Journal: Acta Pharmaceutica Sinica. B

Article Title: Identification of a JAK–STAT–miR155HG positive feedback loop in regulating natural killer (NK) cells proliferation and effector functions

doi: 10.1016/j.apsb.2025.02.034

Figure Lengend Snippet: MiR155HG promotes NK cell proliferation, survival and effector functions. (A) Quantitative real-time polymerase chain reaction (RT-qPCR) assays of miR155HG levels in miR155HG knockdown (shmiR155HG) and control (Ctrl) NK92 cells with or without cytokine stimulation for 24 h. (B) Flowcytometry analysis of CD16, NKP30, NKP46 and KIR2DL5A expression on shmiR155HG and Ctrl NK92 cells after stimulated with or without cytokines for 24 h. gMFI: geometric mean fluorescence intensities. (C) Cell counting assay of shmiR155HG and Ctrl NK92 cells after stimulated with or without cytokines for 72 h. (D) Flowcytometry analysis of Ki67 expression on shmiR155HG and Ctrl NK92 cells after stimulated with or without cytokines for 24 h. (E) Flowcytometry analysis of the ratio of early apoptosis (Annexin V + 7-AAD – ) and late apoptosis (Annexin V + 7-AAD + ) of shmiR155HG and Ctrl NK92 cells after stimulated with cytokines for 48 h. (F) enzyme-linked immunosorbent assay (ELISA) of interferon-gamma (IFN- γ ) in shmiR155HG and Ctrl NK92 cells after stimulated with or without cytokines for 24 h. (G) Flowcytometry analysis of the percentages of granzyme B and perforin in shmiR155HG and Ctrl NK92 cells after stimulated with or without cytokines for 24 h. (H) Flowcytometry analysis of the percentages of CD107a on shmiR155HG and Ctrl NK92 cells after co-cultured with K562 cells in the presence or absence of indicated cytokines for 5 h. (I) RT-qPCR assays of miR155HG levels in miR155HG overexpressed (miR155HG) and control (Ctrl) NK92 cells. (J) Flowcytometry analysis of Ki67 expression on miR155HG and Ctrl NK92 cells after stimulated with IL-2 (800 U/mL) for 24 h. (K) ELISA of IFN- γ in miR155HG and Ctrl NK92 cells after stimulated with IL-2 (800 U/mL) for 24 h. (L) Flowcytometry analysis of the percentages of granzyme B and perforin in miR155HG and Ctrl NK92 cells after stimulated with IL-2 (800 U/mL) for 24 h. (M) Flowcytometry analysis of the percentages of live (Annexin V – 7-AAD – ) and early apoptosis (Annexin V + 7-AAD – ) of K562 cells after co-cultured with shmiR155HG or Ctrl NK92 cells with or without cytokines for 5 h. (N) Mouse models of human HCC were established by subcutaneous injection of HCC-LM9 into NCG-IL15 mice. shmiR155HG and Ctrl NK92 cells were administered intravenously to recipients every 7 days starting at 7 days after HCC-LM9 inoculation ( n = 8 mice/group). The relative tumor volume and the weight of excised tumors are shown. The relative values shown are fold change of tumor volume/weight at indicated times relative to the mean volume of shCtrl group on Day 7. The data from at least three independent experiments are presented as mean ± SEM (A–N); P values were assessed by unpaired (A–N) Student's t -test or two-way ANOVA (N). ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001; ∗∗∗∗ P < 0.0001; ns, not significant.

Article Snippet: The following antibodies were used in flow cytometry analysis: CD45 (#560178; BD Biosciences; #304037; BioLegend), CD56 (#562780, BD Biosciences; #392406, BioLegend; #IM2474, Beckman Coulter, Miami, FL, USA), CD3 (#300316; BioLegend), NKp46 (#331914; BioLegend), CD16 (#302012; BioLegend), CD34 (#343516; BioLegend), CD43 (#343206; BioLegend), IFN- γ (#502530; BioLegend), Ki67 (#350530; BioLegend), granzyme B (#561142; BD Biosciences), perforin (#353314; BioLegend), CD107a (#555801; BD Biosciences), 7-AAD antibodies (#559763; BD Bioscience), NKP30 (#130-112-430; Miltenyi, Bergisch Gladbach Germany), KIR2DL5A (#566330; BD Biosciences).

