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: STAR Protocols

Article Title: Preparation of mouse pancreatic tumor for single-cell RNA sequencing and analysis of the data

doi: 10.1016/j.xpro.2021.100989

Figure Lengend Snippet:

Article Snippet: Dead Cell Removal Kit , Miltenyi Biotec , , 130-090-101.

Techniques: Recombinant, Saline, Modification, Staining, Red Blood Cell Lysis, Multiplex Assay, Sequencing, Generated, Software, Sterility

Journal: STAR Protocols

Article Title: Preparation of mouse pancreatic tumor for single-cell RNA sequencing and analysis of the data

doi: 10.1016/j.xpro.2021.100989

Figure Lengend Snippet: 1 × Binding Buffer (Dead cell removal kit)

Article Snippet: Dead Cell Removal Kit , Miltenyi Biotec , , 130-090-101.

Techniques: Binding Assay, Concentration Assay, Sterility

Journal: iScience

Article Title: SPI1-KLF1/LYL1 axis regulates lineage commitment during endothelial-to-hematopoietic transition from human pluripotent stem cells

doi: 10.1016/j.isci.2024.110409

Figure Lengend Snippet: Multi-omics analysis reveals potential regulatory roles of SPI1 in EHT (A) Heatmap of 575 expressed TFs during the differentiation of Pluripotent Stem Cells (PSC) into Hematopoietic Progenitor Cells (HPC). Four expression clusters (TC1-TC4) are identified and are denoted by color and number. (B) Line graph of variable accessible chromatin elements during PSC to HPC differentiation, again with four identified clusters (C1-C4). (C) Enriched TF motifs in peaks of clusters C1-C4. (D) Heatmap showing variations in TF activity among the top 50 active transcription factors during differentiation. (E) Pearson correlation analysis of TF activity and gene expression for five key TFs. (F) Visualization of ATAC-seq footprint for motifs of the five representative TFs across different developmental stages. See also Figure S2 and .

Article Snippet: CD34 MicroBeads, human , Miltenyi Biotec , Cat# 130-046-702.

Techniques: Biomarker Discovery, Expressing, Activity Assay, Gene Expression

Journal: iScience

Article Title: SPI1-KLF1/LYL1 axis regulates lineage commitment during endothelial-to-hematopoietic transition from human pluripotent stem cells

doi: 10.1016/j.isci.2024.110409

Figure Lengend Snippet: Knockdown of SPI1 at the in vitro -generated HECs stage severely impairs HPC generation (A) Schematic illustration of the in vitro differentiation approach. SPI1 siRNA or control siRNA were introduced on D4 of EHT, with evaluations from D5 to D8. (B) Flow cytometric sorting strategy for HECs treated with siRNA. (C) qPCR results showing SPI1 expression from D5 to D8, using two distinct siRNA sequences targeting SPI1 . (D) Western blot analysis of PU.1 expression in siCtrl and siSPI1_1/2 groups from D5 to D8, with GAPDH as the internal normalization control. (E) Representative flow cytometry density plots showing CD34 + CD43 + cells among total cells in siCtrl and siSPI1_1/2 groups, from D5 to D8. (F) Quantification of suspended CD34 + CD43 + HPCs from siCtrl and siSPI1_1/2 groups, from D5 to D8. (G) Microscopy images of cells on D8 for siCtrl and siSPI1 conditions, scale bar = 100 μm. (H) Representative flow cytometry density plots of CD34 + CD43 + HPCs in suspended cells for siCtrl and siSPI1_1/2 groups on D8. Data are from n = 3 independent experiments, and statistical significance was determined using two-tailed unpaired Student’s t-test. Data are presented as means ± SEM. Significance levels: ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. See also Figure S4 , Table S1 and Table S2 .

Article Snippet: CD34 MicroBeads, human , Miltenyi Biotec , Cat# 130-046-702.

Techniques: Knockdown, In Vitro, Generated, Control, Expressing, Western Blot, Flow Cytometry, Microscopy, Two Tailed Test

Journal: iScience

Article Title: SPI1-KLF1/LYL1 axis regulates lineage commitment during endothelial-to-hematopoietic transition from human pluripotent stem cells

doi: 10.1016/j.isci.2024.110409

Figure Lengend Snippet: Knockdown of SPI1 during EHT does not alter HPC proliferation or apoptosis but affects differentiation potential (A) Flow cytometry sorting strategy for D8 suspended CD34 + CD43 + HPCs (FAM +/− ). (B and C) Representative flow cytometry density plots (B) and frequency plot (C) for cell cycle, G0/G1 (Edu − Hoechst low ), S (Edu + Hoechst low/high ) and G2/M (Edu − Hoechst high ) on D8 suspended CD34 + CD43 + HPCs (FAM +/− ) in siCtrl and siSPI1_1/2 groups. (D and E) Representative flow cytometry density plots (D) and frequency plot (E) of early apoptosis (Annexin V + DAPI − ) and late apoptosis (Annexin V + DAPI + ) on D8 suspended CD34 + CD43 + HPCs (FAM +/− ) in siCtrl and siSPI1_1/2 groups. Significance determined by two-tailed unpaired Student’s t-test. (F) Colony Forming Unit (CFU) assay of FAM − CD34 + CD43 + HPCs generated on D8 under siCtrl and siSPI1_1/2 conditions. Unless otherwise stated, data are from n = 3 independent experiments, and statistical significance was determined using two-way ANOVA. Data are presented as means ± SEM. Significance levels: ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ns, no significance. See also Figure S5 .

Article Snippet: CD34 MicroBeads, human , Miltenyi Biotec , Cat# 130-046-702.

Techniques: Knockdown, Flow Cytometry, Two Tailed Test, Colony-forming Unit Assay, Generated

Journal: iScience

Article Title: SPI1-KLF1/LYL1 axis regulates lineage commitment during endothelial-to-hematopoietic transition from human pluripotent stem cells

doi: 10.1016/j.isci.2024.110409

Figure Lengend Snippet: Transcriptional regulatory network suggests LYL1 and KLF1 as potential downstream targets of SPI1 (A) Heatmap displaying reordered normalized trans -regulation score (TRS) at the HPC stage. Identified modules from nonnegative matrix factorization (NMF) are represented in different colors. (B) Boxplots showing gene expression levels in different modules from A, comparing Endothelial Progenitor Cells (EPC) and HPC. p values, two-tailed paired Student’s t test. (C) All SPI1 Target Genes (TGs) are ordered descendingly according to their TRS, with a focus on the top 10 transcription factors (TFs) within these TGs. (D) Violin plots showing expression levels of the top 10 TFs from SPI1 TGs in both in vivo AGM hematopoiesis (left) and the in vitro system (right). (E) qPCR results showing the expression of the top 10 transcription factors from SPI1 target genes in D8 CD34 + CD43 + HPCs, excluding non-expressed genes SNAI3 and RUNX3 , in siCtrl and siSPI1_1/2 groups. Data are from n = 3 independent experiments, with significance assessed using two-tailed unpaired Student’s t test. (F) Enrichment analysis of Gene Ontology (GO) terms and KEGG signaling pathways of SPI1 ’s TGs that have p -value <0.001. Data are presented as means ± SEM. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ns, no significance.

