Journal: bioRxiv

Article Title: LRRC23 loss-of-function impairs radial spoke 3 head assembly and causes defective sperm motility underlying male infertility

doi: 10.1101/2023.02.25.530050

Figure Lengend Snippet: A bi-allelic splicing donor site variant in LRRC23 was identified from asthenozoospermia patients. ( A ) A consanguineous pedigree with two infertile males (IV-1 and IV-2). IV-1 was subjected for WES (arrow). Genotypes of the variant (blue) in all attended family members (III-1, III-2, IV-1, IV-2, IV-3, and IV-4) are confirmed by Sanger sequencing. +, wild-type allele. An infertile female sibling (IV-4) is marked in black circle. ( B ) Papanicolaou-stained sperm from the infertile male (IV-2). ( C ) Mapping of the LRRC23 variant. Mutation of G to A at the splicing donor site in the 5 th intron is predicted to prevent LRRC23 mRNA from splicing. ( D ) Sequencing chromatograms presenting the LRRC23 variant in the infertile male (IV-1) and his father (III-2). The variant is underlined and normal splicing donor site (GT) is boxed. ( E-F ) Minigene assay for testing altered splicing of LRRC23 by the variant. ( E ) Minigene constructs expressing LRRC23 ORF containing the 5 th intron (sashed) with wild-type (WT) or mutant (Mut, red) splicing donor site were generated. The constructs are tagged with FLAG and HA at N- and C-termini, respectively. ( F ) RT-PCR of the 293T cells transfected with the minigene constructs reveals the 5 th intron is not spliced out and retained by the variant. Intron-spanning primers, F1 and R1, are used. Three times biological replicated.

Article Snippet: cDNA clones of Human LRRC23 (HG24717-UT; SinoBiological) human RSPH3, RSPH6A , and RSPH9 (616166, 5270908, and 5296237, respectively; Horizon Discovery), and RSPH22 (OHu31347; GenScript) were purchased. cDNA clones were subcloned into phCMV3 or pGEX-6P2 vector to generate mammalian or bacterial expression constructs using Q5 Hot Start High-Fidelity 2X Master Mix (NEB) and NEBuilder HiFi DNA Assembly Kit (NEB).

Techniques: Variant Assay, Sequencing, Staining, Mutagenesis, Mini Gene Assay, Construct, Expressing, Generated, Reverse Transcription Polymerase Chain Reaction, Transfection

Journal: bioRxiv

Article Title: LRRC23 loss-of-function impairs radial spoke 3 head assembly and causes defective sperm motility underlying male infertility

doi: 10.1101/2023.02.25.530050

Figure Lengend Snippet: Lrrc23 mutant mice mimicking human splice variant phenocopy male infertility and reduced sperm motility. ( A-B ) Immunoblotting of LRRC23 in testis ( A ) and epididymal sperm ( B ) from mutant male mice. Truncated LRRC23 (arrowheads) is detected from testis microsome fraction (filled), but not in mature sperm (empty), of heterozygous (+/Δ) and homozygous (Δ/Δ) males. Acetylated tubulin (AcTub) is a loading control. ( C ) Confocal images of immunostained LRRC23 in Lrrc23 +/Δ and Lrrc23 Δ/Δ epididymal sperm. Samples from WT were used for positive or negative control of normal or truncated LRRC23 ( A, B , and C ). ( D ) Epididymal sperm counts. n.s., not significant. ( E ) Pregnancy rate of Lrrc23 +/Δ and Lrrc23 Δ/Δ males. ( F ) Number of litters from fertile females mated with Lrrc23 +/Δ and Lrrc23 Δ/Δ males. ( G ) Swimming trajectory of Lrrc23 +/Δ and Lrrc23 Δ/Δ sperm in viscous media (0.3% methylcellulose). Swimming trajectory for 2 seconds is overlaid. ( H ) Flagellar waveforms of Lrrc23 +/Δ and Lrrc23 Δ/Δ sperm before (0 minute) and after (90 minutes) inducing capacitation. Flagellar movements for two beat cycles are overlaid and color coded in time. Circles indicate sperm counts from individual males ( D ) and pup numbers from each litter ( F ). Data represented as mean ± SEM ( D and F ). Statistical comparison was perfomed by Mann-whiteny U test (D) or Student’s t-test (F). Experiments were repeated three times with biological replications ( A, B, C, G , and H ).

Article Snippet: cDNA clones of Human LRRC23 (HG24717-UT; SinoBiological) human RSPH3, RSPH6A , and RSPH9 (616166, 5270908, and 5296237, respectively; Horizon Discovery), and RSPH22 (OHu31347; GenScript) were purchased. cDNA clones were subcloned into phCMV3 or pGEX-6P2 vector to generate mammalian or bacterial expression constructs using Q5 Hot Start High-Fidelity 2X Master Mix (NEB) and NEBuilder HiFi DNA Assembly Kit (NEB).

