Journal: Acta Neuropathologica

Article Title: Epigenetic loss of RNA-methyltransferase NSUN5 in glioma targets ribosomes to drive a stress adaptive translational program

doi: 10.1007/s00401-019-02062-4

Figure Lengend Snippet: Transcriptional silencing of NSUN5 by promoter CpG island hypermethylation in human glioma cells. a Percentage of NSUN5 methylation in the Sanger panel of cancer cell lines by tumor type. b NSUN5 methylation is associated with loss of the transcript in the glioma cell lines from Sanger ( n = 48). Correlation analysis between methylation beta values and expression Z -score are shown. The P value corresponding to Spearman’s rank correlation test and the associated rho coefficient are indicated in the figure. c Bisulfite genomic sequencing of NSUN5 promoter CpG Island in glioma cells lines and brain white matter. CpG dinucleotides are represented as short vertical lines and the transcription start site (TSS) is represented as a long black arrow. Single clones are shown for each sample. Presence of an unmethylated or methylated cytosine is indicated by a white or black square, respectively, and percentage of methylation is indicated on the right. d DNA methylation profile of the CpG island promoter for the NSUN5 gene analyzed by the 450 K DNA methylation microarray. Single CpG absolute methylation levels (0–1) are shown. Green, unmethylated; red, methylated. Data from the studied six glioma cell lines, brain white matter and nineteen normal brain samples are shown. e NSUN5 expression levels in glioma cell lines determined by real-time PCR (data shown represent mean ± S.D. of biological triplicates) and western blot. f Expression of the NSUN5 RNA transcript and protein was restored in the A172, LN229 and KS-1 cells by treatment with the demethylating drug 5-aza-2′-deoxycytidine (AZA). Data shown represent the mean ± S.D. of biological triplicates and P values were obtained by the Mann–Whitney test. ** P < 0.01

Article Snippet: Human glioma cell lines DBTRG-05MG, M059J, A172, and LN229 were purchased from the American Type Culture Collection; CAS-1 was obtained from The Biological Bank of the IRCCS Azienda Ospedaliera Universitaria San Martino—IST Istituto Nazionale per la Ricerca sul Cancro; and KS-1 was purchased from the Japanese Collection of Research Bioresources Cell Bank.

Techniques: Methylation, Expressing, Genomic Sequencing, Clone Assay, DNA Methylation Assay, Microarray, Real-time Polymerase Chain Reaction, Western Blot, MANN-WHITNEY

Journal: Acta Neuropathologica

Article Title: Epigenetic loss of RNA-methyltransferase NSUN5 in glioma targets ribosomes to drive a stress adaptive translational program

doi: 10.1007/s00401-019-02062-4

Figure Lengend Snippet: Restoration of NSUN5 impairs glioma tumor growth in vivo. a Western blot to show efficient restoration of NSUN5 protein expression upon stable transfection in A172 and LN299 glioma cells and efficient depletion of NSUN5 protein expression in NSUN5-shRNA DBTRG-05MG glioma cells. EV empty vector. An equal number of the indicated A172 and LN299 cells populations were stereotactically inoculated into the brain of athymic mice. The size of the tumors was estimated at 10 and 17 days post-inoculation (DPI) by the quantification of luciferase activity in the tumor cells. b Scatter plots showing the individual size of the indicated LN229 and A172 tumors after 10 and 17 DPI. c Representative images of the luciferase signal from mice inoculated with the indicated LN229 and A172 tumors after 17 DPI. d LN229-EV and LN229-NSUN5 cells were injected in the left or right flank of 10 mice, respectively. Tumor volume measured over time ( left panel ) and tumor weight upon sacrifice ( right panel ) are shown. P values obtained by Student’s t test. Error bars show means ± s.d. e Scramble and NSUN5-shRNA-depleted DBTRG-05MG cells were injected in the left or right flank of 10 mice, respectively. Tumor volume measured over time (left panel) and tumor weight upon sacrifice (right panel) are shown. P values obtained by Student’s t test. Error bars show means ± s.d

Article Snippet: Human glioma cell lines DBTRG-05MG, M059J, A172, and LN229 were purchased from the American Type Culture Collection; CAS-1 was obtained from The Biological Bank of the IRCCS Azienda Ospedaliera Universitaria San Martino—IST Istituto Nazionale per la Ricerca sul Cancro; and KS-1 was purchased from the Japanese Collection of Research Bioresources Cell Bank.

