6 A regulators (LUAD tumor tissues vs. normal lung tissues) in TCGA and GTEx databases. FC represented the expression fold change of T-median/N-median. WilcoxTest. * P < 0.05, ** P < 0.01, *** P < 0.001 and not significant (ns) P > 0.05. B RT-qPCR showed the relative mRNA expression levels of METTL3, METTL14, ALKBH5, FTO, and IGF2BP1/3 in eight paired LUAD tissues (Tumor) and adjacent non-tumor tissues (Normal). Paired t test. ** P < 0.01 and not significant (ns) P > 0.05. C, D Western blots showed the protein expression of METTL3, METTL14, FTO, ALKBH5, and IGF2BP1/3 in LUAD tissues ( C ) and cell lines ( D ), respectively. E The Pearson correlation analysis of m 6 A regulators’ expression levels in 872 samples in TCGA and GTEx databases. The number in the circle represented the Pearson coefficient, which determined the size of circle. The cross in the circle represented no correlation ( P > 0.001). F The PPI network analysis of m 6 A regulators. Line thickness indicated the strength of data support

Article Snippet: The primary antibodies were as follows: β-actin (4970S, 1:1000, Cell Signaling Technology (CST), Beverly, MA, USA), METTL3 (86132S, 1:2000, CST, Beverly, MA, USA), METTL14 (26158-1-AP, 1:2000, Proteintech, Wuhan, China), ALKBH5 (80283S, 1:1000, CST, Beverly, MA, USA), FTO (27226-1-AP, 1:2000, Proteintech, Wuhan, China), IGF2BP1 (EPR26408-18, 1:2000, Abcam, Cambridge, UK), and IGF2BP3 (14642-1-AP, 1:2000, Proteintech, Wuhan, China).

Techniques: Expressing, Quantitative RT-PCR, Western Blot