Techniques: Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Knockdown, Control, Expressing, Fluorescence, Cell Counting, Enzyme-linked Immunosorbent Assay, Cell Culture, Injection

Journal: Scientific Reports

Article Title: Reporter cell assay for human CD33 validated by specific antibodies and human iPSC-derived microglia

doi: 10.1038/s41598-021-92434-2

Figure Lengend Snippet: CD33 reporter cell line constructs. ( a ) Schematic drawing of the two CD33-DAP12 constructs. Both, the full CD33 ecto-domain (CD33M) and the ecto-domain lacking the sialic acid binding domain (CD33 ΔE2 ) were fused to TYROBP/DAP12. ( b ) Schematic drawing of the readouts for the CD33 reporter cell line. CD33 can be activated by ligands or antibodies, which results in increased SYK phosphorylation and consequently increased intracellular calcium levels. ( c ) Gel electrophoresis image of CD33-DAP12 constructs cloned into pcDNA5/FRT after digestion by EcoRI. ( d ) Successful exchange of the viral CMV promoter with the human EEF1A1 promoter was indicated by a second band after digestion with XhoI at 1,339 bp (CD33M) or 958 bp (CD33 ΔE2 ). ( e ) Gel electrophoresis image of pcDNA5/FRT-CD33-DAP12-GCaMP6m plasmids after digestion with XhoI. GCaMP6m positive clones exhibited three bands compared to the control (Ctrl) with only two bands. Gel images were cropped for better visualization. Supplementary Fig. shows the uncropped full-length gel.

Article Snippet: Subsequently, the samples were incubated with the CD33 antibody clones 1c7/1 (Cedarlane), WM53 (Abcam) or P67.6 (Santa Cruz) in PBS (all 5 μg/ml) for 1 h on ice followed by 30 min incubation with the secondary antibody PerCP/Cy5.5-conjugated (BioLegend) or PE-conjugated (Invitrogen) anti-mouse at 5 μg/ml in PBS in darkness on ice.

Techniques: Construct, Binding Assay, Phospho-proteomics, Nucleic Acid Electrophoresis, Clone Assay, Control

Journal: Scientific Reports

Article Title: Reporter cell assay for human CD33 validated by specific antibodies and human iPSC-derived microglia

doi: 10.1038/s41598-021-92434-2

Figure Lengend Snippet: CD33-specific and control antibodies.

Article Snippet: Subsequently, the samples were incubated with the CD33 antibody clones 1c7/1 (Cedarlane), WM53 (Abcam) or P67.6 (Santa Cruz) in PBS (all 5 μg/ml) for 1 h on ice followed by 30 min incubation with the secondary antibody PerCP/Cy5.5-conjugated (BioLegend) or PE-conjugated (Invitrogen) anti-mouse at 5 μg/ml in PBS in darkness on ice.

Techniques: Control, Imaging

Journal: Scientific Reports

Article Title: Reporter cell assay for human CD33 validated by specific antibodies and human iPSC-derived microglia

doi: 10.1038/s41598-021-92434-2

Figure Lengend Snippet: Flow cytometric analysis of CD33 surface expression. ( a ) Schematic drawing of the CD33-DAP12 constructs. Both, the full CD33 ecto-domain (CD33M) and the ecto-domain lacking the sialic acid binding domain (CD33 ∆E2 ) were fused to TYROBP/DAP12. CD33 ∆E2 can be identified by binding of the antibody clone 1c7/1 (blue) but not WM53 or P67.6 (red), whereas all three antibody clones can bind CD33M. ( b ) The CD33-DAP12 and CD33-DAP12-GCaMP6m cells were stained for CD33 surface expression with the antibody clones 1c7/1, WM53 and P67.6. A representative flow cytometry histogram plot for the CD33M-DAP12-GCaMP6m cells is shown (left side). All three tested antibodies were able to stain full-length CD33 on the cell surface. Expression of variant 2 CD33 from CD33 ∆E2 -DAP12 and CD33 ∆E2 -DAP12-GCaMP6m cells was only detected by antibody clone 1c7/1. A representative flow cytometry histogram plot for the CD33 ∆E2 -DAP12-GCaMP6m cells is shown (right side). ( c ) Quantification of CD33 staining showed a high percentage of CD33 expressing cells in the CD33M-DAP12-GCaMP6m line for all three tested antibody clones but only the CD33 antibody clone 1c7/1 was able to detect CD33 in CD33 ∆E2 -DAP12-GCaMP6m expressing cells. The antibody clones WM53 and P67.6 did not show any staining of CD33 ∆E2 -DAP12 expressing cells. ( d ) Quantification of CD33 staining revealed a high percentage of cells in the CD33M-DAP12 line expressed CD33, and was detected by all three antibody clones. CD33 in CD33 ΔE2 -DAP12 expressing cells was only detected by antibody clone 1c7/1. Data are shown as mean + SEM of three to five independent experiments; *** p ≤ 0.001 compared to Secondary Control determined by Welch ANOVA followed by Games-Howell post hoc test.

