Journal: Nature Communications

Article Title: TRIB2 confers resistance to anti-cancer therapy by activating the serine/threonine protein kinase AKT

doi: 10.1038/ncomms14687

Figure Lengend Snippet: ( a ) Matched isogenic TRIB2 cell line FACS analysis following the knockdown of FOXO3a and subsequent exposure to BEZ235, BAY236 (copanlisib) or BAY439 (BAY1082439) ( n =6). P values are indicated for each comparison by two-way ANOVA and data represent the mean±s.d. ( b ) Quantitative real time PCR (qRT-PCR) analysis of FOXO3a-dependent gene expression after FOXO3a knockdown and isogenic cell line treatment for 24 h with PI3K inhibitors ( n =6), (* P ≤0.05, ** P ≤0.01 and were analysed by two-way ANOVA). Data represent the mean±s.d. ( c ) Matched isogenic TRIB2 cell line FACS analysis after 72 h exposure to various chemotherapeutics ( n =6). P values are indicated for each comparison and data represent the mean±s.d. ( d ) I Representative immunoblot analysis showing 50 μg (MDM2), 100 μg (MDM2-Ser166), 50 μg (p53) total protein lysate per lane separated by 6–10% SDS–PAGE. ( e ) p53-dependent gene expression was evaluated following TRIB2 isogenic cell line treatment with each indicated chemotherapeutic agent for 24 h. P values are shown for comparison by 2-way ANOVA (* P ≤0.05, ** P ≤0.001, *** P ≤0.0001) and data shown indicates mean±s.d. ( f ) Representative immunoblot analysis showing TRIB2 protein expression (100 μg total protein loaded per lane) following exposure to each indicated PI3K inhibitor. ( g ) FACS analysis of 293T cells after transfection of each TRIB2 construct and subsequent exposure to BEZ235, BAY236 (BAY 80-6946) or BAY439 (BAY1082439) ( n =6) for 72 h. P values are indicated for each comparison by two-way ANOVA and data represent the mean±s.d. P values are shown for each comparison where no significant difference was noted. Data was analysed by 2-way ANOVA * P ≤0.05, ** P ≤0.001. ( h ) Representative immunoblot analysis showing 50 μg (GFP), 100 μg (AKT-Ser473), 100 μg (FOXO3a-Ser253), 100 μg (MDM2-Ser166) protein expression 48 h post-transfection of the indicated GFP tagged TRIB2 plasmid constructs.

Article Snippet: Dacarbazine (Sigma, PT), gemcitabine hydrochloride (Eli Lilly #VL7502), AKT inhibitor VIII (Calbiochem, US), BEZ235 (Novartis, US), BAY236, (BAY 80-6946) BAY439 (BAY1082439), BAY1001931 (a gift from Bayer AG, Germany), rapamycin (SIGMA, PT), actinomycin D (Sigma, PT), and cyclohexamide (Sigma, PT) and MG132 (Sigma, PT) were used at concentrations described in the text.

Techniques: Knockdown, Comparison, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Gene Expression, Western Blot, SDS Page, Expressing, Transfection, Construct, Plasmid Preparation

Journal:

Article Title: Ferrihydrite-Dependent Growth of Sulfurospirillum deleyianum through Electron Transfer via Sulfur Cycling

doi: 10.1128/AEM.70.10.5744-5749.2004

Figure Lengend Snippet: Ferrihydrite reduction in growth experiments with S. deleyianum supplied with acetate, formate, ferrihydrite, and 0.01% inoculum. One of the following was added: cysteine (2 mM), cysteine (2 mM) plus thiosulfate (0.05 mM), or cystine (0.5 mg ml−1). The inoculum had been grown with fumarate as the substrate and cysteine as the sulfur source. Means of two determinations are shown for duplicate experiments.

Article Snippet: S. deleyianum (DSM 6946 T ) was obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany.

Techniques:

Journal:

Article Title: Ferrihydrite-Dependent Growth of Sulfurospirillum deleyianum through Electron Transfer via Sulfur Cycling

doi: 10.1128/AEM.70.10.5744-5749.2004

Figure Lengend Snippet: Growth experiments with S. deleyianum supplied with acetate, formate, cysteine, and 0.01% inoculum. (A) Growth with fumarate (20 mM) or thiosulfate (2 mM) as electron acceptor in the absence or presence of tungstate (5 mM). Data from representative cultures are shown. (B) Ferrihydrite reduction in growth experiments supplied with ferrihydrite and thiosulfate. Except for the control experiment, different concentrations of tungstate were added. Means of duplicate determinations are shown for representative cultures.

