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

Journal: International Journal of Biological Sciences

Article Title: CD137L-macrophage induce lymphatic endothelial cells autophagy to promote lymphangiogenesis in renal fibrosis

doi: 10.7150/ijbs.66781

Figure Lengend Snippet: Expression of CD137L on macrophages in UUO mice. Mice were subjected to unilateral ureteral obstruction (UUO) by left ureteral ligation for 7 and 14 days. CD137L mRNA and protein expressions were detected by Real-time PCR (A) and Elisa (B). (C) Scheme of the cell-sorting approach. (D) Flow cytometric analysis and quantification of CD137L + macrophages in sham and UUO mice. kidney cell suspensions from the obstructed kidneys. (E) Immunofluorescence staining showing CD137L (green) expression and colocalization (white arrows) with F4/80 (red) in UUO kidneys. The error bars represent the SEM. ***P < 0.001. Original magnification, Scale bar, 20µm. n= 6 per group. The data were pooled from three independent experiments.

Article Snippet: Recombinant CD137L (Cat: HY-P7446) was purchased from MedChemExpress (Shanghai, China).

Techniques: Expressing, Ligation, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, FACS, Immunofluorescence, Staining

Journal: International Journal of Biological Sciences

Article Title: CD137L-macrophage induce lymphatic endothelial cells autophagy to promote lymphangiogenesis in renal fibrosis

doi: 10.7150/ijbs.66781

Figure Lengend Snippet: VEGF-C promotes CD137L production in macrophages in vitro. (A)Relative mRNA expression of CD137L was measured in BMDMs treated with VEGF-C (0, 20, 50, 100, 1000 ng/mL) subpopulations by real-time PCR. Flow cytometric analysis and quantification showing the percentages of CD137L + MPMs (B, C) and BMDMs (D, E) with VEGF-C (100 ng/mL) and SAR131675 (23 nM). The data shown were representative FACS profiles. (F) Flow cytometric analysis showing the percentages of CD137L + BMDMs with TGF-β(10ng/mL), VEGF-C (100 ng/mL), L-1β (25ng/mL) and mixture of cytokines. The data shown were representative FACS profiles. (G) BMDMs were grown in complete medium and treated with VEGF-C (100 ng/mL) with or without SAR131675 (23 nM, 46 nM) for 48 h. Western blot analysis was performed to measure the expression of TLR4, p-P65, P65, IKK-β and GAPDH. (H) The histograms show the relative intensity for each marker normalized to GAPDH. The error bars represent the SEM. *P< 0.05, **P < 0.01; ***P < 0.001 versus control. The data were pooled from three independent experiments.

Article Snippet: Recombinant CD137L (Cat: HY-P7446) was purchased from MedChemExpress (Shanghai, China).

Techniques: In Vitro, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Marker, Control

Journal: International Journal of Biological Sciences

Article Title: CD137L-macrophage induce lymphatic endothelial cells autophagy to promote lymphangiogenesis in renal fibrosis

doi: 10.7150/ijbs.66781

Figure Lengend Snippet: CD137L promotes the proliferation, migration and tube formation of lymphatic endothelial cells. (A) The proliferation of LECs treated with CD137L or CD137L and inhibitory for 24 h, 48 h, and 72 h was examined by CCK-8 assay. The migration of LECs was measured with the cell scratch test (B) and Transwell migration assay (D) after CD137L or CD137L and inhibitory treatment for 12 h. The histograms represent migrated (C, E) cells per field. (F) A tube formation assay was used to test the lymphangiogenesis capacity of LECs after CD137L or CD137L and inhibitory treatment for 3 h (image magnification, 100×). The histograms represent the branch number per field. (G, H, I). The error bars represent the SEM. *P< 0.05, **P < 0.01; ***P < 0.001. The data were pooled from three independent experiments.

Article Snippet: Recombinant CD137L (Cat: HY-P7446) was purchased from MedChemExpress (Shanghai, China).

Techniques: Migration, CCK-8 Assay, Transwell Migration Assay, Tube Formation Assay

Journal: International Journal of Biological Sciences

Article Title: CD137L-macrophage induce lymphatic endothelial cells autophagy to promote lymphangiogenesis in renal fibrosis

doi: 10.7150/ijbs.66781

Figure Lengend Snippet: Transcriptome analysis of LECs after CD137L treatment (A) Eleven significantly activated KEGG pathway in SVEC4-10 cell line after treatment with CD137L. (B) Enriched KEGG pathway for differentially expressed genes in SVEC4-10 cells with or without CD137L treatment. The terms enriched from upregulated genes in CD137L treated SVEC4-10 cells are marked by red, and terms enriched from down regulated genes are marked by blue. (FDR≤0.01) (C) Top20 significantly activated biological processes in SVEC4-10 cell line after treatment with CD137L, as indicated in GO analysis (FDR≤0.01). GO, gene ontology; DEGs, differentially expressed genes; FDR, false discovery rate; KEGG, Kyoto Encyclopedia of Genes and Genomes.

Article Snippet: Recombinant CD137L (Cat: HY-P7446) was purchased from MedChemExpress (Shanghai, China).

Techniques:

Journal: International Journal of Biological Sciences

Article Title: CD137L-macrophage induce lymphatic endothelial cells autophagy to promote lymphangiogenesis in renal fibrosis

doi: 10.7150/ijbs.66781

Figure Lengend Snippet: CD137L induced autophagy of lymphatic endothelial cells via mTOR pathway. (A) Representative electron micrographs of CD137L-induced autophagosome in SVEC4-10 cells that were either stimulated with inhibitory, or with 3-MA for 24 h. (B) The protein expression levels of autophagy-associated proteins (Atg5, Atg7 and Atg12) and autophagic substrates (LC3 and p62) were analyzed in SVEC4-10 cells after CD137L, inhibitory or 3-MA treatment for 24 h by Western blot. (C) SVEC4-10 cells were infected with RFP-GFP-LC3-expressing lentivirus. Cells were untreated or treated with CD137L, inhibitory or 3-MA for 24 h. Fluorescence was examined by confocal microscopy. Transwell migration assay (D), scratch test (E) and tube formation assay (F) stimulated with CD137L for 12 h, and with or without 3-MA treatment. (G) The protein expression levels of phosphorylated PI3K, Akt and mTOR were analyzed in SVEC4-10 cells after CD137L or inhibitory or 3-MA treatment for 24 h by Western blot. (H) SVEC4-10 cells were knocked down Atg5 and Atg7 by using siRNAs, and stimulated with CD137L for 24h, the protein expression levels of autophagy-associated proteins (Atg5, Atg7 and Atg12) and autophagic substrates (LC3 and p62) were detected by Western blotting.

Article Snippet: Recombinant CD137L (Cat: HY-P7446) was purchased from MedChemExpress (Shanghai, China).

Techniques: Expressing, Western Blot, Infection, Fluorescence, Confocal Microscopy, Transwell Migration Assay, Tube Formation Assay