Article Snippet: Subsequently, the samples were incubated with the CD33 antibody clones 1c7/1 (Cedarlane), WM53 (Abcam) or P67.6 (Santa Cruz) in PBS (all 5 μg/ml) for 1 h on ice followed by 30 min incubation with the secondary antibody PerCP/Cy5.5-conjugated (BioLegend) or PE-conjugated (Invitrogen) anti-mouse at 5 μg/ml in PBS in darkness on ice.

Techniques: Expressing, Construct, Binding Assay, Clone Assay, Staining, Flow Cytometry, Variant Assay, Control

Journal: Scientific Reports

Article Title: Reporter cell assay for human CD33 validated by specific antibodies and human iPSC-derived microglia

doi: 10.1038/s41598-021-92434-2

Figure Lengend Snippet: pSYK detection in CD33M-DAP12 cell lines. ( a ) pSYK detection in CD33M-DAP12 reporter cells treated with CD33-specific antibodies. Addition of CD33 antibodies P67.6 and 1c7/1 resulted in increased pSYK levels, whilst P67.6 F(ab), WM53 as well as the different isotype IgG1 control antibodies did not show any change in endogenous pSYK levels. ( b ) 1c7/1 and P67.6 dose–response curve in CD33M-DAP12 reporter cells. Addition of CD33 antibody clones 1c7/1 and P67.6 resulted in an increase in endogenous pSYK levels measured 30 min after the treatment. Data are presented as mean ± or + SD; ** p ≤ 0.01 compared to untreated determined by one-way ANOVA analysis followed by Dunnett’s post hoc test.

Article Snippet: Subsequently, the samples were incubated with the CD33 antibody clones 1c7/1 (Cedarlane), WM53 (Abcam) or P67.6 (Santa Cruz) in PBS (all 5 μg/ml) for 1 h on ice followed by 30 min incubation with the secondary antibody PerCP/Cy5.5-conjugated (BioLegend) or PE-conjugated (Invitrogen) anti-mouse at 5 μg/ml in PBS in darkness on ice.

Techniques: Control, Clone Assay

Journal: Scientific Reports

Article Title: Reporter cell assay for human CD33 validated by specific antibodies and human iPSC-derived microglia

doi: 10.1038/s41598-021-92434-2

Figure Lengend Snippet: Calcium imaging in CD33-DAP12-GCaMP6m reporter cell lines. ( a ) Schematic time line of image acquisition and compound handling. ( b , c ) Calcium imaging analyzed as ΔF/F(t) in CD33-DAP12-GCaMP6m lines. Addition 100 µM dATP led to a strong increase in intracellular calcium levels in both cell lines, CD33M- and CD33 ∆E2 -DAP12-GCaMP6m, with a peak at around 20–25 s. The CD33 antibody clones 1c7/1 and P67.6 evoked a selective intracellular calcium response only in CD33M-DAP12-GCaMP6m cells. The CD33 antibody clones WM53 and P67.6 F(ab) as well as the isotype IgG1/F(ab’)2 antibodies did not show a change in intracellular calcium levels. ( d , e ) The area under the curve as well as the maximum ΔF/F(t) signal calculated from independent experiments showed a significant increase in dATP treated samples in both CD33-DAP12-GCaMP6m lines and a selective increase in CD33M-DAP12-GCaMP6m expressing cells if treated with the CD33 antibody clone P67.6 or 1c7/1. Data are presented mean + SEM; n = 3–6; *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05 compared to 10 µg/ml IgG1 determined by Welch ANOVA followed by Games-Howell post hoc test.

Article Snippet: Subsequently, the samples were incubated with the CD33 antibody clones 1c7/1 (Cedarlane), WM53 (Abcam) or P67.6 (Santa Cruz) in PBS (all 5 μg/ml) for 1 h on ice followed by 30 min incubation with the secondary antibody PerCP/Cy5.5-conjugated (BioLegend) or PE-conjugated (Invitrogen) anti-mouse at 5 μg/ml in PBS in darkness on ice.