Techniques: Mutagenesis, Variant Assay, Western Blot, Control, Negative Control, Comparison

Journal: bioRxiv

Article Title: LRRC23 loss-of-function impairs radial spoke 3 head assembly and causes defective sperm motility underlying male infertility

doi: 10.1101/2023.02.25.530050

Figure Lengend Snippet: C-terminal truncation of human LRRC23 by the splicing site mutation prevents its interaction with radial spoke (RS) head. ( A ) Sub-tomogram averaging images of RSs from Chlamydomonas reinhardtii ( left ), Trypanosoma brucei ( middle ), and mouse sperm ( right ). Original data from Electron Microscopy Data Bank was rendered. ( B ) Structure of RS in C. reinhardtii . A schematic cartoon shows the RS1 and 2. The structure of RS2 stalk is shown in inset (PDB Id: 7JRJ). ( C - D ) Purification of normal (hLRRC23 WT ) and the mutant human LRRC23 (hLRRC23 Mut ) by the splicing site mutation (c.621+1G>A) in this study. ( C ) Diagrams for the purified recombinant normal and mutant proteins tagged with tagged with GST and HA at N- and C-termini, respectively. ( D ) Purified proteins by Coomassie blue staining ( left ) and immunoblotting with a-HA ( middle ) and a-LRRC23 ( right ). Proteins matched to the predicted size were marked with asterisks. ( E ) A cartoon of the RSPH-trap approach to test LRRC23 interaction with RS proteins. Individual human RS proteins tagged with FLAG (RSPH-FLAG) are expressed in 293T cells and enriched by α-FLAG resin from cell lysates. The recombinant RSPH proteins were incubated with the purified hLRRC23 WT or hLRRC23 Mut and subjected to immunoblotting. ( F ) Interaction of hLRRC23 to a RS head component, RSPH9. The purified hLRRC23 were incubated with the RSPH-Trap (RS head, RSPH6A and RSPH9; stalk, RSPH3 and RSPH22) and subjected to immunoblotting. 5% amount of the hLRRC23s used for the trap assay were loaded as inputs. White lines in individual α-HA blot images indicate marker information (75 kDa, left ; 50 kDa, right ). Experiments were repeated four times. Purified GST was used for negative control ( SI Appendix , Fig. S4 B ). ( G ) A phylogenetic tree constructed by Maximum-likelihood analysis of the protein sequences of the C. reinhardtii RSP15 and the orthologs of LRRC23 and LRRC34. LRR37, the first LRRC23 ortholog identified in Ciona intestinalis is marked in bold. ( H ) Comparison of the reported RSP15 from C. reinhardtii and the predicted structure of LRRC23 and LRRC34 from human. Atomic structure of the C. reinhardtii RS2 containing RSP15 are represented by ribbon (RS2) and surface (RSP15) diagram ( left , PDB Id: 7JU4). Ribbon diagrams of C. reinhardtii RSP15 and AlphaFold-predicted human LRRC23 ( middle ) and LRRC34 ( right ) are shown for structural comparison. Secondary structures are color-coded. Different from C. reinhardtii RSP15 and LRRC34, LRRC23 does not display repeated α-helix (magenta) between β-sheets (gold).

Article Snippet: cDNA clones of Human LRRC23 (HG24717-UT; SinoBiological) human RSPH3, RSPH6A , and RSPH9 (616166, 5270908, and 5296237, respectively; Horizon Discovery), and RSPH22 (OHu31347; GenScript) were purchased. cDNA clones were subcloned into phCMV3 or pGEX-6P2 vector to generate mammalian or bacterial expression constructs using Q5 Hot Start High-Fidelity 2X Master Mix (NEB) and NEBuilder HiFi DNA Assembly Kit (NEB).

Techniques: Mutagenesis, Electron Microscopy, Purification, Recombinant, Staining, Western Blot, Incubation, TRAP Assay, Marker, Negative Control, Construct, Comparison

Journal: bioRxiv

Article Title: LRRC23 loss-of-function impairs radial spoke 3 head assembly and causes defective sperm motility underlying male infertility

doi: 10.1101/2023.02.25.530050

Figure Lengend Snippet: Head of the third radial spoke is absent in Lrrc23 Δ/Δ sperm flagella. ( A - B ) Sub-tomogram averaging (STA) to analyze structural defects at radial spoke (RS) of WT ( A ) and Lrrc23 Δ/Δ sperm ( B ). Shown are STA images resulted from 96-nm doublet repeats from WT and Lrrc23 Δ/Δ sperm. RS2 and 3 are magnified and density to represent RS3 head and the bridge between RS2 and RS3 (red circle) is missed in Lrrc23 Δ/Δ sperm specifically. ( C ) Overwrapped STA images from 96 nm-doublet repeats from WT (gray) and Lrrc23 Δ/Δ (gold) sperm, and Chlamydomonas reinhardtii (cyan). ( D ) A proposed model of impaired sperm motility and male infertility by the LRRC23 loss of function.

Article Snippet: cDNA clones of Human LRRC23 (HG24717-UT; SinoBiological) human RSPH3, RSPH6A , and RSPH9 (616166, 5270908, and 5296237, respectively; Horizon Discovery), and RSPH22 (OHu31347; GenScript) were purchased. cDNA clones were subcloned into phCMV3 or pGEX-6P2 vector to generate mammalian or bacterial expression constructs using Q5 Hot Start High-Fidelity 2X Master Mix (NEB) and NEBuilder HiFi DNA Assembly Kit (NEB).

Techniques:

Journal: bioRxiv

Article Title: LRRC23 loss-of-function impairs radial spoke 3 head assembly and causes defective sperm motility underlying male infertility