Techniques: In Vivo, Western Blot, Expressing, Stable Transfection, shRNA, Plasmid Preparation, Luciferase, Activity Assay, Injection

Journal: Acta Neuropathologica

Article Title: Epigenetic loss of RNA-methyltransferase NSUN5 in glioma targets ribosomes to drive a stress adaptive translational program

doi: 10.1007/s00401-019-02062-4

Figure Lengend Snippet: NSUN5 epigenetic inactivation abrogates the methylation of the C3782 position of human 28S rRNA. a Top , RNA sequence alignment of the conserved human 28S rRNA C3782 position (black square) in the corresponding 26S, 25S and 28S rRNAs orthologues from C. elegans , S. cerevisiae and M. musculus . Below , Protein sequence alignment of human NSUN5 with its recognized rRNA 5-methylcytosine RNA-methyltransferase orthologues in C. Elegans , S. Cerevisiae and M. musculus . Highlighted in black and grey the identical and physicochemically similar (scoring > 0.5 in the Gonnet PAM 250 matrix) residues, respectively. The aligned region includes the RNA-methyltransferase domain. b NSUN5 interaction with 28S rRNA. Total extracts from LN229 cells, either transfected with empty vector (EV) or NSUN5 were immunoprecipitated with an anti-Flag antibody ( left panel ), followed by analysis of the retrieved RNA by quantitative RT-PCR ( right panel ). c RNA bisulfite sequencing of the 28S rRNA in glioma cells lines according to NSUN5 epigenetic status. Cytosines are represented as short vertical lines and the C3782 site is represented as a long black arrow. Single clones are shown for each sample. Presence of an unmethylated or methylated cytosine is indicated by a white or black square, respectively. d RNA bisulfite sequencing of the 28S rRNA in empty-vector (EV) and NSUN5-transfected LN229 and A172 glioma cells. e RNA bisulfite sequencing of the 28S rRNA in scramble and NSUN5-shRNA-depleted DBTRG-05MG and CAS-1 glioma cells. For CAS-1, western-blot to show efficient NSUN5 depletion is shown above

Article Snippet: Human glioma cell lines DBTRG-05MG, M059J, A172, and LN229 were purchased from the American Type Culture Collection; CAS-1 was obtained from The Biological Bank of the IRCCS Azienda Ospedaliera Universitaria San Martino—IST Istituto Nazionale per la Ricerca sul Cancro; and KS-1 was purchased from the Japanese Collection of Research Bioresources Cell Bank.

Techniques: Methylation, Sequencing, Transfection, Plasmid Preparation, Immunoprecipitation, Quantitative RT-PCR, Methylation Sequencing, Clone Assay, shRNA, Western Blot

Journal: Acta Neuropathologica

Article Title: Epigenetic loss of RNA-methyltransferase NSUN5 in glioma targets ribosomes to drive a stress adaptive translational program

doi: 10.1007/s00401-019-02062-4

Figure Lengend Snippet: NSUN5 epigenetic loss is associated with depletion of global protein synthesis and the emergence of a stress-response translational program. a NSUN5 unmethylated glioma cell lines DBTRG-05MG, MO59J, and CAS-1 show higher overall protein synthesis assessed by OP-Puro under oxidative stress (100 mM H 2 O 2 ) than the NSUN5 methylated cells (A172, LN229 and KS-1). b Restoration of NSUN5 function by transfection in epigenetically inactive LN299 cells increases overall protein synthesis under oxidative stress (100 mM H 2 O 2 ) assessed by OP-Puro. Enhancement of global protein synthesis upon NSUN5 recovery in LN299 cells is also observed by the [3H] leucine incorporation assay. c Similar results were obtained upon nutrient deprivation. d Comparison of the total RNA (RNA-seq) and ribosome-protected RNA (Ribo-seq) deep-sequencing profiles to identify those RNAs with enhanced translational efficiency in NSUN5 deficient cells. 1987 RNAs that did not change in the RNA-seq of LN229 cells upon NSUN5-transfection were upregulated in the Ribo-seq of empty-vector-transfected cells indicating enhanced translational efficiency. e NSUN5 affects both CAP-dependent and CAP-independent translation according to the use of a reporter plasmid encoding for Firefly (IRES) and Renilla (CAP) luciferases. f Gene set enrichment analysis (GSEA) of the RNAs with increased translational efficiency in NSUN5 deficient cells (hypergeometric test with a FDR adjusted P value < 0.05)

Article Snippet: Human glioma cell lines DBTRG-05MG, M059J, A172, and LN229 were purchased from the American Type Culture Collection; CAS-1 was obtained from The Biological Bank of the IRCCS Azienda Ospedaliera Universitaria San Martino—IST Istituto Nazionale per la Ricerca sul Cancro; and KS-1 was purchased from the Japanese Collection of Research Bioresources Cell Bank.