Techniques: Imaging, Clone Assay, Expressing

Journal: Scientific Reports

Article Title: Reporter cell assay for human CD33 validated by specific antibodies and human iPSC-derived microglia

doi: 10.1038/s41598-021-92434-2

Figure Lengend Snippet: Activation of endogenous CD33 in iPSdMiG by CD33 agonistic antibodies. ( a ) pSYK analysis in TREM2 + DAP12 reporter cells. Addition of anti-TREM2 antibody AF1828 resulted in an increase in endogenous pSYK levels measured 30 min after the treatment only in TREM2 + DAP12 but not DAP12 expressing control reporter cells. Data are presented mean ± SD. ( b ) CD33 antibodies P67.6 and 1c7/1 were able to decrease the increased pSYK/tSYK levels triggered by TREM2 activation in WT iPSdMiG (left). In CD33 −/− (middle) and CD33 ΔE2 (right) iPSdMiG none of the tested antibodies was able to modulate pSYK/tSYK levels after TREM2 activation. Data are presented mean + SEM; n = 3–6; ** p ≤ 0.01 compared to IgG1 (anti-CD33 Ctrl) determined by Welch ANOVA followed by Games-Howell post hoc test. ( c ) CD33 antibodies P67.6 and 1c7/1 decreased the phagocytic uptake of pHrodo S. aureus BioParticles in WT iPSdMiG (left). In CD33 −/− (middle) and CD33 ΔE2 (right) iPSdMiG none of the tested antibodies was able to modulate pHrodo S. aureus BioParticle phagocytosis. Data are presented mean + SEM; n = 3; ** p ≤ 0.01 and * p ≤ 0.05 compared to IgG1 determined by ANOVA followed by Dunnett’s post hoc test.

Article Snippet: Subsequently, the samples were incubated with the CD33 antibody clones 1c7/1 (Cedarlane), WM53 (Abcam) or P67.6 (Santa Cruz) in PBS (all 5 μg/ml) for 1 h on ice followed by 30 min incubation with the secondary antibody PerCP/Cy5.5-conjugated (BioLegend) or PE-conjugated (Invitrogen) anti-mouse at 5 μg/ml in PBS in darkness on ice.

Techniques: Activation Assay, Expressing, Control

Journal: Cell Reports Medicine

Article Title: Antitumor immunity induced by antibody-based natural killer cell engager therapeutics armed with not-alpha IL-2 variant

doi: 10.1016/j.xcrm.2022.100783

Figure Lengend Snippet:

Article Snippet: Anti-human NKp30 APC , Miltenyi Biotec , Cat# 130-121-995, RRID:AB_2784148.

Techniques: Multiplex Assay, Recombinant, Membrane, Expressing, Saline, Polymer, Blocking Assay, Cell Isolation, cDNA Synthesis, RNA HS Assay, Software, Luminex

Journal: Frontiers in Microbiology

Article Title: Single-Molecule RNA Sequencing Reveals IFNγ-Induced Differential Expression of Immune Escape Genes in Merkel Cell Polyomavirus–Positive MCC Cell Lines

doi: 10.3389/fmicb.2021.785662

Figure Lengend Snippet: Overview of all differentially expressed genes categorized according to their activity in cancer biology.

Article Snippet: For antibody staining, 1 × 10 6 cells of each MCC cell line were cultured in a six-well plate in the presence or absence of IFNγ (3,000 U/ml) for 72 h. Cells were washed with fluorescence-activated cell sorting (FACS) buffer (dulbecco’s phosphate-buffered saline with 1% FBS and 0.2% sodium azide) and stained for the following markers: STAT1α/β, Indoleamine-2,3-dioxygenase (IDO), C-X-C motif chemokine 10 (CXCL10), PD-L1, bone marrow stromal antigen 2 (BST2), HLA class II histocompatibility antigen gamma chain (CD74), and HLA A, B, and C (HLA-ABC) with the following monoclonal antibodies: anti-human STAT1 (phycoerythine; PE, clone REA272, Miltenyi Biotec), anti-IDO1 (PE, clone eyedio, Invitrogen), anti-human CXCL10 (PE, clone REA334, Miltenyi Biotec), anti-CD274 (Pe-Cy7, clone MIH1, BD Biosciences), anti-human BST2 (PE, clone REA202, Miltenyi Biotec), anti-CD74 (PE, clone 5-329, Miltenyi Biotec), and anti–HLA-ABC (fluorescein-5-isothiocyanat, BD Biosciences).