doi: 10.1101/2023.02.25.530050

Figure Lengend Snippet: LRRC23 mutation disrupts the third radial spoke (RS) in sperm flagellum. ( A ) Immunostaining of flagellar proteins in different compartments. Shown are midpiece (TOM20), annulus (SEPT4 and SEPT12), fibrous sheath (AKAP4), outer dense fiber (ODF2), and axoneme (acetylated tubulin, AcTub) in Lrrc23 +/Δ ( top ) and Lrrc23 Δ/Δ ( bottom ) sperm. Magnified insets are represented for annulus proteins (scale bars in insets = 2μm). Fluorescence and corresponding DIC images are merged. Sperm heads were counter stained with Hoechst. Experiments were performed with three biological replications. (B) Transmission electron microscopy images of Lrrc23 +/Δ ( left ) and Lrrc23 Δ/Δ ( right ) sperm. Shown are longitudinal section of sperm flagella. M, mitochondria; ODF, outer dense fiber; AX, axoneme; CP, central pair; MT, microtubule; FS, fibrous sheath. ( C ) Cryo-electron tomography (cryo-ET) of WT and Lrrc23 Δ/Δ sperm flagella. Shown are representative tomographic slices from WT ( left ) and Lrrc23 Δ/Δ sperm ( right ). The 9+2 axonemal structure are shown in both WT and Lrrc23 Δ/Δ in cross-sectional view ( left ). Axonemal structures are shown with proximal side of the flagellum on the left in longitudinal view ( right ). Magnified insets ( bottom ) reveal that RS1, 2, and 3 are shown in WT sperm ( left , filled arrowheads) but RS3, especially head part, is not clearly visible ( right , red arrowheads) in Lrrc23 Δ/Δ sperm. Lrrc23 +/Δ ( A and B ) or WT ( C ) sperm were used for positive control.

Article Snippet: cDNA clones of Human LRRC23 (HG24717-UT; SinoBiological) human RSPH3, RSPH6A , and RSPH9 (616166, 5270908, and 5296237, respectively; Horizon Discovery), and RSPH22 (OHu31347; GenScript) were purchased. cDNA clones were subcloned into phCMV3 or pGEX-6P2 vector to generate mammalian or bacterial expression constructs using Q5 Hot Start High-Fidelity 2X Master Mix (NEB) and NEBuilder HiFi DNA Assembly Kit (NEB).

Techniques: Mutagenesis, Immunostaining, Fluorescence, Staining, Transmission Assay, Electron Microscopy, Tomography, Positive Control

Journal: Frontiers in Oncology

Article Title: 3D CRISPR screen in prostate cancer cells reveals PARP inhibitor sensitization through TBL1XR1-SMC3 interaction

doi: 10.3389/fonc.2022.999302

Figure Lengend Snippet: CRISPR screen of prostate cancer line in 3D culture. (A) The diagram of steps involved in CRISPR screen in 3D culture. (B) Volcano plot of CRISPR screening analysis. Values on the x-axis show the beta score of each gene and the y axis shows absolute z score (see Methods). Previously identified DNA repair genes were labeled in green and selected novel candidate genes were labeled in red. TBL1XR1 is labeled in blue. (C) Enriched pathways among the top 46 hits (FDR<0.05, beta<-0.2) from negative selection using the BioPlanet 2019 database set of Enrichr (see Methods). (D, E) Selected top candidate hits were validated with individual sgRNAs in 2D (D) and 3D (E) cytotoxicity assays. Data are mean ± s.e.m., n = 3; *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001; n.s., not significant, which were calculated by two-sided t-test between the control (sgNT) and gene-targeting sgRNAs.

Article Snippet: cDNA for human TBL1XR1 was ordered from Sino Biological (Cat. No. HG20220-UT).

Techniques: CRISPR, Labeling, Selection

Journal: Frontiers in Oncology

Article Title: 3D CRISPR screen in prostate cancer cells reveals PARP inhibitor sensitization through TBL1XR1-SMC3 interaction

doi: 10.3389/fonc.2022.999302

Figure Lengend Snippet: TBL1XR1 deficiency sensitizes prostate cancer cells to PARP inhibitor olaparib. (A–F) Increased sensitivity of TBL1XR1 KO cells in 22RV1 (A–C) and OlaR (D–F) cell lines after treatment with olaparib in 2D (A, D) and 3D (B, E) culture. Micrographs show images of growth assays for 22RV1 (C) and OlaR (F) in 3D culture. Data are mean ± s.e.m. normalized to untreated cells. Solid lines show a nonlinear least-squares fit to a four-parameter dose-response model. (G–J) Reduced survival of TBL1XR1 KO cells in 22RV1 (G, I) and OlaR (H, J) cell lines after long-term treatment with olaparib. Data are mean ± s.e.m. normalized to untreated cells. *p < 0.05, **p < 0.01.

Article Snippet: cDNA for human TBL1XR1 was ordered from Sino Biological (Cat. No. HG20220-UT).

Techniques:

Journal: Frontiers in Oncology

Article Title: 3D CRISPR screen in prostate cancer cells reveals PARP inhibitor sensitization through TBL1XR1-SMC3 interaction

doi: 10.3389/fonc.2022.999302

Figure Lengend Snippet: TBL1XR1-deficient sensitizes prostate cancer cells to PARPi partially dependent on PARP trapping and drives the cells to suffer more serious DNA replication stress. (A–D) siRNA of PARP1 knockdown PARP1 protein efficiently (A) and both WT and TBL1XR1-deficient cells reverse sensitivity to olaparib (B) , niraparib (C) and talazoparib (D) when transfected with PARP1 siRNA. (E, F) DNA fiber assays of WT and TBL1XR1-deficient cells under vehicle or 10μM olaparib treatment for 4h (as the diagram in E). Representative images of DNA fibers of different groups are shown in (E) . Quantification of red fiber (CldU labeled) length from the ongoing replication was done using the ImageJ software (NIH). At least 200 fibers were measured for each group. P values calculated by two-tailed Student’s t-test. N.S. indicated not significant.

Article Snippet: cDNA for human TBL1XR1 was ordered from Sino Biological (Cat. No. HG20220-UT).