Techniques: Methylation, Transfection, Comparison, RNA Sequencing, Sequencing, Plasmid Preparation

Journal: Acta Neuropathologica

Article Title: Epigenetic loss of RNA-methyltransferase NSUN5 in glioma targets ribosomes to drive a stress adaptive translational program

doi: 10.1007/s00401-019-02062-4

Figure Lengend Snippet: NSUN5 epigenetic silencing activates stress-related protein and confers growth inhibition sensitivity to NQO1-targeting molecules. a Validation of NSUN5 translational regulation of the identified stress-related target NQO1 expression at the RNA level determined by RNA-seq counts (left) and real-time quantitative PCR (middle) do not change upon NSUN5 transfection, but NQO1 expression decreased at the protein level (right) upon NSUN5 restoration. b qPCR shows enrichment of the NQO1 transcript in the polysome fraction of the empty-vector LN229 cells in comparison to NSUN5 transfected-LN229 cells. c NQO1 expression levels in glioma cell lines determined by western blot according to NSUN5 methylation status. d IC50 determination using the SRB assay in the glioma cell lines grouped by NSUN5 methylation status. Black dashed curves represent the 95% confidence band for each group. Glioma cells harboring NSUN5 methylation-associated NQO1 overexpression (A172, LN229 and KS-1) show increased sensitivity to deoxynyboquinone (DNQ) and IB-DNQ in comparison to NSUN5 unmethylated cells (DBTRG-05MG, MO59 J, and CAS-1). Drug-response curves were generated using GraphPad Prism software and analyses were performed with the drc R package. For each cell line and the drug, we fit a four-parameter generalized log-logistic model. Comparison of the IC50 values calculated from the slopes were obtained by means of a z test ( P < 0.0001). e IC50 determination using the SRB assay in NSUN5-transfected LN299 cells in comparison with empty-vector-transfected cells (EV) shows enhanced resistance to DNQ-mediated growth inhibition in the cells with restored NSUN5 expression. Comparison of the IC50 values calculated from the slopes was obtained by means of a z test ( P < 0.001). f Kaplan–Meier analysis of Survival according treatment conditions (IB-DNQ treated vs mock group) in a set of mice models with implanted tumors derived from the NSUN5 methylated cell line A172 (left) or the NSUN5 unmethylated cell line DBTRG (right). Significance of the log-rank test is shown. Results of the univariate Cox regression analysis are represented by the hazards ratio (HR) and 95% confidence interval (CI)

Article Snippet: Human glioma cell lines DBTRG-05MG, M059J, A172, and LN229 were purchased from the American Type Culture Collection; CAS-1 was obtained from The Biological Bank of the IRCCS Azienda Ospedaliera Universitaria San Martino—IST Istituto Nazionale per la Ricerca sul Cancro; and KS-1 was purchased from the Japanese Collection of Research Bioresources Cell Bank.

Techniques: Inhibition, Biomarker Discovery, Expressing, RNA Sequencing, Real-time Polymerase Chain Reaction, Transfection, Plasmid Preparation, Comparison, Western Blot, Methylation, Sulforhodamine B Assay, Over Expression, Generated, Software, Derivative Assay

Journal: Molecular cell

Article Title: Harnessing BET Inhibitor Sensitivity Reveals AMIGO2 as a Melanoma Survival Gene

doi: 10.1016/j.molcel.2017.11.004

Figure Lengend Snippet: (A) Immunofluorescence of SKmel147 cells stably expressing AMIGO2-GFP (green), stained with AMIGO2 antibody (red) and Hoechst 33342 (blue). Scale bar, 20 μm. (B) Functional annotation of AMIGO2-interacting proteins detected by GFP pull-down followed by MS in SKmel147 cells stably expressing AMIGO2-GFP (see Table S4). (C) PTK7 and GFP immunoblots following GFP pull-down from 501MEL cells stably expressing AMIGO2-GFP. (D) Full-length PTK7 (FL-PTK7), C-terminal fragments CTF1- and CTF2-PTK7, and FOXM1 immunoblots of 501MEL cells 72 hr post-infection with shSCR or shPTK7 (shP7 #1 and #2). Actin was used as a loading control. (E) Relative growth curves of 501MEL (left) and SKmel147 (right) cells stably transduced with shSCR or shPTK7 (shP7 #1 and #2). Values are normalized to seeding control (n = 3). (F) Percent Annexin V-positive cells 6 days post-transduction for same cells as in (E). (G) FL-PTK7, CTF-PTK7, and FOXM1 immunoblots of 501MEL cells 48 hr post-transduction with shSCR or shAMIGO2 (shA2 #1 and #2). Actin was used as a loading control. (H) FL-PTK7, CTF-PTK7, FOXM1, and AMIGO2 immunoblots of 501MEL cells untreated or treated with JQ1 (JQ1[+]) for 72 hr. Tubulin was used as a loading control. (I) CTF2-PTK7 immunoblot of nuclear lysates from same cell as in (G) (left). Lamin B1 was used as loading control. Signal quantification (right), normalized to Lamin B1, relative to shSCR (n = 3). All values and error bars represent mean ± SD or ± SEM. See also Figures S3 and S4.

