Journal: Cellular and Molecular Life Sciences

Article Title: Tumor cell-intrinsic and tumor microenvironmental conditions co-determine signaling by the glycoimmune checkpoint receptor Siglec-7

doi: 10.1007/s00018-023-04816-6

Figure Lengend Snippet: CD43-expressing cells can induce chSiglec-7 signaling under the condition of sufficient sialic acid availability. a HEK293-6E cells were transfected to express CD43 or were mock-transfected and CD43 expression was determined by flow cytometry and compared to the respective isotype control. b Siglec-7 ligand expression was determined by a recombinant human Siglec-7 Fc staining, measured by flow cytometry. c chSiglec-7 or d chSiglec-7R124A expressing Jurkat/MA cells were co-cultured (1:1) with the CD43 or mock transfected HEK293-6E cells and luminescence was assessed using a luciferase assay (n = 5). c , d Nested data are presented of 4 experiments, each performed in duplicate. e Siglec-7 Fc binding to parental IMR-32 cells or CD43 + IMR-32 cells was assessed by flow cytometry and the MFI was quantified. f chSiglec-7 or g chSiglec-7R124A expressing Jurkat/MA cells were co-cultured (1:1) with parental IMR-32 cells or CD43 + IMR-32 cells and luminescence as assessed using a luciferase assay (n = 2). IMR-32 cells were pretreated with Ac 5 Neu5Ac or DMSO as vehicle control for 3 days, which was refreshed on day 2. Representative data are shown of two independent experiments and data present mean ± SD

Article Snippet: The open reading frame of human CD43 was cloned from a pGEM-T vector with the human CD43 open reading frame (SinoBiological, HG13108-G) into the pEGFP-N3 vector, replacing EGFP.

Techniques: Expressing, Transfection, Flow Cytometry, Recombinant, Staining, Cell Culture, Luciferase, Binding Assay

Journal: bioRxiv

Article Title: Altered X-chromosome inactivation predisposes to autoimmune manifestations in mice

doi: 10.1101/2023.04.20.537662

Figure Lengend Snippet: a. Spleen weight of wild-type (WT) and Ftx KO females at 3-months, 1-year and 2-years of age. Median values are shown. ( t-test , * p -values < 0.05). Underneath, representative images of WT and Ftx KO spleens from 3-month, 1-year and 2-year-old females. b. Representative images of hematoxylin-eosin staining on sections of spleens from 1-year-old WT and Ftx KO females. Scale bar; 100 μm. c. Representative flow cytometry analysis of splenic myeloid dendritic cells (m-DC) in WT and Ftx KO 1-year-old females. On the right, percentages of splenic m-DC (CD11b + CD11c + ) and splenic plasmacytoid dendritic cells (p-DC) (CD11c + B220 + SiglecH + ) in leucocytes. Each triangle represents a mouse. Median values are shown. ( t-test , * p -values < 0.05). d. Representative flow cytometry analysis of spontaneously activated B cells (B220 + CD69 + ) (upper panels) or of spontaneously activated T cells (CD4 + CD69 + ) (lower panels) in spleen from 1-year-old WT and Ftx KO females. Percentages in leucocytes are shown on the graphs beneath. Each triangle represents a mouse. Median values are shown. ( t-test , * p -values < 0.05). e. IgG2a natural antibody levels in sera of 1-year- and 2-year-old WT or Ftx KO females measured by ELISA. Each triangle represents a mouse. Median values are shown. ( t-test , * p -values < 0.05). f. Representative flow cytometry analysis of (B220 + CD138 + ) plasma cells in the spleen of 1-year-old WT and Ftx KO females. Percentages in leucocytes are shown on the graphs beneath. Each triangle represents a mouse. Median values are shown. ( t-test , * p -values < 0.05). g. Representative flow cytometry analysis of (CD19 + B220 lo CD5 + ) natural antibody producing B1a in the peritoneal cavity (PerC) of 1-year-old WT and Ftx KO females. Percentages in leucocytes are shown on the graphs beneath. Each triangle represents a mouse. Median values are shown. ( t-test , * p -values < 0.05). h. Cytokines levels in the blood analysed with CBA assays on sera from 3-month-, 1-year-, or 2-year-old WT and Ftx KO females. Each triangle represents a mouse. Median values are shown. ( t-test , * p -values < 0.05). i. Representative flow cytometry analysis of monocyte populations including non-classical (CD11b hi CD43 lo Ly6C + ) scavenger monocytes in the spleen of 1-year-old WT and Ftx KO females. Percentages in leucocytes are shown on the graphs beneath. Each triangle represents a mouse. Median values are shown. ( t-test , * p -values < 0.05).