Techniques: Transfection, Labeling, Software, Two Tailed Test

Journal: Frontiers in Oncology

Article Title: 3D CRISPR screen in prostate cancer cells reveals PARP inhibitor sensitization through TBL1XR1-SMC3 interaction

doi: 10.3389/fonc.2022.999302

Figure Lengend Snippet: (A, B) Micronucleus rate of WT and TBL1XR1-deficient cells under 0μM (vehicle), 3μM or 6μM olaparib treatment for 48h. Micronucleus rate was measured by micronuclei number/total cell nucleus number in one image field (A) . 5 image fields were calculated for each experimental group (B) . P values calculated by two-tailed Student’s t-test. N.S. indicated not significant. (C, D) Comet assay of WT and TBL1XR1-deficient cells with or without olaparib treatment in alkaline condition (C) and neutral condition (D) . NT, no treatment. P values calculated by two-tailed Student’s t-test. N.S. indicated non-significant. (E–G) γH2AX foci in WT and TBL1XR1-deficient cells under olaparib treatment. Representative images of γH2AX foci and EdU staining in cells after 10μM olaparib treated for 4h were shown in (E) . γH2AX foci number per cell in EdU negative or positive cells was calculated by ImageJ software (F) . γH2AX foci number per cell in EdU positive cells under 0μM (vehicle), 1μM, 3μM or 10μM olaparib treatment for 4h (G) . (H) Western blot of DNA damage response proteins in WT and TBL1XR1-deficient cells with 10μM olaparib treatment for 0h (without treatment), 1h, 2h and 4h. * indicated γH2AX and ubiquitied γH2AX (Ub-γH2AX).

Article Snippet: cDNA for human TBL1XR1 was ordered from Sino Biological (Cat. No. HG20220-UT).

Techniques: Two Tailed Test, Single Cell Gel Electrophoresis, Staining, Software, Western Blot

Journal: Frontiers in Oncology

Article Title: 3D CRISPR screen in prostate cancer cells reveals PARP inhibitor sensitization through TBL1XR1-SMC3 interaction

doi: 10.3389/fonc.2022.999302

Figure Lengend Snippet: The interaction between TBL1XR1 and SMC3. (A) Distribution of IP-MS intensity of the two replicates. (B) Pathway analysis of IP-MS signals. (C, D) Co-IP validation of interaction between TBL1XR1 and SMC3 in 22RV1 (C) and 293T (D) cells. (E) SMC3 protein levels in 22RV1 and TBL1XR1-deficient cells after 0h, 1h, 2h and 4h treatment of 10μM olaparib. (F) Nuclear soluble and chromatin-bound SMC3 protein levels in 22RV1 and TBL1XR1-deficient cells under vehicle or olaparib treatment for 4h. The values of SMC3 protein levels are normalized to SP1 (nuclear soluble) or histone H3 (chromatin-bound) and compared to WT without treatment.

Article Snippet: cDNA for human TBL1XR1 was ordered from Sino Biological (Cat. No. HG20220-UT).

Techniques: Co-Immunoprecipitation Assay

Journal: Frontiers in Oncology

Article Title: 3D CRISPR screen in prostate cancer cells reveals PARP inhibitor sensitization through TBL1XR1-SMC3 interaction

doi: 10.3389/fonc.2022.999302

Figure Lengend Snippet: PARPi cytotoxicity and colony formation assay of SMC3 knockdown and TBL1XR1-SMC3 double knockdown (knockout) cells. (A) SMC3 knockdown efficiency in 22RV1 and TBL1XR1-knockout cells. (B–D) Cytotoxicity of olaparib (B) , niraparib (C) and talazoparib (D) in 22RV1_siNT, 22RV1_siSMC3, KO17_siNT and KO17_siSMC3 cells. siNT, non-target siRNA. (E, F) Colony formation assay and cell survival fraction calculation in 22RV1_siNT, 22RV1_siSMC3, KO17_siNT and KO17_siSMC3 cells. siNT, non-target siRNA. ** p <0.01.

Article Snippet: cDNA for human TBL1XR1 was ordered from Sino Biological (Cat. No. HG20220-UT).

Techniques: Colony Assay, Knock-Out

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: Cell reports

Article Title: Mesothelial Cell HIF1α Expression Is Metabolically Downregulated by Metformin to Prevent Oncogenic Tumor-Stromal Crosstalk

doi: 10.1016/j.celrep.2019.11.079

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: CCR2 , Sino Biological , HG16084-NF.

Techniques: Ex Vivo, Isolation, Microarray, Recombinant, Mutagenesis, Enzyme-linked Immunosorbent Assay, Software, Plasmid Preparation

Journal: bioRxiv

Article Title: Cellular electrical impedance to profile SARS-CoV-2 fusion inhibitors and to assess the fusogenic potential of spike mutants

doi: 10.1101/2022.12.13.520307

Figure Lengend Snippet: SARS-CoV-2 spike-transfected cells mimic viral envelope for fusion with ACE2 + target cell membrane. ( A ) Schematic representation of the fusion process for SARS-CoV-2 and a potential target for fusion inhibitors. See text for detailed description of the fusion process. FP, fusion peptide; HR, heptad repeat domain; 6-HB, 6 helix bundle; FI, fusion inhibitor. ( B ) Schematic representation of a split neongreen fusion assay (figure adapted from ). A549.ACE2 + cells (transfected to express the first 10 betasheets of neongreen) were overlayed with HEK293T cells co-transfected with a plasmid encoding the SARS-CoV-2 spike protein and a plasmid encoding the 11 th betasheet of neongreen. Only cell-cell fusion of an A549 cell with a HEK293T cell will result in the assembly of a functional neongreen protein and give a green fluorescence signal as the former expresses spike and the latter human ACE2. Light microscopic picture shows fused cells with neongeen expression (20x magnification). ( C ) Same as in (B). A549.ACE2 + cells were overlayed with HEK293T cells either transfected (TF) with an empty vector (left panels; mock-TF), or with Wuhan-Hu-1 S protein and left untreated (middle) or treated with the fusion inhibitor EK1 (2 μM; right panels). Light microscopic pictures were taken at 3 and 12 hours post overlay (20x magnification). Note that cell-cell fusion in the untreated spike-transfected condition is already visible at 3h post overlay but that neongreen fluorescence is still absent. Cartoons were created with BioRender ( www.biorender.com ).