Article Snippet: LAMIN B1 , Santa Cruz , SC-6217.

Techniques: Immunofluorescence, Stable Transfection, Expressing, Staining, Functional Assay, Western Blot, Infection, Control, Transduction

Journal: Molecular cell

Article Title: Harnessing BET Inhibitor Sensitivity Reveals AMIGO2 as a Melanoma Survival Gene

doi: 10.1016/j.molcel.2017.11.004

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: LAMIN B1 , Santa Cruz , SC-6217.

Techniques: Microarray, Derivative Assay, Recombinant, Magnetic Beads, Flow Cytometry, Caspase Activity Assay, DNA Library Preparation, Blocking Assay, Extraction, TA Cloning, Sequencing, RNA Sequencing, Western Blot, Mass Spectrometry, Expressing, Software

Journal: Science Progress

Article Title: Role of thrombus-derived exosomal lncRNA LOC101928697 in regulating endothelial function via FUS protein interaction in myocardial infarction

doi: 10.1177/00368504251372111

Figure Lengend Snippet: Analysis of the correlation between FUS protein and myocardial infarction. (a) Enrichment Analysis Bar Plot based on differential gene expression profiles in lncRNA microarray analysis.(b) Detection information about lncRNA LOC101928697 binding to FUS proteins in AnnoLnc2 database. (c) Detection information about lncRNA LOC101928697 binding to FUS protein in RBPDP database. (d) Scores in the RPISeq database on the model of lncRNA LOC101928697 binding to FUS protein. (e-g) Prediction information about lncRNA LOC101928697 binding to FUS protein in catRAPID website, (e) Statistical map information about protein and RNA binding sites, (f) Total scoring information, and (g) Interaction map showing the interaction region between protein and RNA. (h-i) Analyses about bioinformatics techniques based on GSE163772 in the GEO database, where (h) is a statistical map of FUS gene expression in endothelial cells of a mouse model of myocardial infarction, and (i) A scatter plot about the correlation between the level of FUS gene expression and the disease state (control vs. myocardial infarction).

Article Snippet: After extensive washing, the bound proteins were eluted, separated by SDS-PAGE, and analyzed by Western blot using anti-FUS antibody (Proteintech, Cat No. 11570-1-AP, dilution 1:5000) to detect the enrichment of FUS protein.

Techniques: Gene Expression, Microarray, Binding Assay, RNA Binding Assay, Control

Journal: Science Progress

Article Title: Role of thrombus-derived exosomal lncRNA LOC101928697 in regulating endothelial function via FUS protein interaction in myocardial infarction

doi: 10.1177/00368504251372111

Figure Lengend Snippet: Interaction of exosomal lncRNA LOC101928697 with FUS proteins. (a and b) The western blot detection of FUS protein expression in each group of cells and the statistical graph. (c) Statistical graph of RT-qPCR to detect the expression of FUS at the mRNA level in each group of cells. (d) The fluorescence graph of fluorescence in situ hybridization (FISH) experiment. In which FUS was labeled with green fluorescence, lncRNA LOC101928697 was labeled with red fluorescence, and the nucleus was labeled with blue fluorescence (20×). (e) Western blot detection of FUS protein following RNA pull-down using sense or antisense LOC101928697 transcripts. (f) Quantification of FUS protein enrichment in sense RNA pull-down versus antisense control, based on densitometric analysis. (g-h) Western blot detection of FUS protein expression in each group of cells after knockdown or overexpression of lncRNA LOC101928697 and the statistical graphs. (i) Statistical graph of mRNA level expression of FUS in each group of cells after knockdown or overexpression of lncRNA LOC101928697 by RT-qPCR assay. a p < 0.05 compared to control group. b p < 0.05 compared to exosome group. c p < 0.05 compared to siRNA + exosome group.

Article Snippet: After extensive washing, the bound proteins were eluted, separated by SDS-PAGE, and analyzed by Western blot using anti-FUS antibody (Proteintech, Cat No. 11570-1-AP, dilution 1:5000) to detect the enrichment of FUS protein.

Techniques: Western Blot, Expressing, Quantitative RT-PCR, Fluorescence, In Situ Hybridization, Labeling, Protein Enrichment, Control, Knockdown, Over Expression

Journal: Frontiers in Molecular Biosciences

Article Title: A systematic review of the barcoding strategy that contributes to COVID-19 diagnostics at a population level

doi: 10.3389/fmolb.2023.1141534

Figure Lengend Snippet: Schematic representation of mechanistic strategies of barcoding. (A–C) Barcodes can be introduced to a template using adaptors through direct ligation (A) , using RT- or PCR primers at the reverse transcription or PCR amplification step (B) , and using hybridizing molecular inversion probes (C) . (D) Schematic representation of the difference between “barcodes” and “sample indexes”. Barcodes aim to correct sequencing errors. For example, a misreading nucleotide, guanosine (G) can be corrected in final consensus sequences for a pool of Sample 1 (top panel). Sample indexes are used to multiplex different sequencing amplicons generated from different pools of samples (Sample 1, 2, and 3) (bottom panel). Panel (A) is modified based on in and panel (C) is modified based on in .

