Journal: bioRxiv

Article Title: Efficient Methods for Target Gene Manipulation in Haematopoietic Stem Cell Derived Human Neutrophils

doi: 10.1101/2023.06.17.545406

Figure Lengend Snippet: A Map of the payload for U6-shRNA vectors. This is a bicistronic construct con-taining U6-shRNA followed by PGK-Puro-T2A-BFP. B Secondary structure of U6-driven shRNA hairpin. The predicted transcript contains a 21nt dsRNA stretch separated by a 9nt hairpin derived from hsa-miR21. C CD11b expression assessed at day 20 of differentiation by flow cytometry. Cells underwent staggered transduction during differentiation with lentivirus carrying three different U6-driven shRNA’s targeting ITGAM, as well as a nontargetign control. D Map of the payload for the tagBFP-miRshRNA vector, with the shRNA in the 3’UTR of tagBFP. In all maps, lentiviral elements up-and downstream of the payload are not shown. E Secondary structures of endogenous hsa-miR-30a (left) and miR30a-ITGAM-sh5 which contains a guide sequence targeted to the 3’LTR of ITGAM . The structure of the synthetic shRNA differs from endogenous in missing two bulges: a 1nt mismatch at position 12, and a 2nt mismatch at positions 30-32. All structures were generated with the RNAfold Webtool . F Experiments showing CD11b expression by flow cytometry at day 20 of differ-entiation after staggered transduction with lentivirus carrying miR-shRNA’s. Five different miR-shRNA’s targeted to ITGAM as well as a nontargeting control were tested. Data for both tagBFP + cells (top) or all live single cells (bottom) are shown. UT, untreated; NT, nontargeting shRNA; ITGsh1-5, shRNA’s 1-5 targeted to ITGAM . For and , individual data from six distinct donors are shown as dots, with horizontal bars indicating the median of each group with the interquartile range depicted by error bars. Asterisks indi-cate statistical significance (Friedman’s test) as detailed by bars between relevant groups: *p ≤ 0.05;**p ≤ 0.01; ***p ≤ 0.001. Gating strategy for all flow cytometry is shown in Fig. S1C, and was obtained on the Attune NxT flow cytometer.

Article Snippet: The following antibodies or staining reagents were used: Mouse-anti human CD11b-FITC (Bear-1, Hycult Biotech), mouse-anti human CD62L-Alexa Fluor 647 (DREG-56, Biolegend), mouse-anti human CD16-Pacific blue (3G8, Biolegend), mouse anti-human CD11b-Alexa Fluor 647 (ICRF44, Biolegend), Annexin V-FITC (Thermofisher Scientific), propidium iodide (Biolegend).

Techniques: shRNA, Construct, Derivative Assay, Expressing, Flow Cytometry, Transduction, Control, Plasmid Preparation, Sequencing, Generated

Journal: bioRxiv

Article Title: Efficient Methods for Target Gene Manipulation in Haematopoietic Stem Cell Derived Human Neutrophils

doi: 10.1101/2023.06.17.545406

Figure Lengend Snippet: A Differentiation timeline with nucleofection (red) after 3 days of StemSpan preconditioning. Nucleofected cells were rested in media containing StemSpan and 5 µ M Z-VAD-FMK, a pan-caspase inhibitor, to mitigate cell losses after nucleofection. Cells were then differentiated G-CSF at 10ng/mL for 14 days. B,C CD11b expression by flow cytom-etry at day 20. MFI (B), and total CD11b - the product of percent positive and MFI of the positive cells (C) are shown. D Genomic indel profiles obtained by Sanger sequencing and Tracking of Indels by DEcomposition (TIDE) analysis comparing ITGAM guide RNA 1 compared to donor-specific nontargeting controls. The Cas9 cut site is shown as 0nt, and insertions (right, positive) and deletions (left, negative) are expressed as a percentage of total sequences. Data at 0nt are indicate no sequence change. Total efficacy is shown at the left of the graph and is the sum of percentage indels. E,F Functional capacities of CRISPR-edited neutrophils. Phagocytosis of serum opsonised pHrodo red conjugated bioparticles of S. aureus (SA) and E. coli (EC) are shown in (E), and oxidative response by DHR fluores-cence are shown in (F). NT, Nontargeting guide; ITGAM KO, ITGAM gRNA1. For and 5E-F, individual data from six distinct donors are shown as dots, with horizontal bars indicating the median of each group with the interquartile range depicted by error bars. Asterisks indicate statistical significance (Friedman’s test) as detailed by bars between rel-evant groups: *p ≤ 0.05;**p ≤ 0.01; ***p ≤ 0.001. Gating strategies are shown in Fig. S1D and all flow data was obtained on the Attune NxT flow cytometer. Example outputs of TIDE analysis are shown in Fig.S2.

