Journal: Frontiers in Molecular Neuroscience

Article Title: Neural mechanism underlies CYLD modulation of morphology and synaptic function of medium spiny neurons in dorsolateral striatum

doi: 10.3389/fnmol.2023.1107355

Figure Lengend Snippet: Key reagents and resources used in the present study.

Article Snippet: C-Myc-GRIA1 , Sino Biological , HG15792-CM.

Techniques: Recombinant, Protein Extraction, Software

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: Nature Communications

Article Title: A genome-wide CRISPR screen identifies host factors that regulate SARS-CoV-2 entry

doi: 10.1038/s41467-021-21213-4

Figure Lengend Snippet: a Sequence alignment of spike protein encompassing the cleavage site between S1 and S2 subunits. The spike proteins of SARS-CoV-2 without (Sfull strain) and with (Sdel strain) deletion were used to compare with that of SARS-CoV. The insertion of multi-basic amino acids in spike protein of SARS-CoV-2 was shown in red. b Comparison of the replication property between Sfull and Sdel strains in different cell lines. The percentage of nucleocapsid (N) protein-positive cells was analyzed by imaging-based analysis following virus infection (two or more experiments; n = 6 except for Calu-3 in which n = 8; one-way ANOVA with Dunnett’s test; mean ± s.d.). c Evaluation of entry efficiency in different cell lines infected with pseudoviruses bearing spike protein Sfull, Sdel, or S mutant (R682S, R685S). Data are normalized to the Sfull of individual experiments (two experiments; n = 6; one-way ANOVA with Dunnett’s test; mean ± s.d.). d Effect of TMPRSS2 serine protease inhibitor Camostat and cysteine protease inhibitor E-64d on Sfull or Sdel infection in different cell lines (two experiments; n = 4 or 6; one-way ANOVA with Dunnett’s test; mean ± s.d.). Data shown were normalized to the untreated group of individual experiments. **** P < 0.0001; n.s. not significant.

Article Snippet: The human TMPRSS2 (Sino Biological #HG13070-CM) and DPP4 (kindly provided by Yaowei Huang, Zhejiang University) genes were cloned by the similar strategy.

Techniques: Sequencing, Imaging, Infection, Mutagenesis, Protease Inhibitor

Journal: Nature Communications

Article Title: A genome-wide CRISPR screen identifies host factors that regulate SARS-CoV-2 entry

doi: 10.1038/s41467-021-21213-4

Figure Lengend Snippet: a Viral load in the tissues of nasal turbinate, trachea, and lung. Tissues were harvested at days 1, 2, and 4 post challenge of Sfull or Sdel virus (n = 6 per day). b Viral RNA in fecal samples. Fresh fecal samples were collected at days 2 and 4 post infection of Sfull or Sdel strain ( n = 6 per day) for qRT-PCR. c Transmission of Sfull or Sdel strain in hamsters by direct contact exposure. Naïve hamsters ( n = 6) were each co-housed with one inoculated donor at day 1 for 3 days. Hamsters were sacrificed and the indicated tissues were harvested for titration. The dashed lines represent the limit of detection by focus-forming assay. Median viral titers ( a – c ): two-tailed Mann–Whitney test; * P < 0.05; ** P < 0.01; n.s. not significant. d H&E staining of lung sections of contact hamsters. Representative images are shown from n = 6 hamsters. Scale bar, 100 μm. e , f RNA ISH of lung and nasal turbinate sections of contact hamsters. Representative images are shown from n = 6 hamsters. Scale bar, 100 μm. g Model of the role of S1/S2 boundary and host factors in regulating cell entry, pathogenicity, and transmissibility of SARS-CoV-2. SARS-CoV-2 enters cells via two pathways. The virus (Sfull) with intact spike protein preferentially enters cells at the plasma membrane (early entry pathway) in airway epithelial cells (Calu-3) or respiratory tract tissues expressing the proteases (e.g., TMPRSS2) to activate the membrane fusion. The deletion at S1/S2 junction site in spike (Sdel), however, propels the virus to enter cells through the endosomal pathway (late entry pathway), which is less efficient than the fusion pathway at the plasma membrane. Host factors such as CTSL, NCP1/2, WDR81/91, and TFE3 are critical for the endosomal entry. Both entry pathways are initiated with virion binding to receptor ACE2 at the cell surface that is regulated by host factors including retromer, CCC, and WASH complexes, etc. The more efficient early entry pathway in respiratory tract with intact spike protein than the late pathway promotes virus production, pathogenesis, and transmission in a hamster model. The SARS-CoV with spike lacking the insertion of multi-basic amino acids may resemble the Sdel virus and enter cell less efficiently than SARS-CoV-2 resulting in relatively low transmissibility.

Article Snippet: The human TMPRSS2 (Sino Biological #HG13070-CM) and DPP4 (kindly provided by Yaowei Huang, Zhejiang University) genes were cloned by the similar strategy.

Techniques: Infection, Quantitative RT-PCR, Transmission Assay, Titration, Focus Forming Assay, Two Tailed Test, MANN-WHITNEY, Staining, Expressing, Binding Assay