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: JCI Insight

Article Title: Virus-induced RGMa expression drives neurodegeneration in HTLV-1–associated myelopathy

doi: 10.1172/jci.insight.184530

Figure Lengend Snippet: ( A ) The concentration of NF-L (pg/mL) in the supernatant when NB-1 cells were cocultured with HAM-PBMCs ( n = 7) or HD-PBMCs ( n = 7). ( B ) The concentration of NF-L (pg/mL) in the supernatant when NB-1 cells were cocultured with HAM-PBMCs ( n = 9) and with mogamulizumab (antiCCR4) in a dose-dependent manner for 72 hours. ( C ) The comparison of RGMA mRNA gene expression levels using DNA microarray among normal CD4 + T cells (HD CD4 + : n = 4), HAM patient–derived CD4 + T cells (HAM CD4 + : n = 4), ACs ( n = 2), and smoldering/chronic-type-ATL patient–derived ( n = 3) HTLV-1–infected CD4 + T cells (Non-HAM infected CD4 + T cells: n = 5), and acute-type-ATL patient–derived HTLV-1–infected CD4 + T cells (Acute ATL infected cells: n = 3). ( D ) The comparison of the expression levels of the genes associated with the inhibition of neuroregeneration ( OMG , MAG , RTN4 , and WNT5A ) between HD CD4 + ( n = 4) and HAM CD4 + T cells ( n = 4). ( E ) The enrichment levels of H3K27me3 –2916 bp upstream from the TSS of the RGMA gene locus in HD CD4 + ( n = 3), HAM CD4 + ( n = 4), and acute-ATL infected cells ( n = 4). Data are shown as mean ± SD. ** P < 0.01; *** P < 0.001 by unpaired t test ( A and D ) or 1-way ANOVA with Dunnett’s multiple-comparison test ( B , C , and E ). NF-L, neurofilament light chain.

Article Snippet: In the experiment analyzing Tax , HBZ , and RGMA gene expression levels in cultured HAM-PBMCs, mRNA was purified using the Magnetic mRNA isolation kit (New England BioLabs).

Techniques: Concentration Assay, Comparison, Gene Expression, Microarray, Derivative Assay, Infection, Expressing, Inhibition

Journal: JCI Insight

Article Title: Virus-induced RGMa expression drives neurodegeneration in HTLV-1–associated myelopathy

doi: 10.1172/jci.insight.184530

Figure Lengend Snippet: ( A ) The validation of RGMA mRNA gene expression levels using qRT-PCR in HD CD4 + ( n = 6) and HAM CD4 + T cells ( n = 6). ( B ) Expression of RGMa protein in CD3 + CD4 + CCR4 + T cells from HAM-PBMCs. Representative dot plots of CCR4 and normal goat IgG (upper) or RGMa expression (bottom) in CD3 + CD4 + gated cells from HD-PBMCs (left) or HAM-PBMCs (right) cultured for 2 days. ( C ) Graph shows the percentage of RGMa protein–expressing cells in CCR4 – cells or CCR4 + cells in CD3 + CD4 + gated cells from HAM-PBMCs ( n = 8) cultured for 2 days, compared with the isotype control, normal goat IgG. ( D ) Graph shows the percentage of RGMa protein–expressing cells among CD3 + CD4 + CCR4 + gated cells from HD-PBMCs ( n = 5) or HAM-PBMCs ( n = 8) cultured for 2 days. Data are shown as mean ± SD. * P < 0.05; ** P < 0.01 by unpaired t test ( A and D ) or 1-way ANOVA with Dunnett’s multiple-comparison test.

Article Snippet: In the experiment analyzing Tax , HBZ , and RGMA gene expression levels in cultured HAM-PBMCs, mRNA was purified using the Magnetic mRNA isolation kit (New England BioLabs).