Article Snippet: Bone marrow, spleen, blood and peritoneal cavity cells were stained using the following antibodies: CD3 PerCP-Vio770 (130-119-656, Miltenyi Biotec), CD4-APC (130-123-207, Miltenyi Biotec), CD5-APC-Vio770 (130-120-165, Miltenyi Biotec), CD8-FITC (130-118-468, Miltenyi Biotec), CD11b APC (553312, BD Pharmingen), CD11c PE-Vio770 (130-110-840, Miltenyi Biotec), CD19-FITC (557398, BD Pharmingen), CD21-APC-Vio770 (130-111-733, Miltenyi Biotec), CD23-PE-Vio770 (130-118-764, Miltenyi Biotec), CD38-PE (130-123-571, Miltenyi Biotec), CD43-PE (130-112-887, Miltenyi Biotec), CD69-PE (130-115-575, Miltenyi Biotec), CD138 PE-Vio615 (130-108-989, Miltenyi Biotec), F4/80 FITC (130-117-509, Miltenyi Biotec), Ter119 PE (130-112-909, Miltenyi Biotec), SiglecH APC-Vio770 (130-112-299, Miltenyi Biotec), B220-APC (130-110-847, Miltenyi Biotec), B220 VioBlue (130-110-851, Miltenyi Biotec), IgM-VioBlue (130-116-318, Miltenyi Biotec), IgD-PE (130-111-496, Miltenyi Biotec), GL7-PE-Cy7 (144619, BioLegend), Ly6C-FITC (130-111-915, Miltenyi Biotec) following recommendations of the manufacturers.

Techniques: Staining, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Clinical Proteomics

Journal: Nucleic Acids Research

Article Title: Multimode drug inducible CRISPR/Cas9 devices for transcriptional activation and genome editing

doi: 10.1093/nar/gkx1222

Figure Lengend Snippet: Design, optimization and cross-comparison of HIT transcription activation systems. ( A–C ) Cartoons illuminating the mechanisms of optimized 4-OHT induced transcription activation using a direct fusion HIT construct (A), the HIT-SAM system (B), and the HIT-SunTag system (C). ( D–I ) Cross-comparison among three optimized HIT transcription activation systems. Transcription activation induced by HIT constructs was examined in the luciferase reporter assay (D), by quantification of relative mRNA level of endogenous expression for Klf4 (E) and Oct4 (F), and by flow-cytometry analyses of CD43 protein level on the cell surface (G–I). Representative plots (G) and quantitative analyses of median CD43 fluorescent intensities (H) and overall CD43 fluorescent intensities (I) of CD43+GFP+ positive population were shown. GFP fluorescence indicates successful transfection. Cells transfected with the same amount of reporter construct or sgRNA only while keeping the total amount of transfection constant were used as negative controls (NC) in the luciferase reporter assay. Cells transfected with an ER T2 tagged GFP construct while keeping the total amount of transfection constant were used as negative control (NC) in qRT-PCR assays. Cells transfected with an unrelated sgRNA (ctl sgRNA) and GFP were used as negative control (NC) in flow-cytometry analyses. Data showed mean ± SD. n = 3 biological replicates. ns: non-significant;* P < 0.05; ** P < 0.01; *** P < 0.001; two tailed t -tests. Three biological replicates means three independently transfected samples throughout this study. The readouts without drug induction were compared in t-tests against the negative controls (NCs) for background detection.

Article Snippet: As for the CD43 activation assay, 48 h after 4-OHT was added, live cells were collected and incubated with a CD43 antibody conjugated with APC (Miltenyi Biotec) according to the manufacturer's recommended protocol.

Techniques: Comparison, Activation Assay, Construct, Luciferase, Reporter Assay, Expressing, Flow Cytometry, Fluorescence, Transfection, Negative Control, Quantitative RT-PCR, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: Multimode drug inducible CRISPR/Cas9 devices for transcriptional activation and genome editing

doi: 10.1093/nar/gkx1222

Figure Lengend Snippet: HIT2: one CRISPR/Cas9 device for simultaneous genome editing and transcriptional activation in a drug inducible manner. ( A ) Cartoon illuminating the mechanism of the optimized drug inducible HIT2 system for simultaneous genome editing and transcriptional activation. ( B ) Simultaneous editing and activation by HIT2 and Cas9–NLS–GCN4 were examined using flow-cytometry. The percentage of GFP positive cells indicated HDR efficiency, while CD43 protein level on the cell surface represented transcription activation. Representative plots (upper panels) and quantitative analyses (lower panels) were shown. Data showed mean ± SD. n = 3 biological replicates. ns: non-significant;* P < 0.05; *** P < 0.001; **** P < 0.0001; two-tailed t -tests.