Techniques: Activity Assay, Ubiquitin Proteomics

Journal: Frontiers in Microbiology

Article Title: Single-Molecule RNA Sequencing Reveals IFNγ-Induced Differential Expression of Immune Escape Genes in Merkel Cell Polyomavirus–Positive MCC Cell Lines

doi: 10.3389/fmicb.2021.785662

Figure Lengend Snippet: Validation of differential gene expression in MCC cell lines on protein level and mRNA level. (A) TPM values were acquired from our sequencing output. The data represent mean values ± SEM from three independent experiments (two for MKL-1 control). (B) After 72 h of treatment with or without IFNγ (3,000 U/ml), the Merkel cell carcinoma cell lines MKL-1, MKL-2, and WaGa were stained for signal transducer and activator of transcription 1-alpha/beta (STAT1), bone marrow stromal antigen 2 (BST2), C-X-C motif chemokine 10 (CXCL10), and HLA class II histocompatibility antigen gamma chain (CD74) and analyzed by flow cytometry. The average MFI of three independent experiments ± standard error is indicated. p -values were determined using the paired student’s t -test. * p < 0.05; ** p < 0.01; ns, not significant.

Article Snippet: For antibody staining, 1 × 10 6 cells of each MCC cell line were cultured in a six-well plate in the presence or absence of IFNγ (3,000 U/ml) for 72 h. Cells were washed with fluorescence-activated cell sorting (FACS) buffer (dulbecco’s phosphate-buffered saline with 1% FBS and 0.2% sodium azide) and stained for the following markers: STAT1α/β, Indoleamine-2,3-dioxygenase (IDO), C-X-C motif chemokine 10 (CXCL10), PD-L1, bone marrow stromal antigen 2 (BST2), HLA class II histocompatibility antigen gamma chain (CD74), and HLA A, B, and C (HLA-ABC) with the following monoclonal antibodies: anti-human STAT1 (phycoerythine; PE, clone REA272, Miltenyi Biotec), anti-IDO1 (PE, clone eyedio, Invitrogen), anti-human CXCL10 (PE, clone REA334, Miltenyi Biotec), anti-CD274 (Pe-Cy7, clone MIH1, BD Biosciences), anti-human BST2 (PE, clone REA202, Miltenyi Biotec), anti-CD74 (PE, clone 5-329, Miltenyi Biotec), and anti–HLA-ABC (fluorescein-5-isothiocyanat, BD Biosciences).

Techniques: Biomarker Discovery, Gene Expression, Sequencing, Control, Staining, Flow Cytometry

Journal: Scientific reports

Article Title: CREB3L2-mediated expression of Sec23A/Sec24D is involved in hepatic stellate cell activation through ER-Golgi transport.

doi: 10.1038/s41598-017-08703-6

Figure Lengend Snippet: Figure 4. COPII-mediated ER to Golgi transport is required for activation of LX-2 cells upon TGF- β1 stimulation. (a) LX-2 cells transfected with the indicated siRNA(s) were cultured for 24 h in DMEM supplemented with 10% FBS. After starvation for 24 h with DMEM supplemented with 0.5% FBS, the cells were untreated or treated with 1 ng/ml TGF-β1 and cultured for 3 days. Proteins were extracted and subjected to SDS-PAGE, followed by western blotting with anti-collagen I, anti-Sec23A, anti-Sec24D, anti-α-SMA and anti-GAPDH antibodies (lysates). Medium was collected for SDS-PAGE followed by western blotting with anti-collagen I antibody (medium). Shown is a representative immunoblot analysis (n = 3). (b and c) LX-2 cells transfected with the indicated siRNA(s) were cultured for 24 h in DMEM supplemented with 10% FBS. After starvation for 24 h with DMEM supplemented with 0.5% FBS, the cells were untreated or treated with 1 ng/ml TGF-β1 and cultured for 3 days. Proteins were extracted and subjected to SDS-PAGE followed by western blotting with anti-Sar1A, anti-Sar1B, anti-α-SMA and anti-GAPDH antibodies. (b) Representative immunoblots. (c) Quantification of immunoblots (n = 3). The band intensities of α-SMA were normalized

Article Snippet: Other antibodies were purchased from following companies: α-SMA (Abcam), CREB3L2/BBF2H7 (Atlas Antibodies), Sar1A (Novus Biological), β-actin (Chemicon), GAPDH (Chemicon), α-SMA-FITC antibody (SIGMA), Sar1B (Abnova), Smad2 (Cell signaling), pSmad2 (Cell signaling).