Techniques: Biomarker Discovery, Gene Expression, Quantitative RT-PCR, Expressing, Cell Culture, Control, Comparison

Journal: JCI Insight

Article Title: Virus-induced RGMa expression drives neurodegeneration in HTLV-1–associated myelopathy

doi: 10.1172/jci.insight.184530

Figure Lengend Snippet: ( A ) Tax (left), HBZ (middle), and RGMA (right) gene expression levels in cultured HAM-PBMCs ( n = 7) in a time-dependent manner. RPL19 was used as an internal control. ( B ) Tax-dependent RGMA mRNA gene induction in Jurkat cells, which were infected with lentivirus carrying the Tax gene. Top: Tax expression in the Jurkat cells was confirmed by Western blotting. β-Actin was measured as an internal control. Bottom: The induction levels of the RGMA gene were evaluated by qRT-PCR in a time-dependent manner ( n = 3). ( C ) Tax -dependent RGMA mRNA gene induction in JPX9 cells treated with 20 μM CdCl 2 in a time-dependent manner. Tax mRNA (upper) and RGMA mRNA (bottom) were measured by qRT-PCR ( n = 3). GAPDH was measured as an internal control. ( D ) Tax-dependent RGMa protein induction in JPX9 cells treated with 20 μM CdCl 2 for 3 days. Dot plots of Tax and normal goat IgG (upper) or RGMa expression (bottom) in JPX9 cells. JPX9(-), untreated JPX9 cells; 20 μM CdCl 2 JPX9, CdCl 2 -supplemented JPX9 cells. Data are shown as mean ± SD. * P < 0.05; ** P < 0.01; *** P < 0.001 by 1-way ANOVA with Dunnett’s multiple-comparison test ( A ), 2-sided Student’s t test ( B ), or an unpaired t test ( C ). Experiments were performed in triplicate ( B and C ).

Article Snippet: In the experiment analyzing Tax , HBZ , and RGMA gene expression levels in cultured HAM-PBMCs, mRNA was purified using the Magnetic mRNA isolation kit (New England BioLabs).

Techniques: Gene Expression, Cell Culture, Control, Infection, Expressing, Western Blot, Quantitative RT-PCR, Comparison

Journal: Journal of cancer science & therapy

Article Title: Blocking PD1/PDL1 Interactions Together with MLN4924 Therapy is a Potential Strategy for Glioma Treatment.

doi: 10.4172/1948-5956.1000543

Figure Lengend Snippet: Figure 1: PDL1 expression in clinical brain tumor samples, glioma cell lines and CSF samples from the immunocompetent glioma mice model. A) Graph (left) represents PDL1 mRNA expression in brain tumors and melanomas (cBioPortal (http://www.cbioportal.org)). Note that the average PDL1 mRNA level in GBM is similar to the average PDL1 mRNA level in melanomas. The PDL1 values in GBM and low-grade gliomas which exceed the average PDL1 mRNA level in melanomas are highlighted in the red box. Kaplan-Meier plot (right) illustrates survival rates of glioma patients with high and low PDL1 expression levels; 9% versus 24% of 2 year survival for high and low PDL1 expression; the difference is significant, P=0.02 (data has been obtained from the Human Pathology Atlas). B) Immunohistochemical detection of PDL1 in the tissue microarray of normal and brain tumor samples. The images were taken at 40x magnification. C) Western blot illustrates PDL1 and Actin protein levels in control and brain tumor clinical samples. Graph represents PDL1 to Actin ratios for corresponding protein samples; 0.87 ± 0.32 (n=7) versus 0.19 ± 0.1 (n=7) for tumor and normal samples, respectively, the difference is significant, P=0.003. Two samples (marked by light grey color) of patients with hemorrhage have been excluded from the average. D) Western blots illustrate PDL1 protein levels in established and PDGx glioma cell lines (left) and in the extracellular media collected from these cell lines (right). The protein content of the collected media was concentrated six folds before an analysis (see method). The graph (right) provides PDL1 concentrations in CSF samples from control and tumor-bearing mice (see method). The immunocompetent glioma mice model with GL261 cells was utilized for this experiment. E) Images illustrate interactions of tumor neuro spheres from XD456 (a) and from two parental U251-IDH1-R132H cell lines with different PDL1 expression levels (b) with primary T-cells. Note that tumor neuro spheres with high PDL1 levels keep their integrity after 48 hours of interaction with primary T-cells (illustrated in the insert). The graph represents the average percent of neuro spheres after 48 hours of interaction with T-cells, 89 ± 7% (n=4) and 14 ± 5% (n=4) for cell lines with high and low PDL1 levels, respectively. The difference is significant, P=0.0002. Primary T-cells were loaded with calcein, AM (green) before experiments for visualization.

