Journal: Scientific Reports

Article Title: Kindlin-3 interacts with the ribosome and regulates c-Myc expression required for proliferation of chronic myeloid leukemia cells

doi: 10.1038/srep18491

Figure Lengend Snippet: ( a ) Western blot analyses of c-Myc expression in wild-type, control siRNA, and kindlin-3 siRNA(#149) cells. GAPDH serves as loading control. Values below protein bands represent the mean fold differences in protein expression levels relative to WT samples (normalized to 1.0) from three independent experiments. ( b ) RT-qPCR analyses of mRNA expression of kindlin-3, c-Myc and cyclin D1. Values represent mean ± S.E.M. of three independent experiments. Wild-type mean value was normalized to 1.0. Two-tailed unpaired t test was performed to compare control siRNA cells with kindlin-3 siRNA(#149) cells. p < 0.05 is considered significant. ( c ) Western blot of cyclin D1 in these cells. Mean fold differences from three independent experiments were determined as in ( a ). ( d ) Cell proliferation rate determination using cell proliferation dye eFluor® 670 and flow cytometry analyses. A representative experiment of three independent experiments is shown. ( e ) Soft agar colony formation assay. Cells were stained with NBT for clearer visualization. Representative microscope images of wells are shown. Scale bar, 250 μm. The area of each tumor colony was determined and plotted. A combined plot of tumor colonies from two independent experiments is shown. Two-tailed unpaired t test was performed. p < 0.05 is considered significant.

Article Snippet: The lentiviral siRNA system was generated by and purchased from Applied Biological Materials Inc, Canada).

Techniques: Western Blot, Expressing, Control, Quantitative RT-PCR, Two Tailed Test, Flow Cytometry, Soft Agar Assay, Staining, Microscopy

Journal: Scientific Reports

Article Title: Kindlin-3 interacts with the ribosome and regulates c-Myc expression required for proliferation of chronic myeloid leukemia cells

doi: 10.1038/srep18491

Figure Lengend Snippet: ( a ) Representative image of tumor formation in Balb/c-Rag−/−IL2Rγ−/− mice transplanted with either control siRNA or kindlin-3 siRNA(#149) K562 cells. ( b ) Plots of tumor volume (cm 3 ) and weight (g). N = 12. Each closed circle or square represents one mouse. Note: there are two overlapping dots for the control siRNA group in the tumor-weight plot. Mann-Whitney test (two-tailed) was performed. p < 0.05 is considered significant. ( c ) Western blot analyses of CD31 and c-Myc expression in tissue samples excised from tumors. Three tumors were examined from each group. GAPDH serves as loading control. Kindlin-3 expression in these tumors was also examined. ( d ) Secreted VEGF-A level in culture supernatant of cells as determined by ELISA. A representative experiment of two independent experiments is shown. Data are means +/− S.D. of technical triplicates. ( e ) RT-qPCR determination of VEGF-A mRNA levels in the two groups of cells. Values represent mean ± S.E.M. of three independent experiments. Wild-type mean value was normalized to 1.0. Two-tailed paired t test was performed to compare control siRNA cells with kindlin-3 siRNA(#149) cells. p < 0.05 is considered significant.

Article Snippet: The lentiviral siRNA system was generated by and purchased from Applied Biological Materials Inc, Canada).

Techniques: Control, MANN-WHITNEY, Two Tailed Test, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: Kindlin-3 interacts with the ribosome and regulates c-Myc expression required for proliferation of chronic myeloid leukemia cells

doi: 10.1038/srep18491

Figure Lengend Snippet: ( a ) (left panel) Flow cytometry analyses of integrin α5ß1 expression in K562 cells expressing either control siRNA or kindlin-3-siRNA(#149). GP: % gated positive; GM: geo-mean fluorescence; EI: expression index = % GP x GM. (right panel) Western blot analysis of kindlin-3 and RACK1 in these cells. Actin serves as loading control. Values below protein bands represent the mean fold differences in protein expression levels relative to WT samples (normalized to 1.0) from three independent experiments. ( b ) Flow assay of cells on immobilized fibronectin (FN) at a shear stress of 0.3 dyn/cm 2 . Data point represents mean of cell number of four different fields analyzed. Representative plots of two independent experiments are shown. ( c ) ECIS measurements of cell adhesion and spreading on immobilized fibronectin. Representative plots of two independent experiments of cell spreading (represented by cell index – impedance readout) against time are shown. Data point represents the mean ± S.D. of technical triplicates. MnCl 2 and function-blocking anti-integrin α5 antibody (Ab) were included. ( d–f ) Western blot analyses of p70S6K and pAkt and their phosphorylated forms under different conditions indicated. The end concentrations of reagents used are: rapamycin (1 μM), soluble fibronectin (20 μg/ml), and MnCl 2 (1 mM). Values below protein bands represent the mean fold differences of phosphor-protein/total protein relative to either WT sample or untreated sample (normalized to 1.0) from three independent experiments. N.D.: Not determined because phosphor-protein band was not detected.

Article Snippet: The lentiviral siRNA system was generated by and purchased from Applied Biological Materials Inc, Canada).

