Journal: Frontiers in Immunology

Article Title: Identification of a distinct cluster of GDF15 high macrophages induced by in vitro differentiation exhibiting anti-inflammatory activities

doi: 10.3389/fimmu.2024.1309739

Figure Lengend Snippet: GDF15 high macrophages exhibited reduced inflammatory activation in vitro . (A) Expression patterns of potential substitute cell surface markers for GDF15 based on the scRNA-seq data. (B) Flow cytometry results showing that rat BMMNC-derived macrophages contained a minor fraction of TNFSF9 high cells, whose expression level was correlated with that of GDF15 (from 3 independent experiments). (C) Flow cytometry verification of the correlation between TNFSF9 and GDF15 expressions in human PBMNC-derived macrophages (from 2 independent experiments). (D) Real-time PCR results showing that GDF15 high macrophages (H) exhibited reduced expressions of TNF-α, IL-1β and IL-6 in response to LPS stimulation (1 μg/mL for 6 hr), as compared to GDF15 low cells (L). Rat BMMNC-derived macrophages were FACS purified using TNFSF9 as a substitute marker for GDF15, and primed with IFN-γ (10 ng/mL for 12 hr). (E) Boyden chamber cell migration assay showing that GDF15 high macrophages (H) exhibited reduced migratory activity as compared to GDF15 low cells (L) in the absence and presence of LPS stimulation. (F) Representative fluorescent microscopic images and quantitative data showing that GDF15 high macrophages exhibited reduced phagocytic activity in the presence of LPS stimulation as compared to GDF15 low cells. Phagocytosis was assessed by internalization of fluorochrome-labeled latex beads (orange color). The macrophages were from CD68pro-GFP rats. Data were mean ± SEM. * P < 0.05, one-way ANOVA. NS, no significance.

Article Snippet: The following antibodies were used: anti-CD68 (#66231-2-Ig), anti-CD86 (#APC-65165), anti-CD80 (#PE-65083), anti-CD206 (#APC-65155) and anti-CD163 (#APC-65169) were from Proteintech; anti-GDF15 (#66231-2-Ig) was from ABclonal; anti-GDF15 (#BM5690) was from Boster (Wuhan); anti-TNFSF9 (tumor necrosis factor superfamily member 9) (#bs-3851R-APC) was from Bioss (Beijing, China); anti-interleukin (IL)-1β (#12-7018-41) was from Thermo Fisher; anti-IL-4 (#500808) was from BioLegend (San Diego, CA, USA).

Techniques: Activation Assay, In Vitro, Expressing, Flow Cytometry, Derivative Assay, Real-time Polymerase Chain Reaction, Purification, Marker, Cell Migration Assay, Activity Assay, Labeling

Journal: Science Advances

Article Title: Uncovering receptor-ligand interactions using a high-avidity CRISPR activation screening platform

doi: 10.1126/sciadv.adj2445

Figure Lengend Snippet: ( A ) FC was used to monitor binding of FITC-labeled 4-1BB magnetic beads to activator cells following up to four rounds of selection using streptavidin–4-1BB magnetic beads. ( B ) FC was used to monitor 4-1BBL expression in activator cells following up to four rounds of enrichment with streptavidin–4-1BB magnetic beads. ( C ) The gRNA enrichment following first, second, third, or fourth round of selection using streptavidin–4-1BB magnetic beads was monitored using −log 10 (score). For each library, the percentages indicate the total gRNA counts for the gene of interest divided by the total number of gRNA counts for all genes (×100). ( D ) Immunoblotting was used to detect myc in 293 cells transfected with siglec-4–myc or 4-1BB–myc. β-Actin (β-act) was included as a loading control. ( E ) FC was used to measure the level of siglec-4 or 4-1BBL on the surface of 293 cells following transfection with pCMV EV (control), pCMV–siglec-4 (siglec-4), or pCMV–4-1BBL (4-1BBL). ( F ) Binding of 4-1BB–HIS to 293 cells transfected with EV (negative control), pCMV–4-1BB (positive control), or pCMV–siglec-4. Data in (A), (B), (D), (E), and (F) were representative of three experiments.