Article Snippet: CD34 MicroBeads, human , Miltenyi Biotec , Cat# 130-046-702.

Techniques: Gene Expression, Two Tailed Test, Expressing, In Vivo, In Vitro, Protein-Protein interactions

Journal: iScience

Article Title: SPI1-KLF1/LYL1 axis regulates lineage commitment during endothelial-to-hematopoietic transition from human pluripotent stem cells

doi: 10.1016/j.isci.2024.110409

Figure Lengend Snippet: Overexpression of LYL1 and KLF1 can rescue SPI1 knockdown phenotypes (A) Co-transfection strategy of SPI1 siRNA with KLF1 / LYL1 overexpression during EHT to assess D8 HPC differentiation potential. (B) Western blot analysis of KLF1/LYL1 from D6 differentiated cells in the siCtrl+OE-Ctrl, siSPI1, and siSPI1+OE-KLF1/LYL1 groups, normalized to GAPDH. (C and D) Flow cytometry analysis of suspended CD34 + CD43 + HPCs on D8: (C) Representative density plot and (D) quantification in siCtrl+OE-Ctrl, siSPI1, siSPI1+OE-KLF1 and siSPI1+OE-LYL1 groups. n = 3 independent experiments. (E) Colony-forming units (CFU) assay on D8 suspended CD34 + CD43 + HPCs across siCtrl+OE-Ctrl, siSPI1, siSPI1+OE-KLF1 and siSPI1+OE-LYL1 groups, with significance assessed using two-way ANOVA. (F) Schematic description of lymphoid differentiation. (G) Representative flow cytometry density plots of the distribution of CD45 -/mid/+ CD5 + CD7 + cells under the normal conditions at D29. (H–I) Analysis of CD45 + CD5 + CD7 + cells: (H) Representative flow cytometry density plots (H) and frequency plot (I) of CD45 + CD5 + CD7 + cells in siCtrl+OE-Ctrl, siSPI1, siSPI1+OE-KLF1 and siSPI1+OE-LYL1 groups. Unless otherwise stated, data are from n = 3 independent experiments, and statistical significance was determined using two-tailed unpaired Student’s t-test. Data are presented as means ± SEM. Significance levels: ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ns, no significance. See also Figures S6 and .

Article Snippet: CD34 MicroBeads, human , Miltenyi Biotec , Cat# 130-046-702.

Techniques: Over Expression, Knockdown, Cotransfection, Western Blot, Flow Cytometry, Colony-forming Unit Assay, Two Tailed Test

Journal: iScience

Article Title: SPI1-KLF1/LYL1 axis regulates lineage commitment during endothelial-to-hematopoietic transition from human pluripotent stem cells

doi: 10.1016/j.isci.2024.110409

Figure Lengend Snippet:

Article Snippet: CD34 MicroBeads, human , Miltenyi Biotec , Cat# 130-046-702.

Techniques: Recombinant, Transfection, Plasmid Preparation, Reverse Transcription, In Vivo, In Vitro, Biomarker Discovery, Software

Journal: Breast cancer research : BCR

Article Title: CXCL17-derived CD11b + Gr-1 + myeloid-derived suppressor cells contribute to lung metastasis of breast cancer through platelet-derived growth factor-BB.

doi: 10.1186/s13058-019-1114-3

Figure Lengend Snippet: Fig. 1 CXCL17 is upregulated in lung metastatic breast cancer cells. a Scheme of the animal model. 4T1 cells (500,000 cells per fat pad) were implanted into the mammary fat pads and allowed to spontaneously metastasize to the lung for 24 days. Whole primary tumor in mammary or tumor nodules in the lungs (n = 3), livers (n = 4), and intestine (n = 4) and lymph nodes (tumor nodules = 7) were subjected to gene profiling. b Specific gene profile of breast cancer metastasized to lung. c The upregulation of CXCL17 protein in lung metastatic 4T1 cells. 4T1 cells were implanted into mice in an orthotic model. The expression of CXCL17 protein of 4T1 cells isolated from primary sites (mammary) or lung tissue (six pairs of 4T1 cell isolated from in mammary and lung) was assessed by ELISA after 48-h incubation. d In the left panel, the box plot generated from Kao Huang Breast GSE20685 dataset of SurvExpress showed the amount of CXCL17 expression in each group. The right panel showed the Kaplan-Meier time to metastasis curve. e The results were from Van De Vijver Nature 2002 dataset of SurvExpress. The left and right panels showed CXCL17 expression and the Kaplan-Meier time to metastasis curve respectively. The group was divided according to the “Maximize Risk Groups” in SurvExpress website. f Kaplan-Meier distant metastasis-free survival via KM plotter database. High and low CXCL17 expression groups were divided according to “Auto select best cutoff” in the KM plotter website. Each value is the mean ± SD of three determinations; *p < 0.05

Article Snippet: CXCL17 levels were assessed by human or mouse CXCL17 ELISA kits (Cusabio Biotech, Wuhan, China).

Techniques: Animal Model, Expressing, Isolation, Enzyme-linked Immunosorbent Assay, Incubation, Generated

Journal: Breast cancer research : BCR

Article Title: CXCL17-derived CD11b + Gr-1 + myeloid-derived suppressor cells contribute to lung metastasis of breast cancer through platelet-derived growth factor-BB.

doi: 10.1186/s13058-019-1114-3

Figure Lengend Snippet: Fig. 2 CXCL17 increased lung metastasis in vivo. a CXCL17 increased the lung metastasis of 4T1 cells in an orthotropic model. The total number of tumor nodule per whole lung lobes was counted and averaged among the animals of each group. b The H&E staining of tumor sections. BALB/c mice were treated with PBS or recombinant mouse CXCL17 protein by intra-tracheal administration for 14 days (1 μg/mouse, 2 times/week, n = 6 per group). 4T1 were implanted into the fat pads of mice. Tumor nodules of 4T1 in the primary site and lungs were collected after 24 days of injections. c CXCL17 increased lung metastasis in human breast MDA-MB-231. Nude mice were treated with PBS or recombinant CXCL17 protein by intra-tracheal administration for 14 days (1 μg/mouse, 2 times/week, n = 6 per group). MDA-MB-231 cells were implanted into mice by tail vein injection. The MDA- MB-231 tumor nodules in the lungs of mice were collected after 90 days of injections. d The H&E staining of tumor sections. e Knockdown of CXCL17 decreased the spontaneous metastatic ability of L4T1 cells. f The H&E staining of tumor sections. CXCL17-knockdown-L4T1 were implanted into the fat pads of mice (n = 6 per group). Tumor nodules in the lungs were collected after 24 days of injections. Representative lung tissue sections were stained with H&E and photographed at × 100 magnification. Each value is the mean ± SEM; Mann-Whitney U test was performed, *p < 0.05. T tumor

Article Snippet: CXCL17 levels were assessed by human or mouse CXCL17 ELISA kits (Cusabio Biotech, Wuhan, China).