Article Snippet: For pCAGGS.SARS-CoV-2_SΔ19 a PCR fragment encoding codon-optimized SARS-CoV-2 Wuhan-Hu-1 spike protein (amplified from pCMV3-C-Myc; VG40589-CM, SinoBiological) with a C-terminal 19 amino acid deletion as described in ( ) was cloned into the pCAGGS backbone.

Techniques: Transfection, Single Vesicle Fusion Assay, Plasmid Preparation, Functional Assay, Fluorescence, Expressing

Journal: bioRxiv

Article Title: Cellular electrical impedance to profile SARS-CoV-2 fusion inhibitors and to assess the fusogenic potential of spike mutants

doi: 10.1101/2022.12.13.520307

Figure Lengend Snippet: Comparison of impedance signal of A549.ACE2 + cells overlayed with mock-transfected versus Wuhan-Hu-1 SARS-CoV-2 spike-transfected HEK293T cells. At time point 0, A549.ACE2 + cells were seeded and impedance was recorded of the proliferating cell monolayer. At 24h post plating (phase #1), empty vector- (grey) and spike-transfected (blue) HEK293T cells were added. The graph depicts the raw impedance signal (expressed as cell index) over time of 4 technical replicates (mean ± SD). Vertical dotted lines 1 to 3 indicate important phases, which are further explained in the text. Note the bigger variation in CI response between the replicates during the disruption of the cell monolayer (starting at phase #2).

Article Snippet: For pCAGGS.SARS-CoV-2_SΔ19 a PCR fragment encoding codon-optimized SARS-CoV-2 Wuhan-Hu-1 spike protein (amplified from pCMV3-C-Myc; VG40589-CM, SinoBiological) with a C-terminal 19 amino acid deletion as described in ( ) was cloned into the pCAGGS backbone.

Techniques: Transfection, Plasmid Preparation

Journal: bioRxiv

Article Title: Cellular electrical impedance to profile SARS-CoV-2 fusion inhibitors and to assess the fusogenic potential of spike mutants

doi: 10.1101/2022.12.13.520307

Figure Lengend Snippet: SARS-CoV-2 spike expression correlates with the intensity and kinetics of impedance signal in CEI quantified cell-cell fusion assay. ( A ) Different ratios of A549.ACE2 + acceptor (A) and trypsinized Wuhan-Hu-1 spike-transfected HEK293T donor (D) cells. In the 1:1 cell ratio, 15,000 cells of acceptor and donor were used. The graph depicts the impedance signal (expressed as cell index) over time, starting at the moment of cell overlay, of 4 technical replicates (mean ± SD), normalized to the mock-transfected condition (grey horizontal curve). ( B ) Different amounts of Wuhan-Hu-1 SARS-CoV-2 S expressing plasmid DNA (as indicated) were added to 200 μl transfection mixture for the transfection of 400,000 HEK293T donor cells. The next day, cells were trypsinized and added to an A549.ACE2 + acceptor cell monolayer. The graph depicts the impedance signal of 2 technical replicates (mean ± SD), normalized to the mock-transfected condition (grey horizontal curve). Bar histograms at the right show the background-substracted mean fluorescence intensity (MFI) values (on a logarithmic scale) for cell surface S staining (Ab R001) of the transfected cells by flow cytometry. See also Supplementary Figure 1D for corresponding flow cytometric histogram plots. ( C ) Comparison of impedance signal of A549.ACE2 + cells either mock-transfected (dark blue) versus TMPRSS2-transfected (light blue) and overlayed by trypsinized Wuhan-Hu-1 SARS-CoV-2 S transfected HEK293T cells. The graph depicts the impedance signal of 2 technical replicates (mean ± SD), normalized to the corresponding mock-transfected HEK293T condition (grey horizontal curve).

Article Snippet: For pCAGGS.SARS-CoV-2_SΔ19 a PCR fragment encoding codon-optimized SARS-CoV-2 Wuhan-Hu-1 spike protein (amplified from pCMV3-C-Myc; VG40589-CM, SinoBiological) with a C-terminal 19 amino acid deletion as described in ( ) was cloned into the pCAGGS backbone.

Techniques: Expressing, Cell-Cell Fusion Assay, Transfection, Plasmid Preparation, Fluorescence, Staining, Flow Cytometry

Journal: bioRxiv

Article Title: Cellular electrical impedance to profile SARS-CoV-2 fusion inhibitors and to assess the fusogenic potential of spike mutants