Article Snippet: For h4-1BB protein binding assay, HEK293 cells were transfected with pCMV6 (Origene, cat. no. PS100001) or pCMV3 (SinoBiological, cat. no. CV011) EV controls, pCMV-human siglec-4 (Origene, cat. no. RC208754), or pCMV-h4-1BBL (SinoBiological, cat. no. HG15693-CM) plasmids using Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific, cat. no. 11668019) according to the manufacturer’s manual.

Techniques: Binding Assay, Labeling, Magnetic Beads, Selection, Expressing, Western Blot, Transfection, Negative Control, Positive Control

Journal: Science Advances

Article Title: Uncovering receptor-ligand interactions using a high-avidity CRISPR activation screening platform

doi: 10.1126/sciadv.adj2445

Figure Lengend Snippet: ( A to C ) FC was used to monitor (A) 4-1BB expression and 4-1BBL–Fc or siglec-4–Fc binding to activated T cells, (B) binding of siglec-4–Fc to stimulated T cells transfected with nonspecific (NS) or 4-1BB knockdown (KD) siRNAs, or (C) binding of siglec-4–Fc to stimulated T cells in the presence of increasing amounts of soluble 4-1BB–HIS protein. MFI, mean fluorescence intensity. Statistical analysis: unpaired t test. ( D ) ELISA was used to measure IFN-γ produced by activated T cells mixed with 293 cells overexpressing EV, siglec-4, 4-1BBL, or siglec-4 plus 4-1BBL. Statistical analysis: unpaired t test; P values correspond to comparisons between groups with or without siglec-4. ( E ) Luciferase assays were used to measure the viability of eGFP-FFLuc–labeled 293 target cells 24 hours after mixing with anti–TEM8–CAR-T cells. TEM8 knockout control cells (293/T8KO) were included as a specificity control. E:T, effector:target cell ratio. Statistical analysis: unpaired t test; P values correspond to comparisons between 293 and 293–Siglec-4 at each E:T cell ratio. ( F to H ) Immunoblotting was used to assess (F) p-c-Jun and c-Jun levels in 293 cells or 293–4-1BB cells following transient transfection with full-length siglec-4–myc or 4-1BB–myc, (G) p-c-Jun and c-Jun levels in unstimulated (U) or stimulated (S) T cells derived from two independent donors, and (H) p-c-Jun and c-Jun levels in T cells cocultured for 1 hour at a ratio of 1:1 with 293 cell transfected with EV (E) or siglec-4 (Sig4). Note that Siglec-4 expression can mediate the down-regulation of c-Jun only if 4-1BB is also present. β-Actin was used as a loading control in (F), (G), and (H). All data or images in (A) to (H) were representative of at least three independent experiments. For (C) to (E), n > = 3 biologically independent samples per group. ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001.

Article Snippet: For h4-1BB protein binding assay, HEK293 cells were transfected with pCMV6 (Origene, cat. no. PS100001) or pCMV3 (SinoBiological, cat. no. CV011) EV controls, pCMV-human siglec-4 (Origene, cat. no. RC208754), or pCMV-h4-1BBL (SinoBiological, cat. no. HG15693-CM) plasmids using Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific, cat. no. 11668019) according to the manufacturer’s manual.

Techniques: Expressing, Binding Assay, Transfection, Fluorescence, Enzyme-linked Immunosorbent Assay, Produced, Luciferase, Labeling, Knock-Out, Western Blot, Derivative Assay

Journal: Science Advances

Article Title: Uncovering receptor-ligand interactions using a high-avidity CRISPR activation screening platform

doi: 10.1126/sciadv.adj2445

Figure Lengend Snippet: ( A ) Depiction of the assembled dCAS9 transcriptional activator gRNA complex. ( B ) Western blot showing dCAS9 expression in the 293-VM-14.7 activator cells. ( C ) FC showing tdTomato expression in parental HEK293 cells and two activator clones following transfection with gRNA tdTomato reporter. ( D ) Schematic representation of the steps used for high–avidity-based CRISPRa screening.

Article Snippet: HEK293 cells were transfected with pCMV–4-1BBL (Sino Biological, cat. no. HG15693-CM) or pCMV–4-1BB (Sino Biological, cat. no. HG10041-CF) plasmids.

Techniques: Western Blot, Expressing, Clone Assay, Transfection