Article Snippet: Primer-associated approach , Sequence-based barcodes , SQK-RBK004: transposase carrying barcodes to the site of the cleavage , - , - , Whole genome , Oxford Nanopore Rapid Barcoding kit (SQK-RBK004) , SARS-CoV-2 patient samples (nasopharyngeal swab) , Oxford Nanopore , Guppy version 3.6.0; ARTIC Network bioinformatics protocol , Multiplex samples , Propose a method to sequence the whole genome of SARS-CoV-2 in a rapid and cost-efficient manner , .

Techniques: Ligation, Reverse Transcription, Amplification, Sequencing, Multiplex Assay, Generated, Modification

Journal: Frontiers in Molecular Biosciences

Article Title: A systematic review of the barcoding strategy that contributes to COVID-19 diagnostics at a population level

doi: 10.3389/fmolb.2023.1141534

Figure Lengend Snippet: Systematic comparison of barcoding strategies used in the category of molecular barcodes.

Article Snippet: Primer-associated approach , Sequence-based barcodes , SQK-RBK004: transposase carrying barcodes to the site of the cleavage , - , - , Whole genome , Oxford Nanopore Rapid Barcoding kit (SQK-RBK004) , SARS-CoV-2 patient samples (nasopharyngeal swab) , Oxford Nanopore , Guppy version 3.6.0; ARTIC Network bioinformatics protocol , Multiplex samples , Propose a method to sequence the whole genome of SARS-CoV-2 in a rapid and cost-efficient manner , .

Techniques: Comparison, Software, Sequencing, Multiplex Assay, CRISPR, Plasmid Preparation, Microarray, Binding Assay, Amplification, Extraction, Ligation, DNA Sequencing, Multiplexing, Generated, Reverse Transcription, Staining, Flow Cytometry, High Throughput Screening Assay, Inhibition, Blocking Assay, Conjugation Assay, RNA Sequencing Assay, Transmission Assay, Incubation, Diagnostic Assay, Next-Generation Sequencing, Infection

Journal: Nutrients

Article Title: Long-Term Consumption of a Sugar-Sweetened Soft Drink in Combination with a Western-Type Diet Is Associated with Morphological and Molecular Changes of Taste Markers Independent of Body Weight Development in Mice

doi: 10.3390/nu14030594

Figure Lengend Snippet: Transcriptome analysis of 59 selected genes associated with oral chemosensation, displayed as a heatmap showing mean fold changes in gene expression of the SSB-fed diet groups in relation to the respective water-fed group (=1) in the form of a color code. The gene expression was analyzed using one customized cDNA microarray per group from pooled RNA samples of the CV from mice that received either a standard diet (chow, n = 10–11) or Western-type diet (WD, n = 7–8) with water (Water) or a sugar-sweetened beverage (SSB) as a drink for 24 weeks.

Article Snippet: The isolated RNA samples per mouse were reverse transcribed to cDNA using the LunaScript RT Supermix Kit (New England Biolabs GmbH, Frankfurt am Main, Germany).

Techniques: Expressing, Microarray, Western Blot

Journal: Human Molecular Genetics

Article Title: Altered keratinocyte differentiation is an early driver of keratin mutation-based palmoplantar keratoderma

doi: 10.1093/hmg/ddz050

Figure Lengend Snippet: Analysis of global gene expression in Krt16-null footpad lesions and comparison to human PC cases. (A) Volcano plot depicting differentially expressed genes in Krt16-null paw skin lesions relative to WT controls. (B) Top 10 genes differentially regulated in Krt16-null paw skin from established paw lesions compared to WT littermate controls. (C) Validation of downregulated genes identified by microarray analysis by RT-qPCR in Krt16-null paw skin lesions. N = 4 mice/genotype. Error bars are SEM. *P < 0.05, **P < 0.01. (D) Overlap in the significantly changed genes that are upregulated between Krt16-null paw skin lesions and KRT16 human cases. (E) Overlap in the significantly changed genes that are downregulated between Krt16-null paw skin lesions and KRT16 human cases. (F) Overlap in the significantly changed genes that are upregulated between Krt16-null paw skin lesions and KRT6 human cases. (G) Overlap in the significantly changed genes that are downregulated between Krt16-null paw skin lesions and KRT6 human cases. Boxes for (D)–(G) list common genes between mouse and human data sets (listed in alphabetical order).