Article Snippet: The PDL1 level in CSF samples was analyzed by using the Mouse PDL1 ELISA Kit (Boster, Pleasanton, CA, USA).

Techniques: Expressing, Immunohistochemical staining, Microarray, Western Blot, Control

Journal: Journal of cancer science & therapy

Article Title: Blocking PD1/PDL1 Interactions Together with MLN4924 Therapy is a Potential Strategy for Glioma Treatment.

doi: 10.4172/1948-5956.1000543

Figure Lengend Snippet: Figure 2: HIF1A accumulation evokes PDL1 up-regulation in glioma cell lines. A) HIF1A accumulations induced by CoCl2 treatment of U251 and U87 cell lines evoke PDL1 up-regulation in protein and mRNA levels. Western blot illustrates HIF1A and PDL1 levels in nuclear and cytoplasmic fractions, respectively, in control and after treatment with CoCl2 (85uM). Lamin A/C and alpha Tubulin were utilized to verify nuclear and cytoplasmic fractions, respectively. Graphs illustrate normalized PDL1/ GAPDH mRNA ratios after cell treatment with CoCl2 (85uM) at different time points. In each experiment, data has been normalized to the corresponding ratios in untreated cells, results are presented as mean ± S.D. B) Transcription initiation complexes encoded by pAC154-dual-dCas9VP160 plasmids, guided by sgRNAs to the HIF1A binding domains in the first intron of the PDL1 gene, evoke PDL1 overexpression. Western blot illustrates PDL1 and Actin protein levels in U251 cells after transfection with plasmids encoding control (scrambled sequence) sgRNA or HIF1A sgRNAs. Note that transcription initiation complexes guided by sgRNAs to the HIF1A binding domains (A), (B), and (A) with (B) simultaneously increased PDL1 expression by 1.5, 3.9 and 1.8 folds, respectively, compared to the control (PDL1 expression in the presence of transcription initiation complexes guided by scrambled sgRNA). In each experiment, PDL1 expression was normalized to the Actin expression.

Article Snippet: The PDL1 level in CSF samples was analyzed by using the Mouse PDL1 ELISA Kit (Boster, Pleasanton, CA, USA).

Techniques: Western Blot, Control, Binding Assay, Over Expression, Transfection, Sequencing, Expressing

Journal: Journal of cancer science & therapy

Article Title: Blocking PD1/PDL1 Interactions Together with MLN4924 Therapy is a Potential Strategy for Glioma Treatment.

doi: 10.4172/1948-5956.1000543

Figure Lengend Snippet: Figure 3: MLN4924 treatment induces up-regulation of HIF1A and PDL1 in glioma cell lines. A) The inhibitory dose- response curves for MLN4924 in established, PDGx and PDGX-stem glioma cell lines. The IC50s are 0.3 ± 0.2 uM (n=4), 2.7 ± 1 uM (n=6), 3 ± 2 uM (n=3), 3 ± 1 uM (n=4), 2.9 ± 0.5 uM (n=4), 0.8 ± 0.2 uM (n =4), 0.2 ± 0.1uM (n=4) for LN221, U251, U87, XD456, JX10, XD456-stem, X14P-stem cell lines, respectively, after treatment with MLN4924 for 5 days. B) Western blots illustrate HIF1A and PDL1 protein levels in nuclear and cytoplasmic fractions in the control and after treatment with MLN4924 (1 uM, 5 days). LaminA/C and alpha Tubulin were utilized to verify nuclear and cytoplasmic fractions, respectively. C) The graph illustrates normalized PDL1/18S mRNA ratios after treatment with MLN4924 (1uM, 5 days) for different cell lines. Note the significant enhancement of the PDL1/18 mRNA ratio for all cell lines after MLN4924 treatment: 8 ± 3, 25 ± 5, 5 ± 1, 8 ± 3, 4.5 ± fold increase compared to the corresponding control values for U251, Ln229, U87, XD456, JX6 cell lines, respectively, P<0.05, n=3.

Article Snippet: The PDL1 level in CSF samples was analyzed by using the Mouse PDL1 ELISA Kit (Boster, Pleasanton, CA, USA).

