Journal: Journal of Human Immunity

Article Title: A heterozygous USB1 variant linked to immunodeficiency

doi: 10.70962/jhi.20250110

Figure Lengend Snippet: The USB1 de novo variant is catalytically active and correctly processes U6 snRNA. (A) Total RNA extracted from the indicated BEBV cell lines were treated with T4 PNK or with buffer only (PNKBuff) in mild acidic conditions. RNA was subsequently treated with poly(A) polymerase (PAP). Nontreated RNA was loaded as a control, ( n = 2). (B) 3′ RACE analysis of U6 oligo(U) tails in the indicated cell lines. At least 24 clones per sample in each experiment ( n = 2) were sequenced. Bars and error bars are averages of the number of U's within U6 oligo(U) tails and SEM from two independent experiments. (C and D) Indicated cell lines were treated with actinomycin D for 0, 4, and 8 h. RNA samples were processed by northern blotting for detection of U6 and 5S ( n = 2). L: marker of known length (67 nucleotides). U6 signals were normalized through the corresponding 5S signals and successively expressed as fold decrease over U6 signal at time 0. Error bars are averages of SEM from two independent experiments. (E and F) U6 relative abundance quantification by qPCR analysis on patients and control cell lines (ctr1 n = 2, ctr2 n = 3, ctr3 n = 1, USB1 −/− n = 3, P1 n = 3) (E), and USB1 −/− cells transduced with the indicated lentiviral constructs ( n = 2) (F). U6 signals were normalized through the corresponding 5S signals and successively expressed as fold decrease over U6 signal at time 0. Error bars are averages of SEM from two independent experiments. EV, empty vector. Source data are available for this figure: .

Article Snippet: Those inserts (with the addition of an HA tag at the C-terminal when indicated) were subcloned into the lentiviral pWPI backbone (RRID:Addgene_12254) by GenScript.

Techniques: Variant Assay, Control, Clone Assay, Northern Blot, Marker, Transduction, Construct, Plasmid Preparation

Journal: Nature Communications

Article Title: Large-scale production of megakaryocytes from human pluripotent stem cells by chemically defined forward programming

doi: 10.1038/ncomms11208

Figure Lengend Snippet: ( a ) TF expression in hPSCs versus cbMKs plotted as a row normalized heatmap with indications of recorded TF internal and chromatin modifiers node numbers and protein interactions among the top 20 TF candidates reported by VisANT. The nine experimentally tested TFs are highlighted in red. ( b ) The H9 hESC line was concurrently transduced with the 9-TFs and maintained in pluripotency medium (FGF2+Activin-A) for 2 days followed by MK medium (TPO+SCF) for a further 5 days. CD41a+ cells generated 7 days after lentiviral transduction (dot plot, mean% ±s.e.m., n =5; FL1: 530/40 nm channel) were sorted by flow cytometry and transgene expression levels quantified by RT-qPCR ( n =1). ( c ) The percentage of CD41a+ cells was monitored by flow cytometry 7 days after transduction of the hiPSC lines #1 and #2 with the 9-TFs or 3-TFs combination. Bar graphs represent the fold increase of CD41a+ cell count relative to the 9-TFs combination (mean ±s.e.m., n =4; ** P< 0.01 by two-tail t -test). NT: non-transduced cells. ( d ) The hiPSC#1 line was transduced with all permutations of the 3-TFs and percentages of CD41a+ cells measured by flow cytometry at day 7. Bar graphs represent the fold increase of CD41a+ cell count relative to the 3-TFs combination (mean ±s.d., n =2; * P =0.06 and P< 0.01 versus G+F and other combination respectively by two-tail t -test). G: GATA1, F: FLI1, T: TAL1, GFP: control vector. ( e ) The endogenous expression of key MK genes was monitored by RT-qPCR from CD41a flow sorted cells 7 days after transduction of the hiPSC lines #1 and #2 (mean ±s.d., n =2). ( f ) The hiPSC lines #1 and #2 were transduced with the 3-TFs and sorted by flow cytometry for expression of CD41a at day 7. The clonogenic potential of sorted cells was tested in methylcellulose semi-solid medium supplemented with TPO and SCF. The number of colonies per 5,000 sown cells was determined after 10 days from duplicate wells (mean±s.e.m., n =4). ( g ) A representative MK colony obtained from a CD41a+ cell co-expressing CD41a and CD42b as detected by immunofluorescence is shown (scale bar, 50 μm).

Article Snippet: The human coding sequences of the nine candidate genes (variants 1 from NCBI Reference Sequence Database) including the 5′ Kozak consensus sequence were generated by PCR using cbMK cDNA, individually cloned into the pWPT lentiviral vector backbone (Dr Trono, Addgene #12255) downstream of the human EF1-alpha ubiquitous promoter and checked for sequence integrity.

Techniques: Expressing, Transduction, Generated, Flow Cytometry, Quantitative RT-PCR, Cell Counting, Control, Plasmid Preparation, Immunofluorescence