Article Snippet: Total RNA isolated from paw skin, epidermis and dermis, of 2 month old Krt16 -null (2 males, 2 females) and WT (2 males, 2 females) littermates (see Biochemical and morphological analyses ) was subjected to microarray analysis by the Johns Hopkins Deep Sequencing Microarray Core using the Affymetrix Mouse Transcriptome Array 1.0 GeneChip.

Techniques: Gene Expression, Comparison, Biomarker Discovery, Microarray, Quantitative RT-PCR

Journal: Cancer gene therapy

Article Title: HN1-mediated activation of lipogenesis through Akt-SREBP signaling promotes hepatocellular carcinoma cell proliferation and metastasis.

doi: 10.1038/s41417-024-00827-y

Figure Lengend Snippet: Fig. 1 HN1 is overexpressed in human HCC and correlates with poor prognosis and promoter methylation. A Kaplan–Meier analysis for the overall survival times of hepatocellular carcinoma patients with high HN1 expression and low HN1 expression in TCGA (left) and the NCI (right) database. TCGA, The Cancer Genome Atlas. NCI, National Cancer Institute. OS, overall survival. B The correlation between HN1 mRNA expression and HN1 DNA methylation was analyzed in HCC patients. C The protein expression of HN1 in paired HCC tissues and adjacent non- tumoral liver tissues from seven patients. T liver tumor tissue. N adjacent non-tumoral liver tissue. D The protein expression of HN1 in unpaired HCC tissues from 7 patients. E The relative levels of HN1 mRNA were examined using qPCR in paired HCC and adjacent non-tumoral liver tissues from seven patients. F The protein levels of HN1 in seven hepatocellular carcinoma cell lines were detected. Data were expressed as the mean ± SEM of three independent experiments. Protein quantification was calculated by ImageJ software. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the corresponding control.

Article Snippet: Control siRNA-A (SC-37007), HN1 siRNA (sc-93940), SREBP-1 siRNA (sc-36557), control shRNA plasmid-A (sc-108060), HN1 shRNA plasmid (sc-93940-SH), control shRNA lentiviral particles (sc-108080), and HN1 shRNA lentiviral particles (sc-93904-V) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: Methylation, Expressing, DNA Methylation Assay, Software, Control

Journal: Cancer gene therapy

Article Title: HN1-mediated activation of lipogenesis through Akt-SREBP signaling promotes hepatocellular carcinoma cell proliferation and metastasis.

doi: 10.1038/s41417-024-00827-y

Figure Lengend Snippet: Fig. 2 Effects of HN1 on cell proliferation and apoptosis in HCC. A WST-1 assay to assess the viability of the HepG2 and SNU449 cell lines after HN1 shRNA knockdown or HN1 overexpression. B A colony staining assay showed the colony formation ability of HepG2 and SNU449 cells with HN1 shRNA knockdown or HN1 overexpression. Colonies were counted at least in five fields. C Apoptosis marker proteins (PARP/ cleaved-PARP and caspase-9/cleaved-caspase-9) were detected in pairs by western blots of HepG2 and SNU449 cells after HN1 shRNA knockdown or HN1 overexpression. GAPDH was used as the internal control. Data were expressed as the mean ± SEM of three independent experiments. Protein quantification was calculated by ImageJ software. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the corresponding control. shCTRL, control shRNA (control). shHN1, HN1 shRNA knockdown. Vector, control vector plasmid (control). HN1 OE HN1 overexpression.

Article Snippet: Control siRNA-A (SC-37007), HN1 siRNA (sc-93940), SREBP-1 siRNA (sc-36557), control shRNA plasmid-A (sc-108060), HN1 shRNA plasmid (sc-93940-SH), control shRNA lentiviral particles (sc-108080), and HN1 shRNA lentiviral particles (sc-93904-V) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: WST-1 Assay, shRNA, Knockdown, Over Expression, Staining, Marker, Western Blot, Control, Software, Plasmid Preparation

Journal: Cancer gene therapy

Article Title: HN1-mediated activation of lipogenesis through Akt-SREBP signaling promotes hepatocellular carcinoma cell proliferation and metastasis.

doi: 10.1038/s41417-024-00827-y

Figure Lengend Snippet: Fig. 3 Effects of HN1 on the cell cycle in HCC. A FACs cell cycle analysis of the HepG2 and SNU449 cell lines with HN1 shRNA knockdown. B Western blot analysis of the G1 cycle-related proteins CDK4, CDK6, Cyclin D1, and p53 in the HepG2 and SNU449 cell lines with HN1 shRNA knockdown. GAPDH was used as the internal control. Data represent the mean ± SEM of three independent experiments. *p < 0.05 and **p < 0.01 compared with the corresponding control. shCTRL control shRNA (control), shHN1 HN1 shRNA knockdown.