Techniques: Western Blot, Control

Journal: Journal of cancer science & therapy

Article Title: Blocking PD1/PDL1 Interactions Together with MLN4924 Therapy is a Potential Strategy for Glioma Treatment.

doi: 10.4172/1948-5956.1000543

Figure Lengend Snippet: Figure 4: Glioma cells treated with MLN4924 decrease T-cell proliferation via utilization of PD1/PDL1 signaling pathway. A) The graph illustrates an inhibition of T-cell proliferation after T-cell encounter with U251 cells (1); with U251 cells in the presence of an inhibitor of PD1/PDL1 interaction (2); with U251 cells treated with MLN4924 (1uM, for 4 days) (3); with U251 cells treated with MLN4924 (1uM, for 4 days) and in the presence of an inhibitor of PD1/PDL1 interaction (4). After MLN4924 treatment, glioma cells were washed and placed in the media with/and without an inhibitor of PD1/PDL1 interaction (4 uM). Note, that glioma cells treated with MLN4924 induce a stronger decrease of T-cell proliferation compared to untreated cells (55 ± 8% (n=4) versus 30 ± 8% (n=4), respectively, the difference is significant with P=0.0005). The reduction of T-cell proliferation induced by glioma cells treated with MLN4924 is inhibited in the presence of an inhibitor of PD1/PDL1 interaction (P=0.0009, n=4). B) The graph illustrates an inhibition of T-cell proliferation after T-cell encounter with XD456 cells (1); with UXD456 cells in the presence of an inhibitor of PD1/PDL1 interaction (2); with XD456 cells treated with MLN4924 (1uM, for 4 days) (3); with XD456 cells treated with MLN4924 (1uM, for 4 days) and in the presence of an inhibitor of PD1/PDL1 interaction (4). After MLN4924 treatment, glioma cells were washed and placed in media with/and without an inhibitor of PD1/PDL1 interaction. Note, that glioma cells treated with MLN4924 induce a stronger decrease of T-cell proliferation compared to untreated cells (74 ± 8% (n=4) versus 41 ± 7% (n=4), respectively, the difference is significant with P=0.0003). The reduction of T-cell proliferation induced by glioma cells treated with MLN4924 is inhibited in the presence of an inhibitor of PD1/PDL1 interaction (P=0.007, n=4).

Article Snippet: The PDL1 level in CSF samples was analyzed by using the Mouse PDL1 ELISA Kit (Boster, Pleasanton, CA, USA).

Techniques: Inhibition

Journal: Cancers

Article Title: Cancer-Associated Fibroblasts Promote Tumor Aggressiveness in Head and Neck Cancer through Chemokine Ligand 11 and C-C Motif Chemokine Receptor 3 Signaling Circuit

doi: 10.3390/cancers14133141

Figure Lengend Snippet: Characterization of CAFs and NFs obtained from clinical surgical tissues from patients with head and neck cancer (HNC) ( a ) Morphological comparisons between CAFs and NFs from a representative HNC case showed that CAFs (right panel) consisted of more cytoplasmic protrusions than NFs (left panel). Photographs were captured at 40× magnification. ( b ) Quantitative PCR (left panel) of the culture medium showed a significantly higher expression of vimentin and α-SMA in CAFs than in NFs. Western blot analysis (right panel) also demonstrated that levels of vimentin and α-SMA were significantly higher in CAFs than in NFs. ( c ) Flow cytometric analysis of the cell surface markers, CD10 and GPR77, showed a marked increase in their expression in CAFs compared to NFs. ( d ) A heat map of the gene microarray of NFs and CAFs showed that there were several differences in the expression profile of the secreted genes. The arrow indicates a marked discrepancy of the relative mRNA levels of CCL11 in CAF compared with NF. ( e ) The RT-PCR (left panel) and ELISA (right panel) analysis showed an increased expression of CCL11 in CAFs compared with that in NFs. ( f ) Western blot analysis showed that the protein level of CCL11 was higher in CAFs than in NFs in cell lysates. ( g ) Western blot analysis showed a higher CCL11 expression in CAF-CM compared to NF-CM. The asterisk indicated a significant difference (*: p < 0.05; **: p < 0.01) between experimental and control groups.