doi: 10.1101/2022.12.13.520307

Figure Lengend Snippet: Optimization of CEI-measured S-induced cell-cell fusion. ( A ) Cell-cell fusion depends both on the expression of ACE2 on A549 cells and SARS-CoV-2 S protein on transfected HEK293T cells. Different combinations of acceptor and donor cells were tested as indicated. The graph depicts the impedance signal of 4 technical replicates (mean ± SD). Bar histograms at the right represent the cell index value at 8:43h post overlay, when the maximum was reached in the positive control. Note that no increase in impedance signal (CI max ∼ 0) was obtained in the conditions in which ACE2 and/or spike were not (over)-expressed. ( B ) Comparison of impedance signal of A549.ACE2 + cells overlayed with SARS-CoV-2 Wuhan Hu-1 S-transfected HEK293T cells, either trypsinized or collected by resuspending. The graph depicts the impedance signal of 2 technical replicates (mean ± SD). ( C ) HEK293T cells were transfected with SARS-CoV-2 Wuhan Hu-1 S. After 6h, transfection reagent was removed and cells were incubated either at 34°C or 37°C for 18h. S-expressing cells were then trypsinized, collected and administered to a A549.ACE2 + cell monolayer, and further incubated at 37°C for the CEI measurement. The graph depicts the impedance signal of 2 technical replicates (mean ± SD). ( D ) Flow cytometric histogram plots of the samples presented in (see figure legend to for experimental details). HEK293T cells were collected 24h post transfection, stained with anti-S Ab (R001) and an AF647-labeled secondary Ab. Of each sample 10,000 cells were analyzed on a FACSCelesta to calculate the mean fluorescence intensity (MFI) value. The grey histogram represents the stained mock-transfected background control sample, whereas the S-transfected cells are indicated in blue. The values in color refer to the respective MFI value.

Article Snippet: For pCAGGS.SARS-CoV-2_SΔ19 a PCR fragment encoding codon-optimized SARS-CoV-2 Wuhan-Hu-1 spike protein (amplified from pCMV3-C-Myc; VG40589-CM, SinoBiological) with a C-terminal 19 amino acid deletion as described in ( ) was cloned into the pCAGGS backbone.

Techniques: Expressing, Transfection, Positive Control, Incubation, Staining, Labeling, Fluorescence

Journal: bioRxiv

Article Title: Cellular electrical impedance to profile SARS-CoV-2 fusion inhibitors and to assess the fusogenic potential of spike mutants

doi: 10.1101/2022.12.13.520307

Figure Lengend Snippet: Validation of CEI cell-cell fusion assay with entry inhibitors of SARS-CoV-2. ( A ) The fusion inhibitor EK1 inhibits cell-cell fusion of A549.ACE2 + acceptor with S-transfected (20A.EU2 strain) donor cells. Inhibitor and donor cells were added simultaneously to the A549.ACE2 + acceptor cells. ( B ) Same as in (A) but for the attachment inhibitor R001, an RBD binding antibody that neutralizes viral entry of authentic SARS-CoV-2 virus, and with SARS-CoV-2 Wuhan-Hu-1 S. ( C ) Same as in (A) but for the entry inhibitor UDA, a carbohydrate-binding small monomeric plant lectin from stinging nettle rhizomes. The graphs on the left depict the impedance signal of 2 technical replicates (mean ± SD), normalized to the mock-transfected condition (grey horizontal curve). Bar histograms on the right show the inhibition of impedance response relative to the untreated control sample, calculated from the maximum CI values obtained for each treated sample.

Article Snippet: For pCAGGS.SARS-CoV-2_SΔ19 a PCR fragment encoding codon-optimized SARS-CoV-2 Wuhan-Hu-1 spike protein (amplified from pCMV3-C-Myc; VG40589-CM, SinoBiological) with a C-terminal 19 amino acid deletion as described in ( ) was cloned into the pCAGGS backbone.

Techniques: Cell-Cell Fusion Assay, Transfection, Binding Assay, Inhibition

Journal: bioRxiv

Article Title: Cellular electrical impedance to profile SARS-CoV-2 fusion inhibitors and to assess the fusogenic potential of spike mutants

doi: 10.1101/2022.12.13.520307

Figure Lengend Snippet: Validation of CEI cell-cell fusion assay with entry inhibitors of SARS-CoV-2. ( A ) Fusion inhibitor EK1 inhibits concentration-dependently the cell-cell fusion of S-transfected (Wuhan-Hu-1 strain) HEK293T donor with A549.ACE2 + acceptor cells. Inhibitor and donor cells were added simultaneously to the A549.ACE2 + acceptor cells. The graph on the left shows the impedance signal of 2 technical replicates (mean ± SD), normalized to the mock-transfected condition. Concentration-response curve on the right shows the inhibition of impedance response relative to the untreated control sample, calculated from the CI values obtained at the time point when maximum CI was reached in the positive control. The calculated 50% inhibitory concentration (IC 50 ) is given in the boxed insert. ( B ) The S-binding attachment inhibitor Ab R001 delays the the cell-cell fusion of S-transfected (Wuhan-Hu-1 strain) HEK293T donor with A549.ACE2 + acceptor cells transiently transfected with TMPRSS2. R001 (10 μg/ml) and donor cells were added simultaneously to the A549.ACE2 + .TMPRSS2 + acceptor cells. The graph shows the impedance signal of 2 technical replicates (mean ± SD), normalized to the mock-transfected condition. Bar histograms on the right show the maximum CI values (mean ± SD; n=2). ( C ) RBD peptide from SARS-CoV-2 Wuhan-Hu-1 S delays the cell-cell fusion of S-transfected (Wuhan-Hu-1) HEK293T donor with A549.ACE2 + acceptor cells. RBD (81 nM) and donor cells were added simultaneously to the A549.ACE2 + acceptor cells (red curve). In parallel, RBD (81 nM) was administered to a monolayer of A549.ACE2 + cells in the absence of spike-expressing cells to measure the (small) morphological changes induced by RBD binding to the ACE2 receptor (green curve). The graph shows the impedance signal of 4 technical replicates (mean ± SD), normalized to the respective mock-transfected or untreated condition. Bar histograms on the right show the maximum CI values (mean ± SD; n=4).

Article Snippet: For pCAGGS.SARS-CoV-2_SΔ19 a PCR fragment encoding codon-optimized SARS-CoV-2 Wuhan-Hu-1 spike protein (amplified from pCMV3-C-Myc; VG40589-CM, SinoBiological) with a C-terminal 19 amino acid deletion as described in ( ) was cloned into the pCAGGS backbone.