Techniques: In Vivo, Staining, Recombinant, Injection, Knockdown, MANN-WHITNEY

Journal: Breast cancer research : BCR

Article Title: CXCL17-derived CD11b + Gr-1 + myeloid-derived suppressor cells contribute to lung metastasis of breast cancer through platelet-derived growth factor-BB.

doi: 10.1186/s13058-019-1114-3

Figure Lengend Snippet: Fig. 3 CXCL17 increases the recruitment of MDSCs in metastatic lungs of mice. The effect of CXCL17 in the recruitment of CD11b+Gr-1+ MDSCs (a), CD11b+Gr-1−MDSCs (b), and CD11b+F4/80+ macrophages (c) in the lungs of mice. BALB/c mice were treated with PBS or recombinant mouse CXCL17 protein by intra-tracheal administration for 14 days (1 μg/mouse, 2 times/week, n = 6 per group). Various immune cells were isolated from the lungs of mice by antibody conjugated magnetic beads. Each value is the mean ± SEM; *p < 0.05. CXCL17 increased the migration (d) and transendothelial migration (e) of CD11b+Gr-1+ MDSCs in vitro. GPR35 inhibitor decreased the migration (f) and transendothelial migration (g) of CD11b+Gr-1+ MDSCs induced by CXCL17. CD11b+Gr-1+ MDSCs were isolated from the lungs of normal mice (n = 3). PKH26-labeled CD11b+Gr-1+ MDSCs cells were seeded onto inserts (1 × 105 cells in 3-μm pore insert for migration analysis). For transendothelial migration analysis, C166 cells were seeded in 3-μm pore collagen-coated inserts for confluent monolayer, and PKH26-labeled CD11b+Gr-1+ MDSCs cells (1 × 105/insert) were seeded onto C166 confluent monolayer inserts, and the migration of cancer cells was assessed by fluorescence microscope. CXCL17 (1 ng/ml) were added in bottom well as chemoattractant. For blocking experiment, GPR35 inhibitor (CID2745687, 2 μM) was added in the inserts. Results are representative of at least three independent experiments, and each value is the mean ± SD of three determinations. *Significant difference between the two test groups (p < 0.05)

Article Snippet: CXCL17 levels were assessed by human or mouse CXCL17 ELISA kits (Cusabio Biotech, Wuhan, China).

Techniques: Recombinant, Isolation, Magnetic Beads, Migration, In Vitro, Labeling, Fluorescence, Microscopy, Blocking Assay

Journal: Breast cancer research : BCR

Article Title: CXCL17-derived CD11b + Gr-1 + myeloid-derived suppressor cells contribute to lung metastasis of breast cancer through platelet-derived growth factor-BB.

doi: 10.1186/s13058-019-1114-3

Figure Lengend Snippet: Fig. 4 CXCL17 increases angiogenesis in lung metastatic niche by recruiting CD11b+Gr-1+ MDSCs. a CXCL17 increased CD31+ cells in the lungs of mice. Digital images of tissues were captured and analyzed with ImageJ software to calculate the percentage of positive cells (high positive + positive + low positive cells). b Conditioned medium (CM) (50%) of CD11b+Gr-1+ MDSCs isolated from the lungs of CXCL17-treated mice (n = 5) increased tube formation of C166 cells. The effect of CXCL17 in the PDGF-AA (c), PDGF-BB (d), VEGF-A (e), and EGF basic (f) secretions of CD11b+Gr-1+ MDSCs in vivo. BALB/c mice were treated with PBS or recombinant mouse CXCL17 protein by intra-tracheal administration for 14 days (1 μg/mouse, 2 times/ week, n = 6 per group). The lungs of these mice were harvested and constrained by CD31 antibody and H&E. Alternatively, CD11b+Gr-1+ MDSCs were isolated from lungs of PBS or CXCL17 treated mice (n = 6 per group) by antibody conjugated magnetic beads, and CM was collected after culturing for 24 h. The expression of various angiogenic factors was assessed by Luminex Assays. g CXCL17 increased the expression of PDGF-BB in CD11b+Gr- 1+ MDSCs isolated from the lungs of normal mice. CD11b+Gr-1+ MDSCs were isolated from the lungs of normal mice (n = 5) by antibody conjugated magnetic beads and treated with rmCXCL17 (10 ng/ml) for 24 h. The expression of PDGF-BB was assessed by Luminex Assays. h Inhibition of PDGFR-β by specific inhibitor (DMPQ-2HCl, 5 μM) prevents CD11b+Gr-1+ MDSC-mediated C166 tube formation. Results are representative of at least three independent experiments in vitro studies, and each value is the mean ± SD of three determinations. *Significant difference between the two test groups (p < 0.05)

Article Snippet: CXCL17 levels were assessed by human or mouse CXCL17 ELISA kits (Cusabio Biotech, Wuhan, China).

Techniques: Software, Isolation, In Vivo, Recombinant, Magnetic Beads, Expressing, Luminex, Inhibition, In Vitro

Journal: Breast cancer research : BCR

Article Title: CXCL17-derived CD11b + Gr-1 + myeloid-derived suppressor cells contribute to lung metastasis of breast cancer through platelet-derived growth factor-BB.

doi: 10.1186/s13058-019-1114-3

Figure Lengend Snippet: Fig. 5 CD11b+Gr-1+ myeloid cells in lungs metastatic niche promote cancer cell colonization. a CXCL17 increased cancer cell extravasation into the lungs of mice. Athymic nude mice were treated with recombinant CXCL17 protein (1 μg/mouse, 2 times/week, n = 6 per group) for 14 days; MDA-MB-231-RFP-Luc cells were injected into mice by tail vein. The lungs of these mice were harvested after 48-h injection, then examined by a confocal microscope. b Scheme of CD11b+Gr-1+ depletion in the animal model. Depletion of CD11b+Gr-1+ cells decreased CXCL17-mediated cancer extravasation (c) and tumor nodules formation (d) in the lungs of mice. e The H&E staining of tumor sections of lungs. For MDSC depletion studies, mice (n = 6 per group) were treated with isotype or anti-Gr-1 antibodies (Bio X Cell) at 100 μg/mouse intraperitoneal injections every 4th day, and 4T1 cells were injected via tail vein into the mice (n = 6 per group). The control group received intraperitoneal injection of purified rat immunoglobulins. PKH26-labeled 4T1 cells were injected into mice by tail vein for indicated times (24 days for lung metastasis and 48 h for extravasation). The lungs of these mice were harvested and examined by a confocal microscope. Each value is the mean ± SEM; *p < 0.05. White arrows indicate cancer cells

Article Snippet: CXCL17 levels were assessed by human or mouse CXCL17 ELISA kits (Cusabio Biotech, Wuhan, China).