Article Snippet: The following antibodies or staining reagents were used: Mouse-anti human CD11b-FITC (Bear-1, Hycult Biotech), mouse-anti human CD62L-Alexa Fluor 647 (DREG-56, Biolegend), mouse-anti human CD16-Pacific blue (3G8, Biolegend), mouse anti-human CD11b-Alexa Fluor 647 (ICRF44, Biolegend), Annexin V-FITC (Thermofisher Scientific), propidium iodide (Biolegend).

Techniques: Expressing, Sequencing, Functional Assay, CRISPR, Flow Cytometry

Journal: Biomedicines

Article Title: Targeting the Glutamine Transporter SLC1A5 Enhances Sensitivity of Acute Myeloid Leukemia to MLN4924

doi: 10.3390/biomedicines14030667

Figure Lengend Snippet: Effect of MLN4924 in combination with SLC1A5 inhibitor (V9302) on AML cell proliferation and cell differentiation. ( A – C ) Evaluate the interference effect of shRNA on SLC1A5 in AML cells through RT-qPCR and Western blot. ( D , E ) The effect of SLC1A5 knockdown on the sensitivity to MLN4924 of AML cells was detected through CCK8 assay (24 h after treatment). ( F , G ) Kasumi-1 and MOLM-13 cells were treated with V9302 for 24 h and IC50 was detected through CCK8 assay. ( H – M ) CCK8 assay was used to verify the effect of MLN4924 combined with V9302 on AML cell viability. ( N – V ) CD11b, CD14, and CD16 expression on AML cell surface treated with MLN4924 and V9302 for 48 h was detected through flow cytometry. ( W ) Wright–Giemsa staining to detect morphological changes of AML cells treated with MLN4924 and V9302 for 24 h captured using an oil immersion lens (1000×). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Antibody against CD11b (Anti-Human CD11b Mouse IgG2a Recombinant Antibody, FITC-65582) was purchased from Proteintech (China).

Techniques: Cell Differentiation, shRNA, Quantitative RT-PCR, Western Blot, Knockdown, CCK-8 Assay, Expressing, Flow Cytometry, Staining

Journal: Journal of Biological Chemistry

Article Title: Cholesterol-binding protein TSPO2 coordinates maturation and proliferation of terminally differentiating erythroblasts

doi: 10.1074/jbc.ra119.011679

Figure Lengend Snippet: Figure 3. TSPO2 mutations causative of the HK RBC phenotype are detrimental to the TSPO2 function. A, copy numbers of transfected cTSPO2 cDNAs (indicated by open bars) and the contents of cTSPO2 mRNA (indicated by gray bars) in stably transfected K562 cells were estimated by quantitative PCR and quantitative RT-PCR, respectively, for the wild-type (WT), C40Y (C40Y), and VFT (VFT) cTSPO2. The cellular contents of 16-kDa cTSPO2 were quantitated by densitometric scanning of the immunoblots using the anti-cTSPO2 antibody and normalized with actin (43 kDa, indicated by black bars). A representative immunoblot from 3 independent experiments is shown in the right panel (an entire blot is shown in Fig. S1). Data are expressed as the means ± S.D. (n =3). ** P < 0.01. The migrating positions of the size markers are shown in kDa. B, fluorescent signals of incorporated NBD-cholesterol (NBD- cholesterol), non-esterified cholesterol stained with filipin III (Filipin), cholesteryl esters (CEs) stained with Nile Red (Nile Red), and incorporated FITC-transferrin (FITC-Tf) in K562 cells stably transfected with the empty vector (Control) and wild-type (WT), C40Y (C40Y), or VFT (VFT) cTSPO2 cDNA are shown. Bars, 20 μm. C, lipid droplets stained with Nile Red in B were counted and the numbers of vesicles larger than 1 μm in the major axis were compared. Data are expressed as the means ± S.D. (n = 22~28). ***P < 0.001. D, cholesterol accumulation in the ER was assessed by an LDLR-promoter luciferase assay. Activities relative to the control cells are expressed as the means ± S.D. (n =3). ** P < 0.01, ***P < 0.001. E, total cholesterol contents in the cells are expressed as the means ± S.D. (n =3).

