Journal: PLoS ONE

Article Title: The effects of tissue fixation on sequencing and transcript abundance of nucleic acids from microdissected liver samples of smallmouth bass ( Micropterus dolomieu )

doi: 10.1371/journal.pone.0236104

Figure Lengend Snippet: (A) Mean (+ standard error) of RNA and (B) DNA concentrations (μg/mm 3 ) of microdissected smallmouth bass liver samples fixed in 10% neutral buffered formalin (NBF), Z-Fix ® (ZF), and PAXgene® (PG) for 24 hours, 48 hours, 7 and 14 days. Samples preserved in 95% alcohol (ETOH) and RNALater® were included as controls.

Article Snippet: Tissues preserved in PG were removed from the PAXgene® Tissue FIX (Product # 765312, QIAGEN) after 4 hrs at RT, placed in the PAXgene® Tissue STABILIZER solution (Product # 765512, QIAGEN), and stored at 4°C for 24 hrs, 48 hrs, seven and 14 days.

Techniques:

Journal: PLoS ONE

Article Title: The effects of tissue fixation on sequencing and transcript abundance of nucleic acids from microdissected liver samples of smallmouth bass ( Micropterus dolomieu )

doi: 10.1371/journal.pone.0236104

Figure Lengend Snippet: (A) Mean RIN values of RNA and (B) fragment size (bp) of DNA from samples fixed in 10% neutral buffered formalin (NBF), Z-Fix (ZF) and PAXgene (PG) for 24 hours, 48 hours, 7 and 14 days. Samples preserved in 95% alcohol (ETOH) and RNAlater were included as controls.

Article Snippet: Tissues preserved in PG were removed from the PAXgene® Tissue FIX (Product # 765312, QIAGEN) after 4 hrs at RT, placed in the PAXgene® Tissue STABILIZER solution (Product # 765512, QIAGEN), and stored at 4°C for 24 hrs, 48 hrs, seven and 14 days.

Techniques:

Journal: Acta Naturae

Article Title: A Highly Productive CHO Cell Line Secreting Human Blood Clotting Factor IX

doi:

Figure Lengend Snippet: Analysis of target gene integrity in the genomic DNA and cDNA from producer cell lines by PCR, RT-PCR, Southern blotting and pseudo-Northern blotting. DNA ladder bands are shown as bps. Panel A – amplification of whole ORF regions of the target genes in genomic DNA (gDNA) by PCR. The annealing temperature gradient is shown with red triangles; the linear shift in annealing temperature was from 53 to 68 oC. The expected amplification products of the FIX , furin, and VKORC1 genes are shown with red arrows. Panel B – RT-PCR products for total mRNA with primers towards the 5’- and 3’- ends of the FIX ORF; the amplification product of expected size is shown with a red arrow. Panel C – Southern blotting of gDNA with biotin-labeled probes. The IDbla probe is the probe targeting the IRES and DHFR regions, the ampicillin resistance gene (the bla gene) and the region of plasmid origin of replication (ori). The FIX probe is the probe targeting the FIX ORF region. Sizes of the detected target restriction fragments are shown with arrows. Negative agarose gel images; the membrane images were contrast-enhanced to add visibility. Panel D – Analysis of cDNA by pseudo-Northern blotting. Denotation is the same as that in panel C. The actual position and size of FIX cDNA are shown with an arrow

Article Snippet: The commercially available clone of human FIX cDNA, pCMV6-XL4/NM_000133.2 (sc126517, Origene, USA), and adapter primers AD-9-AbsF and AD-9- NheR ( ) were used as a source of FIX ORF.

Techniques: Reverse Transcription Polymerase Chain Reaction, Southern Blot, Northern Blot, Amplification, Labeling, Plasmid Preparation, Agarose Gel Electrophoresis, Membrane

Journal: International Journal of General Medicine

Article Title: AK3 as a Hypoxia-Angiogenesis–Related Prognostic Biomarker and Therapeutic Target in Clear Cell Renal Cell Carcinoma

doi: 10.2147/IJGM.S552108

Figure Lengend Snippet: Single cell analysis and RT-PCR validation. ( A ) Violin plots depicting the expression levels of RPL36A, AK3, LGALS1, TIMP1, and VIM across various cell types within ccRCC tissues, based on the single-cell dataset GSE14526 . ( B ) Comparative analysis of RNA expression levels of RPL36A, AK3, LGALS1, TIMP1, and VIM between normal and tumor tissues from ccRCC patients. ( C ) Forest plots summarizing the univariate and multivariate Cox proportional hazards regression analyses of the prognostic value of RPL36A, AK3, LGALS1, TIMP1, and VIM in the TCGA ccRCC cohort. The red box highlights AK3, which was identified as an independent protective prognostic factor in the multivariate analysis. Data were shown as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The antibodies used include β-Actin antibody, AK3 antibody, AKT antibody, Phospho-AKT (Ser473) antibody, GSK3β antibody (Proteintech, China).