Article Snippet: Control siRNA-A (SC-37007), HN1 siRNA (sc-93940), SREBP-1 siRNA (sc-36557), control shRNA plasmid-A (sc-108060), HN1 shRNA plasmid (sc-93940-SH), control shRNA lentiviral particles (sc-108080), and HN1 shRNA lentiviral particles (sc-93904-V) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: Cell Cycle Assay, shRNA, Knockdown, Western Blot, Control

Journal: Cancer gene therapy

Article Title: HN1-mediated activation of lipogenesis through Akt-SREBP signaling promotes hepatocellular carcinoma cell proliferation and metastasis.

doi: 10.1038/s41417-024-00827-y

Figure Lengend Snippet: Fig. 4 Effects of HN1 on cell migration and invasion in HCC. A Wound-healing assay to assess the cell migration rate in the HepG2 and SNU449 cell lines after HN1 shRNA knockdown or HN1 overexpression. Representative images were obtained at 0, 24, and 48 h. Migration ability was quantified by measuring the gap distance. B Matrigel transwell assay to assess the cell invasion ability of the HepG2 and SNU449 cell lines after HN1 shRNA knockdown or HN1 overexpression for 72 h. The migration-related molecular uPA and vimentin protein levels (C) and mRNA levels (D) were detected by western blotting and rt-qPCR, respectively, in HepG2 and SNU449 cells after HN1 shRNA knockdown or HN1 overexpression. GAPDH was used as the internal control. Data are expressed as the mean ± SEM of three independent experiments. Protein quantification was calculated by ImageJ software. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the corresponding control. shCTRL control shRNA (control), shHN1 HN1 shRNA knockdown, Vector control vector plasmid (control), HN1 OE HN1 overexpression.

Article Snippet: Control siRNA-A (SC-37007), HN1 siRNA (sc-93940), SREBP-1 siRNA (sc-36557), control shRNA plasmid-A (sc-108060), HN1 shRNA plasmid (sc-93940-SH), control shRNA lentiviral particles (sc-108080), and HN1 shRNA lentiviral particles (sc-93904-V) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: Migration, Wound Healing Assay, shRNA, Knockdown, Over Expression, Transwell Assay, Western Blot, Quantitative RT-PCR, Control, Software, Plasmid Preparation

Journal: Cancer gene therapy

Article Title: HN1-mediated activation of lipogenesis through Akt-SREBP signaling promotes hepatocellular carcinoma cell proliferation and metastasis.

doi: 10.1038/s41417-024-00827-y

Figure Lengend Snippet: Fig. 5 Gene expression levels affected by HN1 knockdown in HCC. A cDNA microarray heatmap showing the effects of HN1 shRNA knockdown on AKT signaling pathway-related genes in HCC cells. The data were expressed in matrix format, with rows representing individual genes and columns representing individual samples. Red and green indicate increased and decreased gene expression, respectively. B Gene ontology analysis showed that HN1 knockdown was related to a series of diseases and disorders. shCTRL control shRNA (control), shHN1 HN1 shRNA knockdown.

Article Snippet: Control siRNA-A (SC-37007), HN1 siRNA (sc-93940), SREBP-1 siRNA (sc-36557), control shRNA plasmid-A (sc-108060), HN1 shRNA plasmid (sc-93940-SH), control shRNA lentiviral particles (sc-108080), and HN1 shRNA lentiviral particles (sc-93904-V) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: Gene Expression, Knockdown, Microarray, shRNA, Control

Journal: Cancer gene therapy

Article Title: HN1-mediated activation of lipogenesis through Akt-SREBP signaling promotes hepatocellular carcinoma cell proliferation and metastasis.

doi: 10.1038/s41417-024-00827-y

Figure Lengend Snippet: Fig. 7 HN1 regulated SREBP-1 and SREBP-2 in HCC. A Downstream gene networks from the ingenuity pathway analysis. HN1 knockdown inhibited the SREBF-1 and SREBF-2 genes and their downstream genes. B Precursor and mature SREBP-1c protein expression was examined by western blotting in HepG2 and SNU449 cells after HN1 shRNA knockdown or HN1 overexpression. GAPDH was used as the internal control. C SREBP-1 cell distribution was observed directly by immunofluorescence against the SREBP-1 antibody. Green, SREBP-1. Blue, DAPI (nucleus). The distribution of fluorescence density in the nucleus and protein quantification were measured by ImageJ software. D The mRNA levels of precursor and mature SREBP-1 were measured by rt-qPCR after HN1 shRNA knockdown in the HepG2 and SNU449 cell lines. Data were expressed as the mean ± SEM of three independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the corresponding control. shCTRL control shRNA (control). shHN1 HN1 shRNA knockdown, Vector control vector plasmid (control), HN1 OE HN1 overexpression.