Techniques: Activation Assay, Transfection, Cell Culture, SDS Page, Western Blot

Journal: Glia

Article Title: Deletion of Alzheimer's disease-associated CD33 results in an inflammatory human microglia phenotype.

doi: 10.1002/glia.23968

Figure Lengend Snippet: FIGURE 1 CD33 knockout (CD33−/−) and lentiviral shRNA-mediated PTPN6 knockdown in THP1 macrophages. (a) Exemplary flow cytometry histogram graph showing expression levels of CD33 in wild type (WT) and CD33−/−macrophages. CD33 expression is absent in CD33−/−

Article Snippet: The differentiated THP1 macrophages were treated with either 10 μg/ml anti-FcγRI (CD64) antibody (clone 10.1, Santa Cruz Biotechnology #SC-1184), 10 μg/ml anti-FcγRI antibody combined with 10 μg/ml mouse IgG1 isotype control or combined with 10 μg/ml mouse anti-CD33 antibody (clone 1C7/1, Cedarlane #CL7627AP) for 5 min (SYK detection) or 10 min (ERK1/2 detection) at 37 C. CD33 antibody clone 1C7/1 was previously described as putative agonistic antibody (Taylor et al., 1999).

Techniques: Knock-Out, shRNA, Knockdown, Flow Cytometry, Expressing

Journal: Glia

Article Title: Deletion of Alzheimer's disease-associated CD33 results in an inflammatory human microglia phenotype.

doi: 10.1002/glia.23968

Figure Lengend Snippet: FIGURE 2 Transcriptome analysis of CD33 knockout and PTPN6 knockdown in THP1 macrophages. (a) Principal component analysis (PCA) of THP1 macrophage RNA sequencing. PCA resulted in four clusters representing the four individual groups wild type (WT) shCTRL, WT shPTPN6, CD33−/−shCTRL CD33−/−shPTPN6. (b) Heatmap of the top 200 most varying genes with hierarchical clustering for rows and columns stressed that the most extreme differences were between WT and CD33−/−. Minor changes were observed for PTPN6 knockdown. (c) Dotblot of KEGG pathway enrichment analysis revealed an upregulation of the inflammation-related transcription profile in comparisons 1–3 (i.e., 1. WT shCTRL vs. CD33−/−shCTRL, 2. WT shPTPN6 vs. CD33−/−shPTPN6, 3. WT shCTRL vs. WT shPTPN6) but not in comparison 4 (CD33−/−shCTRL vs. CD33−/−shPTPN6)

Article Snippet: The differentiated THP1 macrophages were treated with either 10 μg/ml anti-FcγRI (CD64) antibody (clone 10.1, Santa Cruz Biotechnology #SC-1184), 10 μg/ml anti-FcγRI antibody combined with 10 μg/ml mouse IgG1 isotype control or combined with 10 μg/ml mouse anti-CD33 antibody (clone 1C7/1, Cedarlane #CL7627AP) for 5 min (SYK detection) or 10 min (ERK1/2 detection) at 37 C. CD33 antibody clone 1C7/1 was previously described as putative agonistic antibody (Taylor et al., 1999).

Techniques: Knock-Out, Knockdown, RNA Sequencing, Comparison

Journal: Glia

Article Title: Deletion of Alzheimer's disease-associated CD33 results in an inflammatory human microglia phenotype.

doi: 10.1002/glia.23968

Figure Lengend Snippet: FIGURE 3 Activation of SYK and ERK1/2 signaling pathways as a consequence of CD33 knockout in THP1 macrophages and human-induced pluripotent stem cell-derived microglia (iPSdMiG). (a) Western blot showing phosphorylated ERK1/2 (pERK1/2, top) and total ERK1/2 (tERK1/2, bottom) in wild type (WT) and CD33−/−macrophages. One representative image of five independent experiments is shown. (b) Quantification of pERK1/2 to tERK1/2 ratio in WT (black) and CD33−/−THP1 macrophages (dark gray). Knockout of CD33 resulted in an increase in pERK1/2 in THP1 macrophages. FcγRI antibody treatment tended to increase pERK1/2 levels slightly in WT THP1 macrophages. CD33 antibody treatment following FcγRI antibody treatment tended to decrease the FcγRI antibody-driven increase in pERK1/2 to tERK1/2 levels only in WT THP1 macrophages. Data are shown as mean + SEM (n = 3–5). *p ≤.05 determined by two-way analysis of variance (ANOVA) followed by Bonferroni post hoc test. (c) Western blot showing phosphorylated SYK (pSYK, top) and total SYK (tSYK, bottom) in WT and CD33−/−macrophages. One representative image of three independent experiments is shown. (d) Quantification of pSYK to tSYK ratio in WT (black) and CD33−/−THP1 macrophages (dark gray). Knockout of CD33 resulted in an increase in pSYK in THP1 macrophages. FcγRI antibody treatment tended to increase pSYK levels slightly and co-treatment with a CD33 antibody tended to counteract this increase, both only in WT THP1 macrophages. Data are shown as mean + SEM (n = 3). *p ≤.05 determined by two-way ANOVA followed by Bonferroni post hoc test. (e) SYK phosphorylation relative to total SYK assessed by AlphaLISA. Untreated iPSdMiG revealed an increased pSYK/tSYK ratio followed by knockout of CD33 (dark gray) or expression of CD33ΔE2 (light gray) compared to WT iPSdMiG (black). Data are shown as mean + SEM (n = 6). **p ≤.01, *p ≤.05 determined by one-way ANOVA followed by Bonferroni post hoc test. (f) Treatment of iPSdMiG with activating antibodies for TREM2 resulted in an increased pSYK/tSYK ratio in WT (black), CD33−/−(dark gray) and CD33ΔE2 iPSdMiG (light gray) compared to isotype antibody-treated controls. Further, pSYK/tSYK was sharply increased in CD33−/−iPSdMiG following anti-TREM2 treatment. Data are shown as mean + SEM (n = 3). ***p ≤.001, *p ≤.05 determined by two-way ANOVA followed by Bonferroni post hoc test