Article Snippet: CAFs and NFs supernatant CCL11concentrations were measured using the CCL11 (human) ELISA kit (R&D system) following the manufacturer’s protocol.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Western Blot, Microarray, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Control

Journal: Cancers

Article Title: Cancer-Associated Fibroblasts Promote Tumor Aggressiveness in Head and Neck Cancer through Chemokine Ligand 11 and C-C Motif Chemokine Receptor 3 Signaling Circuit

doi: 10.3390/cancers14133141

Figure Lengend Snippet: CCL11 produced by CAFs causes increased migration and invasion, and the EMT of HNC cells. ( a ) Comparative analysis of the migration and invasion of HNC cells associated with CCL11. Four test groups were classified for comparative analysis of migration and invasive abilities. FaDu and NPC204 cells cultured with medium containing CAF-induced CCL11 presented greater abilities of migration and invasion, with a statistically significant difference, than three other groups: NF, NF with CCL11, and CAFs treated with CCL11 antibody. The asterisk indicates a significant difference (*: p < 0.05; **: p < 0.01) ( b ) Comparative photographs of the infiltrating behavior of FaDu and NPC204 cells in an organotypic culture in four groups seeded onto a mixture layer containing NFs or CAFs with CCL11 or CCL11 antibody. The arrow(s) indicate infiltration buds from the HNC cells seeded above. ( c ) Representative blots of the EMT-associated markers in FaDu and NPC204 cells, as observed upon Western blotting analysis in five groups, showed that treatment with CAF-conditioned medium or the application of rCCL11 decreased the expression of epithelial-type markers (E-cadherin), and increased the expression of mesenchymal-type markers (fibronectin) and EMT regulators (Snail and Twist). In addition, increased expression of invasion-related MMP2 and MMP9 was also seen in those two groups, compared with other groups. The asterisk indicates a significant difference ( p < 0.05) between experimental and control groups. Results are expressed as mean ± SD.

Article Snippet: CAFs and NFs supernatant CCL11concentrations were measured using the CCL11 (human) ELISA kit (R&D system) following the manufacturer’s protocol.

Techniques: Produced, Migration, Cell Culture, Western Blot, Expressing, Control

Journal: Cancers

Article Title: Cancer-Associated Fibroblasts Promote Tumor Aggressiveness in Head and Neck Cancer through Chemokine Ligand 11 and C-C Motif Chemokine Receptor 3 Signaling Circuit

doi: 10.3390/cancers14133141

Figure Lengend Snippet: Comparative analysis of induction of CSC properties and drug resistance in HNC cells associated with CCL11. ( a ) Four groups were classified for comparative analysis of the ability of sphere formation. Increased ability of sphere formation in two test groups of HNC cells exposed to a CAF medium and the group with treatment of rCCL11 was noted. ( b ) Flow cytometric analysis showed a significant increase in CD44 and CD44/CD24, as well as in CD133 in HNC cells exposed to rCCL11, compared to control HNC cells ( p < 0.05). ( c ) Flow cytometric analysis showed a marked increase in ALDH-1 activity in HNC cells exposed to rCCL11 compared to control HNC cells. ( d ) Western blot analysis showed that CSC-representative markers, Oct-4, Nanog, and Sox-2, were also overexpressed in addition to the increased expression of two important drug resistance genes, ABCG-2 and MDR-1 , in HNC cells exposed to rCCL11. ( e ) Treatment with Cisplatin at 24 h showed a significant increase in chemoresistance in both FaDu and NPC204 cells exposed to rCCL11 compared with control HNC cells. The asterisk indicates a significant difference (*: p < 0.05; **: p < 0.01) between experimental and control groups.

Article Snippet: CAFs and NFs supernatant CCL11concentrations were measured using the CCL11 (human) ELISA kit (R&D system) following the manufacturer’s protocol.

Techniques: Control, Activity Assay, Western Blot, Expressing

Journal: Cancers

Article Title: Cancer-Associated Fibroblasts Promote Tumor Aggressiveness in Head and Neck Cancer through Chemokine Ligand 11 and C-C Motif Chemokine Receptor 3 Signaling Circuit