Article Snippet: As for the CD43 activation assay, 48 h after 4-OHT was added, live cells were collected and incubated with a CD43 antibody conjugated with APC (Miltenyi Biotec) according to the manufacturer's recommended protocol.

Techniques: CRISPR, Activation Assay, Flow Cytometry, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: Multimode drug inducible CRISPR/Cas9 devices for transcriptional activation and genome editing

doi: 10.1093/nar/gkx1222

Figure Lengend Snippet: Comparisons of HIT systems with existing drug inducible designs. ( A–D ) Drug inducible efficiency and background activity of transcription activation was examined head-to-head between HIT systems and existing designs using the luciferase reporter assay (A), in which the expression of luciferase was controlled by a sgRNA target sequence (gLuc sgRNA), and the CD43 activation assay (B-D).Representative plots (B), quantitative analyses of the percentage of CD43 positive cells (C), and median CD43 fluorescent intensities (D) were shown. ( E ) Drug inducible efficiency and background activity of genome editing was examined using the FCR assay in comparison with existing designs. Representative plots (top) and quantifications (right bottom) were shown. NC, cells transfected with an unrelated sgRNA; PC, cells transfected with Cas9-NLS and BFP sgRNA. Data showed mean ± SD. n = 3 biological replicates. ns: non-significant;* P < 0.05; ** P < 0.01; **** P < 0.0001; two-tailed t -tests.

Article Snippet: As for the CD43 activation assay, 48 h after 4-OHT was added, live cells were collected and incubated with a CD43 antibody conjugated with APC (Miltenyi Biotec) according to the manufacturer's recommended protocol.

Techniques: Activity Assay, Activation Assay, Luciferase, Reporter Assay, Expressing, Sequencing, Comparison, Transfection, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: Multimode drug inducible CRISPR/Cas9 devices for transcriptional activation and genome editing

doi: 10.1093/nar/gkx1222

Figure Lengend Snippet: Selective and titratable drug induction of HIT systems. ( A and B ) Dose dependent transcription activation induced by HIT-SunTag (A) and HIT2 (B) with different concentration of β-estradiol or 4OHT was examined using the luciferase reporter assay. ( C and D ) Activation of endogenous gene CD43 was examined using flow cytometry. Quantitative analyses of the percentage of CD43 positive cells (C) and median CD43 fluorescent intensities (D) were shown. ( E ) Dose dependent genome editing activities of HIT2 were examined using the FCR assay upon treatment with different concentration of β-estradiol or 4-OHT. Data showed mean ± SD. n = 3 biological replicates. ns: non-significant; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; two-tailed t -tests. Fold of activation by 4-OHT over the same concentration of β-estradiol was displayed.

Article Snippet: As for the CD43 activation assay, 48 h after 4-OHT was added, live cells were collected and incubated with a CD43 antibody conjugated with APC (Miltenyi Biotec) according to the manufacturer's recommended protocol.

Techniques: Activation Assay, Concentration Assay, Luciferase, Reporter Assay, Flow Cytometry, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: Multimode drug inducible CRISPR/Cas9 devices for transcriptional activation and genome editing

doi: 10.1093/nar/gkx1222

Figure Lengend Snippet: Adaptation of HIT designs to SaCas9. ( A and B ) Drug inducible gene activation by NLS-dSaCas9–GCN4 in conjunction with scFv-2ER T2 -AD was examined in the luciferase reporter assay (A) and the endogenous CD43 activation assay (B). ( C ) Simultaneous genome editing and transcriptional activation by HIT2-SaCas9 were examined by the FCR activity and CD43 activation respectively. Cells transfected with the same amount of reporter construct while keeping the total amount of transfection constant were used as negative controls (NC). ISO represents cells stained with antibody isotype control. Data showed mean ± SD. n = 3 biological replicates. ns: non-significant;* P < 0.05; ** P < 0.01; *** P < 0.001; two-tailed t -tests.

Article Snippet: As for the CD43 activation assay, 48 h after 4-OHT was added, live cells were collected and incubated with a CD43 antibody conjugated with APC (Miltenyi Biotec) according to the manufacturer's recommended protocol.

Techniques: Activation Assay, Luciferase, Reporter Assay, Activity Assay, Transfection, Construct, Staining, Control, Two Tailed Test