Techniques: Recombinant, Injection, Microscopy, Animal Model, Staining, Control, Purification, Labeling

Journal: Breast cancer research : BCR

Article Title: CXCL17-derived CD11b + Gr-1 + myeloid-derived suppressor cells contribute to lung metastasis of breast cancer through platelet-derived growth factor-BB.

doi: 10.1186/s13058-019-1114-3

Figure Lengend Snippet: Fig. 6 CD11b+Gr-1+ MDSCs increased breast cancer extravasation and survival via PDGF-BB. CM of CD11b+Gr-1+ MDSC isolated from the lungs of CXCL17-treated or 4T1-bearing mice (n = 5) increased transendothelial migration (a) and colony formation (b) of 4T1 cells. rmPDGF enhanced 4T1 cell transendothelial migration (c) and colony formation (d). Blockade of PDGFR-β prevented transendothelial migration (e) and colony formation (f) of 4T1 cells induced by CM of CD11b+Gr-1+ MDSCs. CD11b+Gr-1+ MDSC isolation and the collection of their CMs have been described in the legend of Fig. 2. C166 cells were seeded 8-μm pore collagen-coated inserts for confluent monolayer, and PKH26-labeled 4T1 cells (1 × 105/insert) were seeded onto C166 confluent monolayer inserts, and the CM of CD11b+Gr-1+ MDSCs (50%) or rmPDGF-BB protein (20 ng/ml) were placed in the bottom well as chemoattractant. The migration of cancer cells was assessed by a fluorescence microscope. For colony formation analysis, 4T1 cells were treated with different CMs (50%) or PDGF-BB (20 ng/ml), and the colonies counted after staining. g PDGFR inhibitor imatinib decreased lung metastasis in mice (n = 6 per group). h H&E staining of lung sections. Representative lung tissue sections were stained with H&E and photographed at × 100 magnification. Results are representative of at least three independent experiments and each value is the mean ± SD of three determinations. *Significant difference between the two test groups (p < 0.05)

Article Snippet: CXCL17 levels were assessed by human or mouse CXCL17 ELISA kits (Cusabio Biotech, Wuhan, China).

Techniques: Isolation, Migration, Labeling, Fluorescence, Microscopy, Staining

Journal: Breast cancer research : BCR

Article Title: CXCL17-derived CD11b + Gr-1 + myeloid-derived suppressor cells contribute to lung metastasis of breast cancer through platelet-derived growth factor-BB.

doi: 10.1186/s13058-019-1114-3

Figure Lengend Snippet: Fig. 7 A novel mechanism underlying the contribution of primary cancer to lung metastatic niche formation in breast cancer. Primary cancer- secreted CXCL17 increases the accumulation of CD11b+Gr-1+ MDSCs in the lungs, which produce PDGF-BB, resulting in enhanced angiogenesis in the lung tissue before cancer cells’ arrival. In addition, CXCL17 also drives CD11b+Gr-1+ MDSCs to exhibit supportive activity for cancer extravasation and survival by PDGF-BB production under cancer cell arrival

Article Snippet: CXCL17 levels were assessed by human or mouse CXCL17 ELISA kits (Cusabio Biotech, Wuhan, China).

Techniques: Activity Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Therapeutic efficacy, pharmacokinetic profiles, and toxicological activities of humanized antibody-drug conjugate Zt/g4-MMAE targeting RON receptor tyrosine kinase for cancer therapy

doi: 10.1186/s40425-019-0525-0

Figure Lengend Snippet: Generation of humanized Zt/g4 antibody and characterization of RON-targeted antibody-drug conjugates: ( a ) Modeling of CDRs from mouse Zt/g4 in the variable regions of human IgG heavy chain and light chain. The framework of human IgG1 molecule was used for Zt/g4 humanization. The models of Zt/g4 CDRs grafted in the variable regions of human IgG1 heavy chain and light chain were generated by using the software PIGS from Automatic Predictions of Immunoglobulin Structures (Tramontano at University of Rome, Italy). ( b ) Binding of subclone H-Zt/g4 molecules to human RON. Different amounts of individual H-Zt/g4 s were incubated with NIH-3 T3 cells expressing human RON followed by addition of goat anti-human IgG1 antibody coupled with FITC. ( c ) Kinetic characterization of H-Zt/g4 interaction with human RON proteins by Octet RED96 system. Pure RON proteins from lysates of NIH3T3 cells expressing RON were immobilized onto the amine reactive sensor and assayed against individual H-Zt/g4 molecules in duplicate. The data set is analyzed with global fitting to produce the antibody-receptor binding affinity ( K D ). Blue curves represent experimental data and red curves represent the statistical fitting of curves. ( d ) Interaction of H-Zt/g4 H1L3 with RONs from different species. NIH3T3 cells expressing human, monkey, or mouse RON were incubated with H-Zt/g4 H1L3 followed by goat anti-human IgG coupled with FITC. Immunofluorescent intensities from individual samples were determined by flow cytometric analysis. ( e ) Schematic representation of H-Zt/g4-MMAE structure. MMAE was conjugated to H-Zt/g4 by the valine-citruline dipeptide linker according to the manufacturer’s instruction ( www.concortis.com ). ( f ) HIC analysis of MMAE conjugated to H-Zt/g4: Individual Zt/g4-MMAEs with different numbers of MMAE (0 to 8) are marked as P0 to P8. A DAR combining P2, P4, and P6 at 3.77:1 was achieved. ( g ) Free MMAE dissociated from H-Zt/g4-MMAE in human plasma. H-Zt/g4-MMAE at 10 μg per ml was incubated with fresh human plasma at 37 °C for 20 days. The amount of free MMAE in plasma was determined using the LC-MS/MS method with slight modifications. ( h ) Samples from ( g ) were used also for measuring MMAE conjugated H-Zt/g4 as detailed in Materials and Methods. A ratio from free MMAE to the total MMAE in H-Zt/g4-MMAE was calculated to determine the percentages of MMAE dissociated from H-Zt/g4-MMAE

Article Snippet: The amount of MMAE conjugated H-Zt/g4 in plasma was determined by using a MMAE ADC ELISA kit (Eagle Biosciences Inc., Nashua, NH).