Techniques: Flow Cytometry, Expressing, Control, Fluorescence, Western Blot, Shear, Blocking Assay

Journal: Biomolecules

Article Title: Gene Therapy for Lysosomal Storage Disorders: Ongoing Studies and Clinical Development

doi: 10.3390/biom11040611

Figure Lengend Snippet: HSC-GT clinical trials recruiting LSDs patients or currently active (LV: lentiviral vector).

Article Snippet: Avrobio has initiated a phase 1/2 HSC-GT clinical trial (NCT04145037) where autologous CD34+ enriched HSCs are modified with a lentiviral vector and administered in conjunction with a conditioning regime to type 1 Gaucher subjects.

Techniques: Clinical Proteomics, Plasmid Preparation, Transduction

Journal: Nature

Article Title: Immune evasive human islet-like organoids ameliorate diabetes

doi: 10.1038/s41586-020-2631-z

Figure Lengend Snippet: a , Endogenous PD-L1 expression highlighted in red in human islet cells (n= 3245) (β cells are outlined in red). b , Heatmap of top differentially expressed genes between PD-L1 + and PD-L1 - β cells. c , Immunohistochemistry showing overlap of lentiviral-driven PD-L1 expression and insulin promoter-driven GFP expression in wHILOs (scale bar, 100μm). d , Human PD-L1 and human insulin expression in wHILOs with and without lentiviral PD-L1 overexpression, as measured by qPCR (n=3). e , Transplantation of PD-L1 overexpressing wHILOs into the kidney capsule of STZ-induced diabetic mice. f , Blood glucose levels of C57BL6J mice treated with high dose streptozotocin (HD-STZ) prior to transplantation of wHILOs with and without PD-L1 overexpression (500 wHILOs into each kidney, total 1,000 wHILOs) (n=3) g , PD-L1 expression in human islet 12 hours after IFNγ stimulation (n=5). h , PD-L1 expression in wHILOs 12 hours after indicated IFNγ stimulation (n=3). Error bars represent ± SEM. *p<0.05, **p<0.01, ***p<0.001, one-tailed, student’s paired t test. Data were pooled from 3 independent samples (a) or representative of 3 independent experiments (c, d, g, h).

Article Snippet: Lentiviruses were produced using second- or third-generation lentiviral systems in HEK293LTV cell line (Cell Biolabs).

Techniques: Expressing, Immunohistochemistry, Over Expression, Transplantation Assay, One-tailed Test

Journal: Nature Communications

Article Title: Functional screening in human HSPCs identifies optimized protein-based enhancers of Homology Directed Repair

doi: 10.1038/s41467-024-46816-5

Figure Lengend Snippet: A Schematic outlining the lentiviral-based pooled screening platform in HSPCs used to identify protein-based additives to increase HDR at a Cas9-mediated cut site of interest using an AAV6 DNA donor template. Protein variants are encoded in lentiviral libraries; once integrated into the genome the sequences can be amplified from HSPC subpopulations. To functionally quantify homology-based repair in pooled libraries, transduced cells are edited using Cas9 RNP and an AAV6 template that encodes for a GFP insertion at the cut site of interest (e.g. HBB gene). Post editing (3–5 days), cells are sorted via flow cytometry into GFP+ and GFP- populations. Genomic DNA is extracted from each sorted cell population, sequenced via NGS, and analyzed to determine the distribution of variants relative to a control. B Residues targeted for mutagenesis were chosen by their proximity to the binding interface between with 53BP1 and i53 and are shown in red (T12, T14, L67, H68). C Example enrichment of residues following screening with a saturation mutagenesis (NNK) library at position L67 (parent: i53, library size = 32 unique codons encoding 20 variants). n = 3 separate pooled analyses and mean ± SD depicted. Each bar represents a unique codon for that amino acid. n.s. not significant; * p < 0.05; ** p < 0.01; *** p < 0.001. Two-tailed t -test with Holm-Šídák correction for multiple comparisons. Exact p -values are reported on Source Data file. Of the 19 new variants tested, one (L67R) was found to be significantly enriched relative to parent i53 (L67), although L67H was borderline significant and was also moved on to subsequent validation. D Example enrichment of residues following a combinatorial library at positions T12 and T14 (parent: L67H), library size = 324 variants for which all replicates were enriched over parent are highlighted in red (16 variants, or 5%). n = 3 separate pooled analyses; bars represent mean ± SEM. Selected top hits were subsequently validated in focused libraries and experiments with purified recombinant protein. E Dot plot representation of variant fold change enrichment in combinatorial analysis, clustered by amino acid properties. Variations of residues 12 and 14 shown on the x-axis and y-axis, respectively. Additional information for this library shown in Supplementary Fig. . C – E Source data are provided as a Source Data file.

Article Snippet: The construct for the shRNA knockdown of PolQ was adapted from the previously reported pLKO and cloned from a third-generation lentiviral plasmid (Lenti SFFV) purchased from Twist Biosciences.

Techniques: Amplification, Flow Cytometry, Control, Mutagenesis, Binding Assay, Two Tailed Test, Biomarker Discovery, Purification, Recombinant, Variant Assay