Article Snippet: The differentiated THP1 macrophages were treated with either 10 μg/ml anti-FcγRI (CD64) antibody (clone 10.1, Santa Cruz Biotechnology #SC-1184), 10 μg/ml anti-FcγRI antibody combined with 10 μg/ml mouse IgG1 isotype control or combined with 10 μg/ml mouse anti-CD33 antibody (clone 1C7/1, Cedarlane #CL7627AP) for 5 min (SYK detection) or 10 min (ERK1/2 detection) at 37 C. CD33 antibody clone 1C7/1 was previously described as putative agonistic antibody (Taylor et al., 1999).

Techniques: Activation Assay, Protein-Protein interactions, Knock-Out, Derivative Assay, Western Blot, Phospho-proteomics, Expressing

Journal: Glia

Article Title: Deletion of Alzheimer's disease-associated CD33 results in an inflammatory human microglia phenotype.

doi: 10.1002/glia.23968

Figure Lengend Snippet: FIGURE 4 Inflammatory cytokine gene transcription is elevated after CD33 knockout or PTPN6 knockdown. (a) Semiquantitative real-time polymerase chain reaction (PCR) of TNFA gene transcription in THP1 macrophages. Gene transcription levels of TNFA were unchanged in untreated wild type (black) and CD33−/−(dark gray) THP1 macrophages regardless of PTPN6 knockdown (shPTPN6). Following LPS treatment TNFA levels were increased. (b) IL1B mRNA levels in THP1 macrophages determined by semiquantitative real-time PCR. Knockout of CD33 increased gene transcription of IL1B in THP1 macrophages (dark gray), which was further elevated by treatment with LPS. IL1B levels tended to be increased after PTPN6 knockdown (shPTPN6) in wild-type THP1 macrophages (black). (c) IL8 gene transcription in THP1 macrophages analyzed via semiquantitative real-time PCR. IL8 mRNA levels were increased after knockout of CD33 (dark gray) and elevated in all cell lines after LPS treatment. PTPN6 knockdown (shPTPN6) attenuated the increase in IL8 transcription in CD33−/−THP1 macrophages. (d) Semiquantitative real-time PCR of IL10 mRNA levels in THP1 macrophages. Gene transcription levels of IL10 were increased in CD33−/−THP1 macrophages (dark gray). Knockdown of PTPN6 decreased IL10 gene transcription below the control vector levels (shCTRL) in both, untreated and LPS-treated THP1 macrophages. Data are shown as mean + SEM (n = 3–7). ***p ≤.001, **p ≤.01, *p ≤.05 determined by two-way analysis of variance (ANOVA) followed by Bonferroni post hoc test. (e–i) Gene transcription levels of the inflammatory cytokines TNFA, IL1B, IL8, IL10, and IL6 analyzed in wild type (WT) (black), CD33−/−(dark gray) and CD33ΔE2 human-induced pluripotent stem cell-derived microglia (iPSdMiG) (light gray) by semiquantitative real-time PCR. Knockout of CD33 as well as expression of CD33ΔE2 in iPSdMiG resulted in constitutively elevated mRNA levels of (e) TNFA, (f) IL1B, (g) IL8, (h) IL10, and (i) IL6. TNFA, IL10 and IL6 mRNA levels were decreased in CD33ΔE2 iPSdMiG compared to CD33−/−