Techniques: Cell-Cell Fusion Assay, Concentration Assay, Transfection, Inhibition, Positive Control, Binding Assay, Expressing

Journal: bioRxiv

Article Title: Cellular electrical impedance to profile SARS-CoV-2 fusion inhibitors and to assess the fusogenic potential of spike mutants

doi: 10.1101/2022.12.13.520307

Figure Lengend Snippet: Plant lectin UDA inhibits CEI quantified cell-cell fusion through binding to SARS-CoV-2 S. ( A ) A monolayer of A549.ACE2 + cells were pretreated with UDA (2μM) for 1h at 37°C, washed and overlaid with S-transfected (Wuhan-Hu-1) HEK293T cells without additional compound administration. ( B ) At 24h post transfection, S-transfected (Wuhan-Hu-1) HEK293T cells were first pretreated with UDA (2μM) for 1h at 4°C, trypsinized, collected and washed. Cells were resuspended in culture medium and overlaid on a monolayer of A549.ACE2 + cells without additional compound administration. Graph show the impedance signal of 2 technical replicates (mean ± SD), normalized to the mock-transfected condition.

Article Snippet: For pCAGGS.SARS-CoV-2_SΔ19 a PCR fragment encoding codon-optimized SARS-CoV-2 Wuhan-Hu-1 spike protein (amplified from pCMV3-C-Myc; VG40589-CM, SinoBiological) with a C-terminal 19 amino acid deletion as described in ( ) was cloned into the pCAGGS backbone.

Techniques: Binding Assay, Transfection

Journal: bioRxiv

Article Title: Cellular electrical impedance to profile SARS-CoV-2 fusion inhibitors and to assess the fusogenic potential of spike mutants

doi: 10.1101/2022.12.13.520307

Figure Lengend Snippet: CEI measures the alteration in fusogenic potential of SARS-CoV-2 S variants. ( A ) Comparison of impedance signal of A549.ACE2 + cells overlayed with HEK293T cells transfected with plasmid DNA coding for SARS-CoV-2 S either from Wuhan-Hu-1, carrying D614 (blue) or a mutant with G614 (red) as found in the Nextstrain clade 20A and its descendants. In both conditions, 2.5 μg S expressing plasmid DNA was added to 200 μl transfection mixture for the transfection of 400,000 HEK293T donor cells. Graph on the left depicts the impedance signal of 2 technical replicates (mean ± SD), normalized to the mock-transfected condition (grey horizontal curve). Bar histograms on the right show the maximum CI values (mean ± SD). ( B ) Same as in (A) but for the comparison between Wuhan-Hu-1 and Omicron. ( C ) Fusion-inhibitory effect of UDA (2 μM) on different N-glycosylation deletion mutants. Mutants of Wuhan-Hu-1 S that contained two deletions of adjacent N-glycosylation sites in the S2 subunit were generated (by N into Q conversion) and analyzed in a CEI-based cell-cell fusion assay for their sensitivity to UDA. Graphs show the impedance signal of 2 technical replicates (mean ± SD), normalized to the mock-transfected condition.

Article Snippet: For pCAGGS.SARS-CoV-2_SΔ19 a PCR fragment encoding codon-optimized SARS-CoV-2 Wuhan-Hu-1 spike protein (amplified from pCMV3-C-Myc; VG40589-CM, SinoBiological) with a C-terminal 19 amino acid deletion as described in ( ) was cloned into the pCAGGS backbone.

Techniques: Transfection, Plasmid Preparation, Mutagenesis, Expressing, Generated, Cell-Cell Fusion Assay

Journal: bioRxiv

Article Title: Cellular electrical impedance to profile SARS-CoV-2 fusion inhibitors and to assess the fusogenic potential of spike mutants

doi: 10.1101/2022.12.13.520307

Figure Lengend Snippet: CEI measures the alteration in fusogenic potential of SARS-CoV-2 S variants. ( A ) Same as in but with transfection of Omicron S (BA.1 variant). ( B ) Transfected HEK293T samples (each with 2.5 μg plasmid DNA) from were collected 24h post transfection, stained with anti-S Ab (R001) and an AF647-labeled secondary Ab. Bar histograms represent the background-corrected mean fluorescence intensity (MFI) values (on a logarithmic scale), calculated from 10,000 cells analyzed by a FACSCelesta flow cytometer. ( C ) Untreated control samples from were plotted together in one graph to compare the fusion efficiency of N-glycosylation mutants of S. ( D ) Transfected HEK293T samples from (C) were collected 24h post transfection, stained with anti-S Ab (MM57) and an PE-labeled secondary Ab. Bar histograms represent the background-corrected MFI values (on a logarithmic scale), calculated from 10,000 cells analyzed by a FACSCelesta flow cytometer.

Article Snippet: For pCAGGS.SARS-CoV-2_SΔ19 a PCR fragment encoding codon-optimized SARS-CoV-2 Wuhan-Hu-1 spike protein (amplified from pCMV3-C-Myc; VG40589-CM, SinoBiological) with a C-terminal 19 amino acid deletion as described in ( ) was cloned into the pCAGGS backbone.