Article Snippet: Control siRNA-A (SC-37007), HN1 siRNA (sc-93940), SREBP-1 siRNA (sc-36557), control shRNA plasmid-A (sc-108060), HN1 shRNA plasmid (sc-93940-SH), control shRNA lentiviral particles (sc-108080), and HN1 shRNA lentiviral particles (sc-93904-V) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: Knockdown, Expressing, Western Blot, shRNA, Over Expression, Control, Software, Quantitative RT-PCR, Plasmid Preparation

Journal: Cancer gene therapy

Article Title: HN1-mediated activation of lipogenesis through Akt-SREBP signaling promotes hepatocellular carcinoma cell proliferation and metastasis.

doi: 10.1038/s41417-024-00827-y

Figure Lengend Snippet: Fig. 10 Knockdown of HN1 inhibited tumorigenesis in xenograft mice. The xenograft nude mouse models were established with HepG2 cells transfected with control shRNA or HN1 shRNA. The mice were separated into two groups: (i) control shRNA group and (ii) HN1 shRNA group. A, B Images of mice and tumors. Body weight (C) and tumor volume (E) were measured every 3 days. Tumor weight (D) and images of tumor size (B) were evaluated after euthanasia. F A histopathological analysis of H&E stained tissues and the TUNEL assay were performed to determine the histological characteristics of the mouse tumor tissues. G Tumor tissues were immunostained with HN1 and SREBP-1 antibodies. H Western blot analysis of HN1, precursor and mature SREBP-1C, FAS, and ACC in mouse tumor tissues. GAPDH was used as the cytoplasm internal control. Protein quantification was calculated by ImageJ software. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the control. shCTRL control shRNA (control), shHN1 HN1 shRNA knockdown.

Article Snippet: Control siRNA-A (SC-37007), HN1 siRNA (sc-93940), SREBP-1 siRNA (sc-36557), control shRNA plasmid-A (sc-108060), HN1 shRNA plasmid (sc-93940-SH), control shRNA lentiviral particles (sc-108080), and HN1 shRNA lentiviral particles (sc-93904-V) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: Knockdown, Transfection, Control, shRNA, Staining, TUNEL Assay, Western Blot, Software

Journal: Cancer gene therapy

Article Title: HN1-mediated activation of lipogenesis through Akt-SREBP signaling promotes hepatocellular carcinoma cell proliferation and metastasis.

doi: 10.1038/s41417-024-00827-y

Figure Lengend Snippet: Fig. 11 Inhibition of SREBP reverses the tumorigenic effect of HN1 in xenograft mice. Xenograft nude mouse models were established using HepG2 cells transfected with either a control or HN1 overexpression vector. The mice were divided into three groups: (i) control group, (ii) HN1 overexpression group, and (iii) HN1 overexpression combined with Fatostatin. A Tumor volume was measured every 3 days. After euthanasia, images of the tumors (B), body weight (C), and tumor weight (D) were evaluated. *p < 0.05, **p < 0.01, and ***p < 0.001: vector vs. HN1 OE. ## p < 0.01 and ###p < 0.001: HN1 OE vs. HN1 OE plus Fatostatin. “Vector” refers to the control vector plasmid, “HN1 OE” to HN1 overexpression, and “Fatostatin” to the SREBP inhibitor.

Article Snippet: Control siRNA-A (SC-37007), HN1 siRNA (sc-93940), SREBP-1 siRNA (sc-36557), control shRNA plasmid-A (sc-108060), HN1 shRNA plasmid (sc-93940-SH), control shRNA lentiviral particles (sc-108080), and HN1 shRNA lentiviral particles (sc-93904-V) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: Inhibition, Transfection, Control, Over Expression, Plasmid Preparation

Journal: Cancer gene therapy

Article Title: HN1-mediated activation of lipogenesis through Akt-SREBP signaling promotes hepatocellular carcinoma cell proliferation and metastasis.

doi: 10.1038/s41417-024-00827-y

Figure Lengend Snippet: Fig. 12 Schematic representation of HN1-mediated inhibition of tumorigenesis of HCC. HN1 suppression deactivates the Akt pathway, leading to reduced levels of mature SREBP in the nucleus, thereby inhibiting lipogenesis and ultimately suppressing hepato- cellular carcinoma (HCC) tumorigenesis.

Article Snippet: Control siRNA-A (SC-37007), HN1 siRNA (sc-93940), SREBP-1 siRNA (sc-36557), control shRNA plasmid-A (sc-108060), HN1 shRNA plasmid (sc-93940-SH), control shRNA lentiviral particles (sc-108080), and HN1 shRNA lentiviral particles (sc-93904-V) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: Inhibition