Article Snippet: S. deleyianum (DSM 6946 T ) was obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany.

Techniques:

Journal:

Article Title: Ferrihydrite-Dependent Growth of Sulfurospirillum deleyianum through Electron Transfer via Sulfur Cycling

doi: 10.1128/AEM.70.10.5744-5749.2004

Figure Lengend Snippet: Ferric iron reduction in growth experiments with S. deleyianum supplied with acetate, formate, cysteine, and 0.01% inoculum. Either ferrihydrite or ferric citrate was added as the electron acceptor; cultures with ferrihydrite were also supplied with thiosulfate (0.05 mM). Means of two determinations are shown for duplicate experiments.

Article Snippet: S. deleyianum (DSM 6946 T ) was obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany.

Techniques:

Journal:

Article Title: Ferrihydrite-Dependent Growth of Sulfurospirillum deleyianum through Electron Transfer via Sulfur Cycling

doi: 10.1128/AEM.70.10.5744-5749.2004

Figure Lengend Snippet: Tubes from agar dilutions inoculated with S. deleyianum after 3 weeks of incubation. The medium was solidified with 1% agar and contained acetate, formate, biologically produced ferrihydrite, cysteine, and thiosulfate (0.05 mM). Arrows point to white zones, which indicate ferrihydrite reduction. Note, white stripes (*) originate from flash light reflections.

Article Snippet: S. deleyianum (DSM 6946 T ) was obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany.

Techniques: Incubation, Produced

Journal: Frontiers in Oncology

Article Title: Tspan9 Induces EMT and Promotes Osteosarcoma Metastasis via Activating FAK-Ras-ERK1/2 Pathway

doi: 10.3389/fonc.2022.774988

Figure Lengend Snippet: The siRNA and shRNA sequences used in this study.

Article Snippet: A FLAG-tagged PGMLV-6946 Tspan9 expression vector was purchased from GenePharma Technology Co., Ltd (Shanghai, China).

Techniques: shRNA, Negative Control

Journal: Frontiers in Oncology

Article Title: Tspan9 Induces EMT and Promotes Osteosarcoma Metastasis via Activating FAK-Ras-ERK1/2 Pathway

doi: 10.3389/fonc.2022.774988

Figure Lengend Snippet: Human OS cells and tumor tissue samples exhibit Tspan9 upregulation. (A–C) Tspan9 mRNA levels were significantly elevated in OS tumor tissues and cell lines in the GSE12865, GSE33383, and GSE42352 datasets relative to levels in normal MSCs and OBs. (D) Relative Tspan9 mRNA levels were markedly increased in HOS cells relative to control hFOB1.19 cells, whereas no changes were evident in U2OS or Mg63 cells as assessed via qRT-PCR. GAPDH served as a normalization control. (E) Western blotting results revealed that Tspan9 protein expression in HOS but not U2OS and Mg63 was significantly higher compared to hFOB1.19 cells. β-actin was used as a loading control and for normalization. Data are means ± SD from two independent experiments. (F) ROC curves and AUC values were determined using the GSE33383 and GSE42352 datasets. * P < 0.05; ** P < 0.01; *** P < 0.001; Student’s t-test. Tspan9, Tetraspanin-9; MSC, mesenchymal stem cell; OB: osteoblast; OS: osteosarcoma; GEO: Gene Expression Omnibus; qRT-PCR, quantitative reverse -transcription polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; SD, standard deviation; ROC, receiver operating characteristic; AUC, area under the curve.

Article Snippet: A FLAG-tagged PGMLV-6946 Tspan9 expression vector was purchased from GenePharma Technology Co., Ltd (Shanghai, China).

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Reverse Transcription Polymerase Chain Reaction, Standard Deviation

Journal: Frontiers in Oncology

Article Title: Tspan9 Induces EMT and Promotes Osteosarcoma Metastasis via Activating FAK-Ras-ERK1/2 Pathway

doi: 10.3389/fonc.2022.774988

Figure Lengend Snippet: Tspan9 knockdown suppresses OS cell proliferation. (A) Tspan9 mRNA levels in siTspan9 cells (siTspan9#1, #2, and #3) were significantly lower than those in siNC cells, as measured via qRT-PCR. (B) The viability of HOS cells in the siNC and siTspan9 HOS cells was assessed via CCK-8 assay. (C) The impact of Tspan9 knockdown on HOS cell proliferation was measured via colony formation assay. Statistical results of colony formation numbers normalized to shNC were presented. * P < 0.05; ** P < 0.01; *** P < 0.001; Student’s t-test. NC, negative control; si, small interfering RNA.