Techniques: Single-cell Analysis, Reverse Transcription Polymerase Chain Reaction, Biomarker Discovery, Expressing, Single Cell, RNA Expression

Journal: International Journal of General Medicine

Article Title: AK3 as a Hypoxia-Angiogenesis–Related Prognostic Biomarker and Therapeutic Target in Clear Cell Renal Cell Carcinoma

doi: 10.2147/IJGM.S552108

Figure Lengend Snippet: Bioinformatics Analysis of AK3 Expression, Clinical Features, and Prognosis in ccRCC. ( A ) Boxplot analysis of AK3 mRNA expression levels across pan-cancers in TCGA. ( B ) Paired analysis of AK3 mRNA expression in normal versus tumor tissues in ccRCC. ( C ) Receiver Operating Characteristic (ROC) curve illustrating the diagnostic accuracy of AK3 expression for ccRCC. ( D ) Boxplot of AK3 protein expression levels in normal and primary tumor tissues from the CPTAC data. ( E ) Boxplots depicting AK3 expression correlation with clinical features, including pathologic M stage, histologic grade, and gender. ( F ) Kaplan-Meier survival curves showing the relationship between AK3 expression and OS, DSS and PFI in ccRCC patients. Data were shown as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The antibodies used include β-Actin antibody, AK3 antibody, AKT antibody, Phospho-AKT (Ser473) antibody, GSK3β antibody (Proteintech, China).

Techniques: Expressing, Diagnostic Assay

Journal: International Journal of General Medicine

Article Title: AK3 as a Hypoxia-Angiogenesis–Related Prognostic Biomarker and Therapeutic Target in Clear Cell Renal Cell Carcinoma

doi: 10.2147/IJGM.S552108

Figure Lengend Snippet: Validation of AK3 Expression Levels in ccRCC Tissue Samples and Cell Lines. ( A ) Western blot analysis of AK3 protein expression in normal control (NC) and tumor tissues from ccRCC. ( B ) Immunohistochemical (IHC) analysis of AK3 expression in a tissue microarray containing normal (N) and tumor (T) samples from ccRCC patients. H-Score quantification of AK3 expression demonstrates significant downregulation in tumor tissues compared to normal tissues. ( C ) Western blot analysis of AK3 protein expression in ccRCC cell lines. ( D ) RT-PCR analysis of AK3 mRNA expression in ccRCC cell lines. ( E ) Immunofluorescence staining of AK3 protein in ccRCC cells. Labeling mitochondria with TOM20 antibody and AK3 protein with AK3 antibody. Data were shown as mean ± SD. * p < 0.05, ** p < 0.01.

Article Snippet: The antibodies used include β-Actin antibody, AK3 antibody, AKT antibody, Phospho-AKT (Ser473) antibody, GSK3β antibody (Proteintech, China).

Techniques: Biomarker Discovery, Expressing, Western Blot, Control, Immunohistochemical staining, Microarray, Reverse Transcription Polymerase Chain Reaction, Immunofluorescence, Staining, Labeling

Journal: International Journal of General Medicine

Article Title: AK3 as a Hypoxia-Angiogenesis–Related Prognostic Biomarker and Therapeutic Target in Clear Cell Renal Cell Carcinoma

doi: 10.2147/IJGM.S552108

Figure Lengend Snippet: Correlation Between AK3 Expression and Immune Infiltration in ccRCC. ( A ) Correlation analysis of AK3 expression with immune cell infiltration using the ssGSEA algorithm across 24 immune cell types. ( B ) Scatter plots depicting the correlation of AK3 expression with the enrichment scores of selected immune cell types: mast cells, T helper cells, Treg cells, and NK CD56bright cells. ( C ) Box plots comparing the expression levels of immune checkpoint genes (CD274, CTLA4, TIGIT, LAG3, PDCD1) between high AK3 (G1), low AK3 (G2), and normal groups. ( D ) Comparison of mutation frequencies of selected genes between high and low AK3 expression groups in the TCGA-KIRC cohort. ( E ) Correlation analysis of AK3 mRNA expression with drug sensitivity using the GSCA database. Data were shown as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The antibodies used include β-Actin antibody, AK3 antibody, AKT antibody, Phospho-AKT (Ser473) antibody, GSK3β antibody (Proteintech, China).