Techniques: Generated, Software, Binding Assay, Incubation, Expressing, Clinical Proteomics, Liquid Chromatography with Mass Spectroscopy

Journal: Journal for Immunotherapy of Cancer

Article Title: Therapeutic efficacy, pharmacokinetic profiles, and toxicological activities of humanized antibody-drug conjugate Zt/g4-MMAE targeting RON receptor tyrosine kinase for cancer therapy

doi: 10.1186/s40425-019-0525-0

Figure Lengend Snippet: Effect of H-Zt/g4-MMAE on RON internalization, cell viability, and death: ( a ) H-Zt/g4-induced cell surface RON internalization. PDAC cell lines BxPC-3, FG and L3.6pl (1 × 10 6 cells per dish) were treated at 37 °C with 5 μg/ml of H-Zt/g4-MMAE, collected at different time points, washed with acidic buffer to eliminate cell surface bound IgG , and then incubated with 2 μg/mL of anti-RON mAb Zt/c1 . Immunofluorescence was analyzed by flow cytometer using FITC-coupled anti-mouse IgG. Immunofluorescence from cells treated with H-Zt/g4 at 4 °C was set as 100%. Internalization efficiency (IC 50 ) was calculated as the time required achieving 50% reduction of cell surface RON. ( b ) Intracellular localization of internalized RON. FG cells in a 6-well plate were treated with 5 μg/ml of H-Zt/g4 at 4 °C or 37 °C for 12 h followed by mouse anti-human IgG1-coupled with FITC. Nuclear DNAs were stained with DAPI. LAMP-1 was used as a marker for protein cytoplasmic localization. Similar results also observed in additional three PDAC cell lines (data not shown). ( c ) Effect of H-Zt/g4-MMAE on viability of PDAC cells. Three PDAC cell lines (8000 cells per well in a 96-well plate in triplicate) were treated with different amounts of H-Zt/g4-MMAE for 96 h. Panc-1 cells served as the negative control. Cell viability was determined by the MTS assay. ( d ) Death of PDAC cells after H-Zt/g4-MMAE treatment. PDAC cells were treated with different amounts of H-Zt/g4-MMAE for 96 h. The percentages of cell death were determined by the trypan blue exclusion method. Data shown in ( c ) and ( d ) are derived from one of three experiments with similar results

Article Snippet: The amount of MMAE conjugated H-Zt/g4 in plasma was determined by using a MMAE ADC ELISA kit (Eagle Biosciences Inc., Nashua, NH).

Techniques: Incubation, Immunofluorescence, Flow Cytometry, Staining, Marker, Negative Control, MTS Assay, Derivative Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Therapeutic efficacy, pharmacokinetic profiles, and toxicological activities of humanized antibody-drug conjugate Zt/g4-MMAE targeting RON receptor tyrosine kinase for cancer therapy

doi: 10.1186/s40425-019-0525-0

Figure Lengend Snippet: Pharmacokinetic profiles of H-Zt/g4-MMAE in both mouse and cynomolgus monkey: ( a ) PK profiles of H-Zt/g4-MMAE in mouse. Tumor-bearing and -nonbearing mice (athymic nude, 5 mice per group) were injected with a single dose of H-Zt/g4-MMAE at 3, 10, and 20 mg/kg, respectively. Collected blood samples were analyzed using the MMAE ADC ELISA kit (Eagle Biosciences, Inc., Nashua, NH). Various PK parameters were calculated using the software provided by Eagle Biosciences. ( b ) Free MMAE dissociated from H-Zt/g4-MMAE in monkey plasma. A single dose of H-Zt/g4-MMAE at 10 or 30 mg/kg was injected into cynomolgus monkey (3 animals per group). Free MMAE from individual blood samples collected at different time intervals were subjected to the LC-MS/MS analysis. ( c ) PK profiles of H-Zt/g4-MMAE in cynomolgus monkey. Blood samples from ( b ) were analyzed for MMAE coupled H-Zt/g4 using the MMAE ADC ELISA kit as described in ( a ) to obtain various PK parameters

Article Snippet: The amount of MMAE conjugated H-Zt/g4 in plasma was determined by using a MMAE ADC ELISA kit (Eagle Biosciences Inc., Nashua, NH).

Techniques: Injection, Enzyme-linked Immunosorbent Assay, Software, Clinical Proteomics, Liquid Chromatography with Mass Spectroscopy

Journal: Journal for Immunotherapy of Cancer

Article Title: Therapeutic efficacy, pharmacokinetic profiles, and toxicological activities of humanized antibody-drug conjugate Zt/g4-MMAE targeting RON receptor tyrosine kinase for cancer therapy

doi: 10.1186/s40425-019-0525-0

Figure Lengend Snippet: Therapeutic efficacy of H-Zt/g4-MMAE in PDAC xenograft tumor models: ( a ) Dose-dependent effect of H-Zt/g4-MMAE: Athymic nude mice (5 mice per group) were subcutaneously inoculated with 5 × 10 6 FG cells. H-Zt/g4-MMAE at 1, 3, 7, 10, and 15 mg/kg was injected through tail vein in the Q6 × 5 regimen after tumors volumes reached to ~ 150 mm 3 . Mice injected with CmIgG-MMAE at 10 mg/kg were used as the control. Xenografts initiated by HT-29 cells served for comparison. ( b ) Effect of H-Zt/g4-MMAE in PDAC xenograft growth and eradication. Individual tumors from different groups described in (A) were collected from euthanized mice. Control mice bearing FG xenografts were sacrificed at day 24 due to rapid growth of tumors. Mice from other groups were killed at day 28 or day 44 dependent on the size of tumors. All tumors were weighted to reach the average tumor weight per group. The number of tumors from individual groups also was counted to determine the eradicating effect of H-Zt/g4-MMAE. NA, no tumors were found in the injected site. ( c ) Effect of H-Zt/g4-MMAE in three PDAC xenograft models: Xenograft tumors in mice (five animals per group) initiated by four PDAC cell lines were used for study. H-Zt/g4-MMAE was used at 20 mg/kg in the Q12 × 2 schedules. To establish the dose-effect relationship, the estimated reduction of H-Zt/g4-MMAE in vivo according to the t½ was marked as red circles. ( d ) Effect of H-Zt/g4-MMAE in tumor growth and eradication: Tumors were collected from mice described in ( b ). Tumor weight, count, and calculation were performed as described in ( b ). NA, no tumors were observed in the injected site

Article Snippet: The amount of MMAE conjugated H-Zt/g4 in plasma was determined by using a MMAE ADC ELISA kit (Eagle Biosciences Inc., Nashua, NH).