doi: 10.3390/cancers14133141

Figure Lengend Snippet: CCL11 and CCR3 expression with associated signal pathway in HNC cell lines and their correlation to clinical outcomes in 104 HNC patients. ( a ) Confocal microscopic images showed CCL11 (green) localized to both cell and nuclear membranes, while CCR3 (red) localized only to the cell membrane in FaDu cells; CCL11 and CCR3 co-localized at the cell membrane (yellow). In NPC204 cells, CCL11 (green) and CCR3 (red) were found to co-localize at protrusions polarized to the cells (yellow). ( b ) Using the crisp technique, higher expression of CCR3, MMP2, and MMP3 was found in over-expressed CCL11 cloned-FaDu and NPC204 cells. Cloned CCL11-overexpressed cells were abolished by adding eotaxin siRNA or CCR3 antibody, which reversed the expression of CCR3 and invasion-related MMP2 and MMP9. ( c ) Higher phosphorylation levels of p38 MAPK and ERK were found in cloned CCL11-overexpressed FaDu and NPC204 cells and were reversed by treatment of the p38 MAPK inhibitor (SB203580) and ERK inhibitor (FR180204), respectively. The phosphorylation level of JNK was kept in low condition before and after treatment of the JNK inhibitor (SP600125). ( d ) Photomicrographs of immunohistochemical staining from tissue microarray showing CCL11 and CCR3 expression in three different representative groups of HNC patients (magnification, ×200). ( e ) Kaplan–Meier survival analysis of patients showed that overexpression of CCL11 and CCR3 were statistically associated with poor overall survival).

Article Snippet: CAFs and NFs supernatant CCL11concentrations were measured using the CCL11 (human) ELISA kit (R&D system) following the manufacturer’s protocol.

Techniques: Expressing, Membrane, Clone Assay, Phospho-proteomics, Immunohistochemical staining, Staining, Microarray, Over Expression

Journal: Cancers

Article Title: Cancer-Associated Fibroblasts Promote Tumor Aggressiveness in Head and Neck Cancer through Chemokine Ligand 11 and C-C Motif Chemokine Receptor 3 Signaling Circuit

doi: 10.3390/cancers14133141

Figure Lengend Snippet: The diagrammatic illustration demonstrates the major mechanism that CAFs secreting CCL11 promotes HNC cell migration and invasion and induces properties of drug resistance and stemness, shown as follows. CAFs in TME secret CCL11 binding to the CCR3 receptors on HNC cells via the paracrine effect. The signal induces overexpression of transcriptional factors, such as Snail and Twist, which regulate EMT and are also responsible for self-induction of CCL11 in an autocrine fashion. As a result, CCL11, via paracrine or autocrine signaling when targeting CCR3 receptors, play a functional role in the induction of EMT and CSC properties, for further tumor progression.

Article Snippet: CAFs and NFs supernatant CCL11concentrations were measured using the CCL11 (human) ELISA kit (R&D system) following the manufacturer’s protocol.

Techniques: Migration, Binding Assay, Over Expression, Functional Assay

Journal: Frontiers in Microbiology

Article Title: Novel Insights Into Cellular Changes in HPV8-E7 Positive Keratinocytes: A Transcriptomic and Proteomic Analysis

doi: 10.3389/fmicb.2021.672201

Figure Lengend Snippet: Transcriptional changes in HPV8-E7 positive keratinocytes. (A) Venn Diagram comparison of two independent cDNA microarray analyses carried out with PHAK expressing HPV8-E7 in the Akgül lab in Cologne and with PHFK expressing HPV8-E7 in the Alonso lab at the DKFZ in Heidelberg. (B) Gene IDs of 4 genes differentially expressed in both array studies. (C,D) GDF15 mRNA expression in isogenic PM1 keratinocytes cultured either in KGM-Gold or RM+, respectively. (E) GDF15 ELISA performed with cell culture supernatants of HPV8-E7 expressing keratinocytes grown in KGM-Gold medium. (F,G) ATF3 mRNA expression in isogenic PM1 keratinocytes cultured either in KGM-Gold or RM+, respectively. (H) GADD34 mRNA expression in PM1 keratinocytes cultured in KGM-Gold. RTqPCRs were normalized to HPRT1 mRNA levels ( n = 3 independent experiments performed in duplicate). Error bars represent standard deviations. Asterisks shown in the figures indicate significant differences of experimental groups in comparison with the corresponding control conditions (* p < 0.05; ** p < 0.01; *** p < 0.001).

Article Snippet: The Human GDF15 Quantikine ELISA Kit (RnD systems) was used to measure GDF15 in cell culture supernatants.