Article Snippet: A FLAG-tagged PGMLV-6946 Tspan9 expression vector was purchased from GenePharma Technology Co., Ltd (Shanghai, China).

Techniques: Quantitative RT-PCR, CCK-8 Assay, Colony Assay, Negative Control, Small Interfering RNA

Journal: Frontiers in Oncology

Article Title: Tspan9 Induces EMT and Promotes Osteosarcoma Metastasis via Activating FAK-Ras-ERK1/2 Pathway

doi: 10.3389/fonc.2022.774988

Figure Lengend Snippet: RNA-seq-mediated identification of the biological roles of Tspan9 in OS cells. (A) Heatmaps demonstrating DEGs identified via RNA-seq in HOS cells in which shTspan9 or shNC were stably expressed. (B) DEGs (n=211) are represented in a volcano plot, including 96 upregulated DEGs (red) and 115 downregulated DEGs (green), with DEGs having been identified using the following criteria: adjusted log fold-change ≥ 1 and P ≤ 0.05. (C, D) GO analyses of DEGs identified following Tspan9 knockdown were conducted, with top enriched biological processes, molecular functions, and cellular components being shown in a bubble chart in which darker coloration is indicative of more significant enrichment. (E, F) KEGG pathway enrichment analyses of identified DEGs were conducted, with the results being shown in a bubble chart in which bubble size is proportional to the number of DEGs in a given pathway, and the bubble color is indicative of P-value significance (red = significant, blue = non-significant). DEGs, differentially expressed genes; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes.

Article Snippet: A FLAG-tagged PGMLV-6946 Tspan9 expression vector was purchased from GenePharma Technology Co., Ltd (Shanghai, China).

Techniques: RNA Sequencing Assay, Stable Transfection

Journal: Frontiers in Oncology

Article Title: Tspan9 Induces EMT and Promotes Osteosarcoma Metastasis via Activating FAK-Ras-ERK1/2 Pathway

doi: 10.3389/fonc.2022.774988

Figure Lengend Snippet: Tspan9 promotes in vitro OS cell migration and invasion. (A) GFP expression was indicative of stable lentiviral transduction of HOS cells with shTspan9 (shTspan9#1 or shTspan9#2) or shNC constructs at near 100% efficiency (fluorescent microscopy). Tspan9 knockdown efficiency was also confirmed via qRT-PCR (lower left panel) and Western blotting (lower right panel). (B) OS cell migration was assessed in a wound-healing assay using cells stably expressing shTspan9 or shNC. (C) The impact of Tspan9 knockdown on OS cell migration and invasion was assessed via a Transwell approach. (D) GFP expression was indicative of successful Tspan9 overexpression, as confirmed via qRT-PCR and Western blotting relative to Mock control. Wound-healing (E) and Transwell assays (F, G) were conducted to assess the impact of Tspan9 on the migratory and invasive activity of OS cells. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; Student’s t-test. GFP, green fluorescent protein; sh, short hairpin RNA.

Article Snippet: A FLAG-tagged PGMLV-6946 Tspan9 expression vector was purchased from GenePharma Technology Co., Ltd (Shanghai, China).

Techniques: In Vitro, Migration, Expressing, Transduction, Construct, Microscopy, Quantitative RT-PCR, Western Blot, Wound Healing Assay, Stable Transfection, Over Expression, Activity Assay, shRNA

Journal: Frontiers in Oncology

Article Title: Tspan9 Induces EMT and Promotes Osteosarcoma Metastasis via Activating FAK-Ras-ERK1/2 Pathway

doi: 10.3389/fonc.2022.774988

Figure Lengend Snippet: Tspan9 regulates EMT, and knocking down of it impairs in vivo OS cell metastasis. (A) EMT marker protein levels and associated transcription factor expression were assessed in HOS and U2OS cells stably expressing shTspan9/OE-Tspan9 or control constructs. (B) Quantification of the Western blotting results presented in (A) . (C, D) HOS cells stably expressing shTspan9 or control constructs were injected via the lateral tail vein into nude mice to establish a model of pulmonary metastasis (n=5/group). Left: representative lung images; Right: representative H&E staining results (1x and 5x). Pulmonary nodules are indicated by red arrows, while normal alveolar tissue is indicated by blue arrows. (E) Numbers of metastatic pulmonary nodules in the indicated groups. (F) Lung weights on day 28 post-HOS tumor cell injection. (G) Murine body weight was assessed every 4 days. * P < 0.05; ** P < 0.01.