Techniques: Drug discovery, Injection, Control, Comparison, In Vivo

Journal: Journal for Immunotherapy of Cancer

Article Title: Therapeutic efficacy, pharmacokinetic profiles, and toxicological activities of humanized antibody-drug conjugate Zt/g4-MMAE targeting RON receptor tyrosine kinase for cancer therapy

doi: 10.1186/s40425-019-0525-0

Figure Lengend Snippet: Therapeutic Effect of H-Zt/g4-MMAE on xenograft tumors mediated by PDAC stem-like cells and primary PDX cells: ( a ) Effect of H-Zt/g4-MMAE on PDAC stem-like cell derived xenografts: Athymic nude mice (five mice per group) were subcutaneously inoculated with 5 × 10 5 PSC + 24/44/ESA prepared from BxPc-3, FG, and L3.6pl cells. H-Zt/g4-MMAE at 20 mg/kg was injected through tail vein in the Q12 × 2 regimen after tumors volumes reached to ~ 150 mm 3 . Mice injected with CmIgG-MMAE at 20 mg/kg were used as the control. ( b ) The eradicating effect of H-Zt/g4-MMAE on PDAC stem-like cell derived xenografts. Tumors were collected from mice as described in Fig. b. Tumor weight, count, and calculation were performed as described in Fig. b. ( c ) Mice were injected with individual primary PDX cell lines at 5 × 10 6 cells in 0.1 ml in PBS. H-Zt/g4-MMAE at 10 mg/kg was injected through tail vein in the Q12 × 2 regimen after tumors volumes reached to 150 to 200 mm 3 . Mice injected with CmIgG-MMAE at 10 mg/kg were used as the control. ( d ) Individual tumors were collected from each group of mice as described in Fig. b. Average tumor weight and number per group were measured to determine levels of inhibition and eradication

Article Snippet: The amount of MMAE conjugated H-Zt/g4 in plasma was determined by using a MMAE ADC ELISA kit (Eagle Biosciences Inc., Nashua, NH).

Techniques: Derivative Assay, Injection, Control, Inhibition

Journal: Journal for Immunotherapy of Cancer

Article Title: Therapeutic efficacy, pharmacokinetic profiles, and toxicological activities of humanized antibody-drug conjugate Zt/g4-MMAE targeting RON receptor tyrosine kinase for cancer therapy

doi: 10.1186/s40425-019-0525-0

Figure Lengend Snippet: Therapeutic effect of H-Zt/g4-MMAE in comparison with H-Zt/g4-DM1 in inhibition of xenograft tumors derived from human pancreatic and colorectal cancer cells a

Article Snippet: The amount of MMAE conjugated H-Zt/g4 in plasma was determined by using a MMAE ADC ELISA kit (Eagle Biosciences Inc., Nashua, NH).

Techniques: Comparison, Inhibition, Derivative Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Therapeutic efficacy, pharmacokinetic profiles, and toxicological activities of humanized antibody-drug conjugate Zt/g4-MMAE targeting RON receptor tyrosine kinase for cancer therapy

doi: 10.1186/s40425-019-0525-0

Figure Lengend Snippet: Toxicological activities of H-Zt/g4-MMAE in mouse and cynomolgus monkey. ( a ) and ( b ) Adverse activities of H-Zt/g4-MMAE in blood leukocytes in cynomolgus monkey. H-Zt/g4-MMAE at 10 or 30 mg/kg in a single dose was injected once into cynomolgus monkey. Monkeys without ADC injection served as the control. Peripheral blood samples were collected at different time intervals. Total numbers of leukocytes ( a ) including neutrophil, lymphocytes, and monocytes from each group were countered accordingly. The percentages of blood leukocytes ( b ) were also determined. ( c ) Adverse effect of H-Zt/g4-MMAE on blood erythrocytes and reticulocytes in cynomolgus monkey. Total numbers of erythrocytes and reticulocytes from blood samples collected from each group as described in ( b ) were counted accordingly. ( d ) Adverse effects of H-Zt/g4-MMAE on various enzymes in plasma of cynomolgus monkey. Six enzymatic activities from each group were quantitatively measured using the blood samples collected from individual monkeys as described in ( b )

Article Snippet: The amount of MMAE conjugated H-Zt/g4 in plasma was determined by using a MMAE ADC ELISA kit (Eagle Biosciences Inc., Nashua, NH).

Techniques: Injection, Control, Clinical Proteomics

Journal: Cell reports

Article Title: The STRIPAK complex is required for radial sorting and laminin receptor expression in Schwann cells

doi: 10.1016/j.celrep.2025.115401

Figure Lengend Snippet:

Article Snippet: The following primary antibodies were used: mouse anti-β-Actin 1:1000 (Santa Cruz sc-47778), mouse anti-β-tubulin 1:2000 (Sigma-Aldrich T4026), mouse anti-β-dystroglycan 1:500 (Leica Biosystems NCL-β-DG), rabbit anti-Ccm3 1:250 (Proteintech 10294–2-AP), rabbit anti-Cdc42 1:250 (Cell Signaling 2462), rabbit anti-Egr2/Krox20 1:500 (kindly shared by D. Meijer), rabbit anti-GAPDH 1:10,000 (Sigma-Aldrich G9545), rabbit anti-GST tag 1:1000 (Proteintech 80006–1-RR), mouse anti-GST tag 1:1000 (Santa Cruz sc-138), rabbit anti-His tag 1:1000 (Cell Signaling 2365S), rabbit anti-His tag 1:50,000 (Cell Signaling 2366T), goat anti-integrin α6 1:250 (Santa Cruz sc-6597), rabbit anti-integrin b1 1:250 (Cell Signaling 4706), rat anti-integrin b4 1:250 (Abcam ab25254), mouse anti-Mob4/Phocein 1:250 (Santa Cruz sc-137229), rabbit anti-Mst1 1:500 (Cell Signaling 3682), rabbit anti-Mst2 1:500 (Abcam ab52641), rabbit anti-p-Mst1/2 1:250 (Proteintech 28953–1-AP), rabbit anti-NF2 1:500 (Cell Signaling 6995), rabbit anti-p-NF2 1:500 (Cell Signaling 1328), rabbit anti-Oct6/Pou3f1 1:500 (kindly shared by D. Meijer), rabbit anti-Pak1 1:500 (Cell Signaling 2602), rabbit anti-p-Pak1 1:500 (Cell Signaling 2601), mouse anti-Rac1 1:250 (EMD Millipore 05–389), mouse anti-Rac1 1:250 (Cytoskeleton, Inc. ARC03), rabbit anti-Rac1 1:500 (Thermo Fisher Scientific PA1–091), rabbit anti-Rac1 1:500 (Proteintech 24072–1-AP), rabbit anti-Sox10 1:250 (Cell Signaling 89356), mouse anti-StrepII tag 1:250 (Thermo Fisher Scientific MA5–37747), mouse anti-STRIP1 1:250 (Origene TA502314), mouse anti-striatin-1 (Santa Cruz sc-136084), rabbit anti-striatin-3 1:100 (Atlas Antibodies HPA004636), mouse anti-striatin-3 (Novus Biologicals NB110–74572), rabbit anti-striatin-4 (GeneTex GTX133282), rabbit anti-Yap 1:250 (Cell Signaling 4912), rabbit anti-Yap 1:250 (Cell Signaling 14074), rabbit anti-Yap/Taz 1:250 (Cell Signaling 8418), rabbit anti-p-Yap 1:250 (Cell Signaling 13008), rabbit anti-p-Taz 1:250 (Cell Signaling 59971).