Techniques: Expressing, Comparison, Mutagenesis

Journal: International Journal of General Medicine

Article Title: AK3 as a Hypoxia-Angiogenesis–Related Prognostic Biomarker and Therapeutic Target in Clear Cell Renal Cell Carcinoma

doi: 10.2147/IJGM.S552108

Figure Lengend Snippet: AK3 Overexpression Inhibits Growth and Migration of ccRCC Cells. ( A ) Western blot analysis showing the successful overexpression of AK3 protein in 786O and OS ccRCC cell lines. ( B ) Relative RNA expression levels of AK3 in 786O and OS cells following transfection with AK3 overexpression plasmids, as determined by RT-PCR. ( C ) Cell growth curves for 786O and OS cells, illustrating a significant decrease in proliferation rates in the AK3 overexpression group compared to controls over a 5-day period. ( D ) Representative images and quantification of colony formation assays in 786O and OS cell lines. ( E ) Wound healing assay images and quantification demonstrating reduced migration of 786O and OS cells following AK3 overexpression. ( F ) Transwell migration assay results showing a significant decrease in the number of migrating 786O and OS cells upon AK3 overexpression. Data were shown as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The antibodies used include β-Actin antibody, AK3 antibody, AKT antibody, Phospho-AKT (Ser473) antibody, GSK3β antibody (Proteintech, China).

Techniques: Over Expression, Migration, Western Blot, RNA Expression, Transfection, Reverse Transcription Polymerase Chain Reaction, Wound Healing Assay, Transwell Migration Assay

Journal: International Journal of General Medicine

Article Title: AK3 as a Hypoxia-Angiogenesis–Related Prognostic Biomarker and Therapeutic Target in Clear Cell Renal Cell Carcinoma

doi: 10.2147/IJGM.S552108

Figure Lengend Snippet: AK3 Overexpression Inhibits the PI3K-AKT/GSK3β Signaling Pathway in ccRCC Cells. ( A ) Volcano plot depicting differential gene expression in 786O cells following transfection with AK3 overexpression plasmids. ( B ) KEGG pathway enrichment analysis highlighting downregulated pathways upon AK3 overexpression. ( C ) KEGG enrichment analysis of upregulated pathways in AK3-overexpressing cells. ( D ) Western blot analysis of key signaling proteins in the PI3K/AKT pathway. ( E ) In vivo analysis of tumor growth volume and weight in nude mice. ( F ) Immunohistochemical analysis of AK3 and Ki67 expression in tumor tissues. Data were shown as mean ± SD. * p < 0.05, ** p < 0.01.

Article Snippet: The antibodies used include β-Actin antibody, AK3 antibody, AKT antibody, Phospho-AKT (Ser473) antibody, GSK3β antibody (Proteintech, China).

Techniques: Over Expression, Gene Expression, Transfection, Western Blot, In Vivo, Immunohistochemical staining, Expressing

Journal: International Journal of General Medicine

Article Title: AK3 as a Hypoxia-Angiogenesis–Related Prognostic Biomarker and Therapeutic Target in Clear Cell Renal Cell Carcinoma

doi: 10.2147/IJGM.S552108

Figure Lengend Snippet: AK3 improves tumor cell sensitivity to oxaliplatin by reducing lipid droplet accumulation and increasing ROS levels. ( A and B ) Dose-response curves depicting cell viability of 786O ( A ) and OS ( B ) cell lines treated with varying concentrations of oxaliplatin, with AK3 overexpression (OE) and negative control (NC). ( C ) Fluorescence microscopy images and quantitative analysis showing increased reactive oxygen species (ROS) levels in AK3-overexpressing cells compared to controls. ( D ) Fluorescence microscopy images and quantitative analysis demonstrating reduced lipid droplet accumulation in AK3-overexpressing cells compared to controls. Data were shown as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The antibodies used include β-Actin antibody, AK3 antibody, AKT antibody, Phospho-AKT (Ser473) antibody, GSK3β antibody (Proteintech, China).

Techniques: Over Expression, Negative Control, Fluorescence, Microscopy