Article Snippet: A FLAG-tagged PGMLV-6946 Tspan9 expression vector was purchased from GenePharma Technology Co., Ltd (Shanghai, China).

Techniques: In Vivo, Marker, Expressing, Stable Transfection, Construct, Western Blot, Injection, Staining

Journal: Frontiers in Oncology

Article Title: Tspan9 Induces EMT and Promotes Osteosarcoma Metastasis via Activating FAK-Ras-ERK1/2 Pathway

doi: 10.3389/fonc.2022.774988

Figure Lengend Snippet: Tspan9-β1 interactions promote FAK-Src-Ras-ERK1/2 pathway signaling and OS metastasis. (A) Interactions between Tspan9 and integrin β1 were detected in a co-IP assay. (B) FAK-Ras-ERK1/2 pathway proteins (FAK Y397 , total-FAK, Ras, pERK1/2, and total-ERK1/2) were analyzed via western blotting, with β-actin as a loading control. (C) Quantification of the Western blotting results presented in (B) . (D) Western blotting was used to assess Ras downstream signaling in OE-Tspan9 U2OS cells following Salirasib treatment (50μM). (E) Following treatment with Salirasib (50μM), OE-Tspan9 cells were analyzed in migration and invasion assays, with representative cells being shown. (F) Schematic presentation of mechanism underlying Tspan9-mediated OS metastasisis. All analyzes were repeated two or three times. Data are means ± SD. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: A FLAG-tagged PGMLV-6946 Tspan9 expression vector was purchased from GenePharma Technology Co., Ltd (Shanghai, China).

Techniques: Co-Immunoprecipitation Assay, Western Blot, Migration

Journal: Blood

Article Title: Array genotyping of transfusion-relevant blood cell antigens in 6946 ancestrally diverse study participants ∗ †

doi: 10.1182/blood.2025028902

Figure Lengend Snippet: Study design. (A) Samples: number of DNA samples provided by the 7 blood services (NHSBT, SANQUIN, NYBC, ARCLB, CBS, FRCBS, SANBS). (B) Array content: bar plot indicating the number of probes per category in the transfusion module. HLA, HEA, HPA, and HNA. (C) Genotyping: 6946 identical DNA samples were genotyped with the UBDT_PC1 Transfusion Array at Sanquin and NYBC, with 3938 of these samples also genotyped using the UKBB_v2.2 GWAS array by NHSBT. (D) QC: Heat map gives the reason for, and number of, samples failing QC for the 3 genotyping laboratories. Venn diagrams show overlap in samples that failed Axiom BP QC, gender-vs-sex discordance (sex discordant), and evidence of contamination (contamination). (E) Venn diagram showing the overlap in samples passing QC. (F) Ancestry: (left) bar plot showing genetically inferred ancestry of samples typed successfully by Sanquin and NYBC (6679 samples). EUR, AFR, AMR, SAS, EAS, OTH are shown. Right: heat map showing concordance between the ancestry inferred from the Sanquin and NYBC genotyping results, respectively. AFR, African; AMR, Admixed American; ARCLB, Australian Red Cross Lifeblood; Axiom BP, Axiom Best Practices; CBS, Canadian Blood Services; EAS, East Asian; EUR, European; FRCBS, Finnish Red Cross Blood Service; OTH, Other; SANBS, South African National Blood Service; SANQUIN, Sanquin Blood Supply Foundation; SAS, South Asian.

Article Snippet: Identical sets of 6946 DNA samples were distributed to Sanquin and NYBC, with a subset of 3938 samples also distributed to NHSBT ( A,C).