Techniques: Comparison, Microarray, Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay, Control

Journal: Translational oncology

Article Title: NF1-RAC1 axis regulates migration of the melanocytic lineage.

doi: 10.1016/j.tranon.2020.100858

Figure Lengend Snippet: Fig. 1. Loss of NF1 reduces RAC1-driven melanoblast migration. A. Scratch-like migration assay representing the percentage of cell coverage after 6 h, 9 h and 12 h using either WT or NF1+/−melanoblasts (MB) in the presence of a RAC1 activator (CN04). B. RAC1 activity was measured by G-lisa in WT and NF1+/−melanoblasts (MB). C. Scratch-like migration assay after 3 h, 6 h, 9 h and 12 h in NF1+/−melanoblasts 48 h-post transfection with either a scramble siRNA (SCR) or with an NF1-specific siRNA (siNF1). D. Expression status of NF1 and expression of phosphorylated and non-phosphorylated ERK and AKT in NF1+/−melanoblasts by western blot. α-actinin was used as a loading control. GTP-RAC1 pulldown and total lysates were blotted with α-RAC1 antibody. E. Scratch-like migration assay representing the percentage of cell coverage after 9 h and 12 h in NF1+/−melanoblasts 48 h-post transfection with either a scramble siRNA (SCR) or with an NF1-specific siRNA (siNF1) and in the presence or absence of a RAC1 activator (CN04). *: SCR vs. siNF1, #: -CN04 vs. +CN04. F. GTP-RAC1 pulldown and total lysates were blotted with α-RAC1 antibody in the presence or absence of a RAC1 activator (CN04). **P < 0.01, *P < 0.05, ns: not significant (unpaired Student's t-test). All error bars represent the SEM of at least three independent experiments.

Article Snippet: The amount of activated RAC1 was determined by western blot using a RAC1 specific antibody (Cytoskeleton inc. Cat. # BK035).

Techniques: Migration, Activity Assay, Transfection, Expressing, Western Blot, Control

Journal: Translational oncology

Article Title: NF1-RAC1 axis regulates migration of the melanocytic lineage.

doi: 10.1016/j.tranon.2020.100858

Figure Lengend Snippet: Fig. 2. Loss of NF1 increases melanoma migration and is associated with increased PREX1 expression. A. NF1 mRNA expression under NF1 silencing with two siRNAs (NF1.6 and NF1.11) in SK-mel-23, Mel501, and SK-mel-103 melanoma cell lines. B. PREX1 mRNA expression under NF1 silencing with two siRNAs in SK-mel-23, Mel501, and SK- mel-103 cell lines. C. Scratch-like migration assay representing the percentage of cell coverage after 6 h, 12 h and 24 h under NF1 silencing in SK-mel-23, Mel501, and SK-mel- 103 cell lines. D. Scratch-like migration assay as in C, after additional transfection with siRNA control (scramble) or with PREX1 siRNA (siPREX1). E. Scratch-like migration assay as in C. in the absence (control) or presence (RAC1 inhibitor) of a RAC1 inhibitor. ***P < 0.001, **P < 0.01, *P < 0.05 (unpaired Student's t-test). All error bars rep- resent the SEM of at least three independent experiments.

Article Snippet: The amount of activated RAC1 was determined by western blot using a RAC1 specific antibody (Cytoskeleton inc. Cat. # BK035).

Techniques: Migration, Expressing, Transfection, Control

Journal: Translational oncology

Article Title: NF1-RAC1 axis regulates migration of the melanocytic lineage.

doi: 10.1016/j.tranon.2020.100858

Figure Lengend Snippet: Fig. 4. PREX is upregulated in low NF1 expressing melanoma metastases. A. Representative microphotographs of Tissue Microarray (TMA) containing primary and metastatic melanoma samples analysed by immunohistochemistry using a specific antibody against NF1, RAC1 and PREX1. Bar, 100 μm. B. Scoring of the immunohistochemistry staining was performed according to our previously described protocol [24]. Duplicates of valid punch samples are represented for each condition. Significance was tested using two-tailed t-test with *P < 0.05 and ns: not significant.

Article Snippet: The amount of activated RAC1 was determined by western blot using a RAC1 specific antibody (Cytoskeleton inc. Cat. # BK035).

Techniques: Expressing, Microarray, Immunohistochemistry, Staining, Two Tailed Test