Article Snippet: Other antibodies used are anti-canine Na,K-ATPase α-subunit (12), anti-human stomatin (13), phycoerythrin (PE)-conjugated anti-mouse CD71 (BD Pharmingen), PE-Cy7-conjugated anti-mouse CD44 and fluorescein isothiocyanate (FITC)-conjugated anti-mouse TER119 (both from Tonbo Biosciences, Japan), allophycocyanin (APC)-conjugated anti-mouse CD45.2, Gr1, and CD11b (all from BioLegend).

Techniques: Transfection, Stable Transfection, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Staining, Plasmid Preparation, Control, Luciferase

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: The Thyroid Hormone Analog GC-1 Mitigates Acute Lung Injury by Inhibiting M1 Macrophage Polarization.

doi: 10.1002/advs.202401931

Figure Lengend Snippet: Figure 2. GC-1 selectively inhibited the M1 macrophage polarization. A) Representative images of IHC staining for macrophage marker F4/80, M1 macrophage marker CD86, or M2 macrophage marker CD206 in lung sections of LPS-induced ALI model mice (n = 3), Scale bars: 100 and 20 μm (insets). B) Quantitative statistical results of IHC-stained positive cells (n = 3). C) Representative pictures of F4/80, CD86, and CD206 IF staining in lung sections from ALI mice induced by HCl (n = 3), Scale bars: 50 μm. Quantitative statistical results of fluorescence intensity from D) F4/80+CD86+ macrophages and E) F4/80+CD206+ macrophages (n = 3). F) Representative flow cytometry plots showing the proportions of M1 (CD11b+CD86+CD206−) and M2 (CD11b+CD86−CD206+) macrophages (n = 3). THP-1 cells were treated with LPS (100 ng mL−1) alone or in combination with GC-1 (100 nm) for 24 h after PMA induction. G) CD11b+CD86+CD206−M1 macrophages and H) CD11b+CD86−CD206+ M2 macrophages quantitative statistical results (n = 3). I) Immunoblot analysis of lysates from THP-1 cells (n = 3). J) Immunoblot gray values statistical quantification (n = 3). The values are shown as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns = not significant.

Article Snippet: Flow Cytometry: According to the instructions, APC Anti-Human CD206 Antibody (E-AB-F1161E, Elabscience Biotechnology), PE AntiHuman CD86 Antibody (E-AB-F1012D, Elabscience Biotechnology), and FITC Anti-Human CD11b Antibody (E-AB-F1081C, Elabscience Biotechnology) were separately added.

Techniques: Immunohistochemistry, Marker, Staining, Cytometry, Western Blot

Journal: Stem Cell Research & Therapy

Article Title: Single-cell RNA sequencing of endometrium uncovers dynamic characteristics and dysregulation of perivascular CD9 + SUSD2 + cells in thin endometrium

doi: 10.1186/s13287-025-04658-y

Figure Lengend Snippet: Single-cell transcriptomic data revealed that CD9 + SUSD2 + cells were likely to be the progenitors in the endometrium. A UMAP of cells with the associated cell types in samples among proliferative ( n = 3), secretory ( n = 3), and thin endometrium ( n = 3). Peri, perivascular cell; Str, stromal cell; pStr, proliferative stromal cell. B Expression of classical marker genes of each cell type in the endometrial samples. C Velocities derived from the dynamical model for endometrial samples are visualized as streamlines in a UMAP-based embedding. D Bar plot shows the representative GO terms of significant gene markers for perivascular CD9 + SUSD2 + cells. E Flow cytometry revealed that perivascular CD9 + SUSD2 + cells were positive for CD73, CD90, and CD105, but negative for HLA-DR, CD34, CD45, CD11b, and CD19. Negative staining results were also generated for corresponding isotype control antibodies. Grey peaks show the isotype control and red peaks display the indicated marker

Article Snippet: Subsequently, the cells were incubated in the dark with fluorescein isothiocyanate (FITC)-labeled anti-CD73 (Cat# 10904-MM07-F, Sino Biological, China), CD90 (Cat# 16897-MM10-F, Sino Biological, China), CD105 (Cat# 561443, BD Pharmingen, USA), CD34 (Cat# 68035-XM01-F, Sino Biological, China), CD45 (Cat# 10086-MM05-F, Sino Biological, China), CD11b (Cat# 03211-50, BioGems, USA), CD19 (Cat# 11880-MM17-F, Sino Biological, China), HLA-DR (Cat# 68038-XM01-F, Sino Biological, China), and Isotype Control FITC (Cat# 44212-50, BioGems, USA) at room temperature for 30 min.

Techniques: Expressing, Marker, Derivative Assay, Flow Cytometry, Negative Staining, Generated, Control