Techniques:

Journal: Blood

Article Title: Array genotyping of transfusion-relevant blood cell antigens in 6946 ancestrally diverse study participants ∗ †

doi: 10.1182/blood.2025028902

Figure Lengend Snippet: Reproducibility of typing results between Sanquin and NYBC for the 6679 DNA samples of the unified data. (A) Genotype reproducibility for 20 681 biallelic probe-variant pairs included in the UBDT_PC1 array design. Reproducibility expressed as percentage of concordant genotype comparisons, and gnomAD MAF for each variant are displayed on the x- and y-axes, respectively. Blue hexagons and red dots on the central scatterplot represent the density of probes with reproducibility of ≥99% and individual probes with <99% concordance, respectively. Marginal histograms show probe counts on a log scale. (B) Correlation of the MAF in EUR study participants vs (non-Finnish) EUR participants from the gnomAD database for each probe-variant pair. Probes with ≥99% and <99% genotype reproducibility are shown in blue and red, respectively. Contour lines represent boundaries of statistical significance with corresponding P values calculated using the χ 2 test. (C) Genotype reproducibility for critical blood antigen types and iron homeostasis probes. Box plots show the percentage reproducibility between genotypes, split across 2 y-axes ranges to highlight high-reproducibility results (99%-100%) and broader distribution patterns (40%-99%). Data are shown for HEAs, HPAs, HNAs, and iron homeostasis variants in blue, orange, green, and red, respectively. Box plots display the median (center line), interquartile range (IQR; box), whiskers (1.5 × IQR), and outliers (black circles). Outlier variants are annotated with relevant antigen types. (D) Reproducibility between HEA types generated by the Sanquin and NYBC laboratories. The reproducibility is given as a percentage between on the y-axis for the 51 HEA types on the x-axis. Results are stratified for the 5 ancestry groups. When the bars for different ancestries are at identical values, only 1 bar is shown in the order of the legend, that is blue for EUR participants in most cases. (E) The percentage of no-type results is given on the y-axis for the 51 HEA types on the x-axis. HEA types with identical percentage of no-type results are visualized according to the principles of panel D. AFR, African; AMR, Admixed American; EAS, East Asian; EUR, European; MAF, minor allele frequency; NFE, non-Finnish European; SAS, South Asian.

Article Snippet: Identical sets of 6946 DNA samples were distributed to Sanquin and NYBC, with a subset of 3938 samples also distributed to NHSBT ( A,C).

Techniques: Variant Assay, Generated

Journal: Blood

Article Title: Array genotyping of transfusion-relevant blood cell antigens in 6946 ancestrally diverse study participants ∗ †

doi: 10.1182/blood.2025028902

Figure Lengend Snippet: Common and rare HEA types. (A) Ancestral differences in frequencies of some common HEA types, which frequently elicit alloantibody formation. Heat map with the ancestry stratified frequencies of the common MNS, Rh, FY, and JK types in the unified set of 6679 DNA samples. Heat map colors range from yellow (0%) to deep blue (100%), showing HEA-type frequencies within each ancestry group. (B) Number of HFA − samples identified in the unified set of 6679 DNA samples with those identified by Sanquin and NYBC on the x- and y-axes, respectively. True negative, false negative in Sanquin, false positive and no-type in NYBC, no-type in Sanquin, no-type in NYBC, no-type in Sanquin and NYBC, and false negative in NYBC are showing in blue, red, green, orange, brown, gray, and magenta, respectively. (C) Number of patients typed negative for 16 HFA identified in the extended unified sample set. Bar plot shows phenotype and the count of negative typing results on the x- and y-axes, respectively. Typing results concordant with clinical type, array detected and confirmed, array detected and unconfirmed, false negative array types, and no-type results are shown in purple, blue, orange, yellow, and green, respectively. (D) Concordance between clinical and array-generated results for DNA samples harboring complex Rh genotypes. A graphical representation of 8 alleles of the RHD gene, in descending order: D + ( RHD∗01 ), weak D type 1 ( RHD∗01W.1 ), weak D type 2 ( RHD∗01W.2 ), weak D type 3 ( RHD∗01W.3 ), D – ( RHD∗01N.01 ), r' S type 1 ( RHD∗03N.01 ), D pseudogene ( RHD∗08N.01 ), and DEL1 ( RHD∗01EL.01 ). Counts on the right show the number of alleles detected, confirmed by clinical type, and discordant in the extended unified sample set in black, green, and orange, respectively. AFR, African; AMR, Admixed American; EAS, East Asian; EUR, European; SAS, South Asian.

Article Snippet: Identical sets of 6946 DNA samples were distributed to Sanquin and NYBC, with a subset of 3938 samples also distributed to NHSBT ( A,C).

Techniques: Generated