Article Snippet: The differentiated THP1 macrophages were treated with either 10 μg/ml anti-FcγRI (CD64) antibody (clone 10.1, Santa Cruz Biotechnology #SC-1184), 10 μg/ml anti-FcγRI antibody combined with 10 μg/ml mouse IgG1 isotype control or combined with 10 μg/ml mouse anti-CD33 antibody (clone 1C7/1, Cedarlane #CL7627AP) for 5 min (SYK detection) or 10 min (ERK1/2 detection) at 37 C. CD33 antibody clone 1C7/1 was previously described as putative agonistic antibody (Taylor et al., 1999).

Techniques: Knock-Out, Knockdown, Real-time Polymerase Chain Reaction, Control, Plasmid Preparation, Derivative Assay, Expressing

Journal: Glia

Article Title: Deletion of Alzheimer's disease-associated CD33 results in an inflammatory human microglia phenotype.

doi: 10.1002/glia.23968

Figure Lengend Snippet: FIGURE 5 Increased gene transcription of INPP5D (SHIP1) caused by loss of CD33.(a + b) Gene transcription of SIRPA measured by semiquantitative real-time polymerase chain reaction (qRT-PCR). SIRPA mRNA levels were increased in CD33−/−(dark gray) and PTPN6 knockdown (shPTPN6) wild type (WT) THP1 macrophages (black). PTPN6 knockdown in CD33−/−

Article Snippet: The differentiated THP1 macrophages were treated with either 10 μg/ml anti-FcγRI (CD64) antibody (clone 10.1, Santa Cruz Biotechnology #SC-1184), 10 μg/ml anti-FcγRI antibody combined with 10 μg/ml mouse IgG1 isotype control or combined with 10 μg/ml mouse anti-CD33 antibody (clone 1C7/1, Cedarlane #CL7627AP) for 5 min (SYK detection) or 10 min (ERK1/2 detection) at 37 C. CD33 antibody clone 1C7/1 was previously described as putative agonistic antibody (Taylor et al., 1999).

Techniques: Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Knockdown

Journal: Glia

Article Title: Deletion of Alzheimer's disease-associated CD33 results in an inflammatory human microglia phenotype.

doi: 10.1002/glia.23968

Figure Lengend Snippet: FIGURE 7 Knockout of CD33 increased radical production in human macrophages and microglia. (a) Production of radicals in THP1 macrophages was assessed by dihydroethidium (DHE) staining and confocal microscopy. Radical production was increased by Aβ1-42 in all four cell lines indicated by DHE staining intensity. Thereby, Aβ1-42-induced radical production in CD33−/−shCTRL macrophages (dark gray) was enhanced compared to the corresponding wild-type (black) shCTRL line. Treatment with scavengers SOD1 and Trolox attenuated the Aβ1-42- driven effect to untreated levels. Data are shown as mean + SEM (n = 4). ***p ≤.001, **p ≤.01, *p ≤.05 determined by two-way analysis of variance (ANOVA) followed by Bonferroni post hoc test. (b) Reactive oxygen species (ROS) production in human-induced pluripotent stem cell- derived microglia (iPSdMiG) analyzed by DHE staining. Wild type (WT) (black), CD33−/−(dark gray) and CD33ΔE2 (light gray) iPSdMiG exhibited similar constitutive ROS production. Treatment of the phagocytes with bacterial particles from S. aureus resulted in an elevated production of ROS in all iPSdMiG lines with a higher magnitude in CD33−/−iPSdMiG. Treatment with the scavenger NAC decreased ROS production to untreated levels. Data are shown as mean + SEM (n = 3–6). ***p ≤.001, **p ≤.01 determined by two-way ANOVA followed by Bonferroni post hoc test

Article Snippet: The differentiated THP1 macrophages were treated with either 10 μg/ml anti-FcγRI (CD64) antibody (clone 10.1, Santa Cruz Biotechnology #SC-1184), 10 μg/ml anti-FcγRI antibody combined with 10 μg/ml mouse IgG1 isotype control or combined with 10 μg/ml mouse anti-CD33 antibody (clone 1C7/1, Cedarlane #CL7627AP) for 5 min (SYK detection) or 10 min (ERK1/2 detection) at 37 C. CD33 antibody clone 1C7/1 was previously described as putative agonistic antibody (Taylor et al., 1999).

Techniques: Knock-Out, Staining, Confocal Microscopy, Derivative Assay