Techniques: Generated, Virus, Recombinant, SYBR Green Assay, Protease Inhibitor, Plasmid Preparation, In Situ, BIA-KA, Mutagenesis, Software

Journal: American Journal of Cancer Research

Article Title: Alpelisib and radiotherapy treatment enhances Alisertib-mediated cervical cancer tumor killing

doi:

Figure Lengend Snippet: Alpelisib enhanced Alisertib-mediated toxicity in cervical cancer cells. A. Hela cells were treated with ALISERTIB at doses ranging from 40 uM to 40 nM in a 96 well plate. Cell viability was measured by the MTT assay at 3-days post-treatment. A dose dependent curve was generated using GraphPad Prism v9 to determine IC50 values. Data points are representative of the mean ± SEM (n = 3). B. Cells were treated with Alpelisib at increasing doses either in the presence or absence of Alisertib (concentration used: 0.5 Alisertib IC50 = 73.25 nM) before measuring cell viability at 3-days post-treatment by the MTT assay. Data is representative of one out of three independent experiments. Data points represent the mean ± SEM. C. Standard isobologram analysis of cell killing by the drug combinations. IC90 values of each drug are plotted on the axes; the solid line represents the additive effect, while the point represents concentrations of each drug resulting in 90% inhibition of growth. The point falls below the line indicating synergism between the drugs. Combination index (CI) at fractional response 0.9 (90% killing) is also shown. CI value is <1, indicative of a synergistic effect. Data is representative of three independent experiments. D. Proteins from cells collected at 24 h were immunoblotted for cleaved PARP. β-actin was used as a loading control. Individual blots shown are representative of three independent experiments. E. HeLa cells exposed to Alisertib, Alpelisib, or a combination of both were stained with annexin V-FITC and PI for FACS analysis after 48 h. Cell populations shown in the lower left quadrant represent living cells; lower right quadrant represents apoptotic cells, upper right quadrant represents necrotic cells and upper left quadrant represents pre-necrotic cells. Data is representative of 3 experiments. F. HeLa cells were treated either alone, with Alisertib (500 nM) or in combination with Alpelisib (20 uM). Images were captured by time-lapse microscopy over 72 h. Still images shown were captured at times indicated using the Hstudio 2.7.5™ live imaging. Data presented are representative of one out of three independent experiments. G. 10 individual cells were tracked from time-lapse microscopy videos using the Hstudio 2.7.5™ live imaging software and the time in and number of cell divisions were plotted on the graph over a 72 h period.

Article Snippet: The percentage of cells undergoing apoptosis was determined by flow cytometry using the Annexin V-FITC Apoptosis Kit (#K101) (BioVision, Milpitas, CA) as per manufacturer’s protocol.

Techniques: MTT Assay, Generated, Concentration Assay, Inhibition, Staining, Time-lapse Microscopy, Imaging, Software

Journal: Cell reports

Article Title: Persistent NF-κB activation in muscle stem cells induces proliferation-independent telomere shortening

doi: 10.1016/j.celrep.2021.109098

Figure Lengend Snippet:

Article Snippet: Samples were permeabilized with 0.5% Triton X100/PBS, underwent heat-mediated antigen retrieval with acidic citrate, and were blocked first with the avidin/biotin blocking kit (Vector Labs), then with 3% BSA/PBS/0.1% Triton X-100.

Techniques: Recombinant, Electron Microscopy, Plasmid Preparation, Avidin-Biotin Assay, Blocking Assay, Lysis, Imaging, Kinase Assay, Staining, Generated, Software, Microscopy

Journal: Nature chemical biology

Article Title: Velcrin-induced selective cleavage of tRNA Leu (TAA) by SLFN12 causes cancer cell death

doi: 10.1038/s41589-022-01170-9

Figure Lengend Snippet: a , Sequence analysis of tRNAs extracted from HeLa cells treated with DMSO or 1 μM DNMDP for 18 h ( n = 2 replicates), based on the mapping to the genomic tRNA database, GtRNAdb 35 . Scatter plot (left), tRNA abundance in DMSO-treated ( X axis) and DNMDP-treated cells ( Y axis). Red dots, tRNA Leu (TAA) isodecoders. b , Leucine tRNA abundance in a panel of cancer cell lines treated with DMSO or 1 μM DNMDP 3 . After demethylation of tRNA, cDNA was synthesized and leucine tRNA levels were measured using qPCR. tRNA Leu (TAA)-1 level is plotted relative to tRNA Leu (TAG)-2 as a control ( n = 3 replicates per condition). Data are represented as mean ± s.e.m. P values were calculated by unpaired two-tailed Student’s t -test.

Article Snippet: RNAs were synthesized from PCR products using the Transcript Aid T7 High Yield Transcription Kit (ThermoFisher) and purified by extracting full-length tRNAs from 15% TBE-urea gels (ThermoFisher).

Techniques: Sequencing, Synthesized, Control, Two Tailed Test

Journal: Nature chemical biology

Article Title: Velcrin-induced selective cleavage of tRNA Leu (TAA) by SLFN12 causes cancer cell death

doi: 10.1038/s41589-022-01170-9

Figure Lengend Snippet: a , b , Preferential digestion of tRNA Leu (TAA) by SLFN12. 0.5 μg of the indicated tRNAs were digested with ( a ) 2 μM SLFN12 ( n = 2 replicates for tRNA Leu (TAA)-1, tRNA Leu (TAA)-3, tRNA Ser (CGA) and tRNA Cys (GCA), and n = 3 replicates for others) or ( b ) 0.25 μM PDE3A and SLFN12 proteins preincubated with 1.25 μM DNMDP ( n = 3 replicates for tRNA Leu (TAA)-1 and tRNA Leu (TAA)-3, and n = 2 replicates for others). The relative amounts of intact tRNAs were measured and plotted. c , The variable loops or acceptor stems of tRNA Leu (TAG)-2 and tRNA Leu (TAA)-3 were swapped to generate six different hybrid leucine tRNAs. Sequences of tRNA Leu (TAG)-2 and tRNA Leu (TAA)-3 are indicated by red and black colors, respectively. d , Each leucine tRNA synthesized by in vitro transcription was digested with 0.25 μM PDE3A and 0.25 μM SLFN12 proteins preincubated with 1.25 μM DNMDP. Cleaved tRNA was analyzed on 15% TBE-Urea gels. The results were reproducible and representative images are shown ( n = 2 for hybrids 2, 3, 5 and 6; n = 3 replicates for others). e , The relative amount of intact tRNA, quantified using ImageJ software, is shown at the bottom of the gels and plotted ( n = 2 for hybrids 2, 3, 5 and 6; n = 3 replicates for others). Data are represented as mean ± s.e.m. P values were calculated by the unpaired two-tailed Student’s t -test.

Article Snippet: RNAs were synthesized from PCR products using the Transcript Aid T7 High Yield Transcription Kit (ThermoFisher) and purified by extracting full-length tRNAs from 15% TBE-urea gels (ThermoFisher).

Techniques: In Vitro, Synthesized, Software, Two Tailed Test

Journal: Nature chemical biology

Article Title: Velcrin-induced selective cleavage of tRNA Leu (TAA) by SLFN12 causes cancer cell death

doi: 10.1038/s41589-022-01170-9

Figure Lengend Snippet: a , Construction of resistant tRNA Leu (CAG:TAA) by mutating the anticodon of tRNA Leu (CAG)-1 to TAA. b , Indicated tRNAs were digested with 0.25 μM PDE3A and 0.25 μM SLFN12 proteins preincubated with 1.25 μM DNMDP. Intact tRNAs are marked with an arrow and were quantified by ImageJ (1.8.0), as reported at the bottom of each lane ( n = 2 replicates). c , HeLa cells were transfected with plasmids expressing tRNA Leu (CAG)-1, tRNA Leu (TAA)-3 or variant tRNA Leu (CAG:TAA) followed by DMSO or 0.5 μM DNMDP treatment. Forty-eight hours after drug treatment, cell viability was assessed by CellTiter-Glo ( n = 3 replicates). Data are represented as mean ± s.e.m. P values were calculated by the unpaired two-tailed Student’s t -test (NS, not significant).

Article Snippet: RNAs were synthesized from PCR products using the Transcript Aid T7 High Yield Transcription Kit (ThermoFisher) and purified by extracting full-length tRNAs from 15% TBE-urea gels (ThermoFisher).

Techniques: Expressing, Transfection, Variant Assay, Two Tailed Test

Journal: Nature chemical biology

Article Title: Velcrin-induced selective cleavage of tRNA Leu (TAA) by SLFN12 causes cancer cell death

doi: 10.1038/s41589-022-01170-9

Figure Lengend Snippet: ( a) Indicated tRNAs were synthesized using a T7 RNA polymerase-mediated transcription reaction. Synthetic tRNAs were treated with the indicated concentrations of SLFN12 at 37 °C for 40 minutes (n = 2 replicates for Leu-TAA-1, Leu-TAA-3, Ser-CGA and Cys-GCA; n = 3 replicates for others). ( b ) 0.25 μM PDE3A and SLFN12 proteins pre-treated with 1.25 μM DNMDP were incubated with synthetic tRNAs at 37 °C for 40 minutes (n = 3 replicates for Leu-TAA-1 and Leu-TAA-3; n = 2 replicates for others). ( c ) 0.5 μM ΔSLFN12 (amino acids 1–347) or PDE3A proteins pre-treated with DMSO, 2.5 μM DNMDP, 2.5 μM trequinsin or 2.5 μM estradiol were incubated with synthetic tRNAs at 37 °C for 40 minutes. After incubation, RNA samples were analyzed on a 15% denaturing polyacrylamide gel (n = 2 replicates). Representative figures are shown here. Intact tRNA is indicated with an arrow. The relative amounts of intact tRNA were quantified using ImageJ software and are shown at the bottom. ( d ) Leucine tRNA abundance was measured by qPCR in purified total RNA treated with 0.25 μM SLFN12 recombinant protein. tRNA-Leu-TAA-1 levels are plotted relative to control tRNA, tRNA-Leu-TAG-2 (n = 3 replicates). Data are represented as mean ± SEM. Statistical significance was calculated by a two-tailed unpaired Student’s t test.

Article Snippet: RNAs were synthesized from PCR products using the Transcript Aid T7 High Yield Transcription Kit (ThermoFisher) and purified by extracting full-length tRNAs from 15% TBE-urea gels (ThermoFisher).

Techniques: In Vitro, Synthesized, Incubation, Software, Purification, Recombinant, Control, Two Tailed Test

Journal: Nature chemical biology

Article Title: Velcrin-induced selective cleavage of tRNA Leu (TAA) by SLFN12 causes cancer cell death

doi: 10.1038/s41589-022-01170-9

Figure Lengend Snippet: ( a ) Sequences of the indicated tRNAs were aligned in R using the msa package. The consensus logo is shown at the top of the figure, with more conserved positions appearing larger. Positions where the sequences do not agree are blank in the consensus. Structural regions of the tRNA are labeled on the bottom of the alignment. ( b ) The computational model of the structure of SLFN12 bound to tRNA was generated using the HADDOCK software. Both the catalytic SLFN12 monomer (green) and the adaptor SLFN12 monomer (magenta) interact with a single molecule of tRNA-Leu-TAA-3 (orange). The catalytic SLFN12 monomer interacts with the tRNA variable loop region (yellow) and the adaptor SLFN12 monomer interacts with the tRNA acceptor stem (cyan). The active site residues of the catalytic SLFN12 monomer are close to the primary tRNA cleavage site in this model.

Article Snippet: RNAs were synthesized from PCR products using the Transcript Aid T7 High Yield Transcription Kit (ThermoFisher) and purified by extracting full-length tRNAs from 15% TBE-urea gels (ThermoFisher).

Techniques: Labeling, Generated, Software