Techniques: Transfection, Variant Assay, Plasmid Preparation, Staining, Labeling, Fluorescence, Flow Cytometry

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Systems-level identification of PKA-dependent signaling in epithelial cells

doi: 10.1073/pnas.1709123114

Figure Lengend Snippet: AQP2 abundance in PKA knockout cells and its rescue by PKA. All observations were made in the presence of dDAVP (0.1 nM continuously). (A, D, and G) Western blots for PKA-Cα, PKA-Cβ, and AQP2 are shown for 12 control clones versus 12 PKA-Cα knockout clones (A), 13 control clones versus 11 PKA-Cβ knockout clones (D), and 12 control clones versus 12 PKA dKO (G). Loading: PKA-Cα blots, 20 μg; PKA-Cβ blots, 30 μg; AQP2 blots, 10 μg; Coomassie, 7 μg. G, glycosylated; nG, nonglycosylated. (B, C, E, F, and H) Band density quantification of the respective immunoblots using beeswarm plus boxplot visualization. Each point is a quantification of a single lane. The heavy horizontal lines represent the median. Band density for AQP2 is summed for glycosylated and nonglycosylated bands. Band densities were normalized by the mean of respective control observations. (I) Immunofluorescence images showing that clusters of PKA dKO cells transfected with PKA-Cα or PKA-Cβ plasmids express AQP2 protein. DAPI labeling shows that the cells are confluent. (Scale bars, 30 μm.) (J and K) Western blot (J) and quantification (K) of protein abundance in transfected cells (n = 3, mean ± SD, *P < 0.05). Values are normalized by band density of AQP2 in vasopressin-treated control cells and expressed as a percentage. Loading: PKA-Cα blots, 20 μg; PKA-Cβ blots, 30 μg; AQP2 blots, 60 μg; Coomassie, 7 μg.

Article Snippet: Control and PKA dKO cells were transfected with a plasmid vector to express AQP2 (Sino Biological; MG57478-UT) and immediately seeded on permeable supports and grown to confluence in the absence of vasopressin (4 to 5 d). dDAVP (0.1 nM) was added to the basolateral medium before cells were prepared for immunofluorescence and Western blotting.

Techniques: Knock-Out, Western Blot, Clone Assay, Immunofluorescence, Transfection, Labeling

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Systems-level identification of PKA-dependent signaling in epithelial cells

doi: 10.1073/pnas.1709123114

Figure Lengend Snippet: RNA-seq–based transcriptomic and SILAC-based quantitative proteomic analysis of PKA dKO versus control cells. (A) Distribution of RNA-seq reads across gene bodies of selected genes for one PKA dKO/control pair: Aqp2 (decreased), Prkaca (decreased), Prkacb (unchanged), Avpr2 (unchanged), Marcks (increased), and Rhcg (increased). Reading direction: blue, left to right; red, right to left. The vertical axis shows binned read counts normalized by total read number (read counts per 107). (B) Volcano plot for 10,190 transcripts expressed in control and PKA dKO cells, quantified for three pairs of PKA dKO vs. control clones. Red points are transcripts with FDR <0.05. Labeled transcripts are discussed in the text. (C) MS1 spectra of representative β-actin, PKA-Cα, PKA-Cβ, and AQP2 peptides showing peaks for control cells (labeled with heavy amino acids) and PKA dKO cells (labeled with light amino acids). Dashed brackets indicate the expected (but not observed) peak locations in the PKA dKO cells (light). The vertical axis shows peptide ion intensity, and the horizontal axis shows the mass-to-charge ratio (m/z). Each peptide has several m/z peaks due to the presence of natural isotopes. (D) The distribution of protein abundance changes for 7,647 proteins in PKA dKO versus control cells (n = 3 pairs). The horizontal axis shows the median log2 (dKO/Ctrl) over the three determinations. Vertical dashed lines indicate mean and ±2 SD (mean, −0.02; SD, 0.87). (E) Volcano plot for the 7,647 proteins quantified in all three pairs of samples. Red points indicate proteins with FDR <0.05. Proteins with FDR <0.05 and absolute value of log2 (dKO/Ctrl) >2 are labeled with the official gene symbol. (F) Correlation between changes in transcript abundance (RNA-seq) and protein abundance (SILAC) in PKA dKO versus control cells. Genes with FDR <0.05 in both analyses are labeled with the official gene symbol. The blue line shows linear regression (with ±SE in gray) calculated from all data.

Article Snippet: Control and PKA dKO cells were transfected with a plasmid vector to express AQP2 (Sino Biological; MG57478-UT) and immediately seeded on permeable supports and grown to confluence in the absence of vasopressin (4 to 5 d). dDAVP (0.1 nM) was added to the basolateral medium before cells were prepared for immunofluorescence and Western blotting.

Techniques: RNA Sequencing Assay, Clone Assay, Labeling

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Systems-level identification of PKA-dependent signaling in epithelial cells

doi: 10.1073/pnas.1709123114

Figure Lengend Snippet: Phosphorylation of aquaporin-2 in PKA dKO versus control mpkCCD cells. (A) AQP2 membrane-spanning topology. AQP2 has six transmembrane domains. A cluster of four vasopressin-dependent phosphorylation sites is present within the terminal 16 amino acids in the C-terminal tail. P, phosphorylation site. (B) The vasopressin-regulated phosphorylation sites are shown. Sequences surrounding Ser256, Ser264, and Ser269 are compatible with phosphorylation by basophilic protein kinases. Ser261 has a proline in position +1 and is presumably phosphorylated by a member of the MAPK family. Vasopressin decreases phosphorylation of Ser261 and increases phosphorylation at the other three sites. (C) Effect of PKA dKO on AQP2 phosphorylation levels. Both control and PKA dKO cells were transfected with AQP2, grown on a solid substratum for 24 h, and then treated with the adenylyl cyclase activator forskolin for 30 min (n = 3). Western blotting was done with phospho-specific antibodies recognizing each of the four phosphorylation sites (Upper) and quantified by densitometry (Lower). The bar graphs show normalized abundances as mean ± SD. Total AQP2 was quantified with a non–phospho-specific AQP2 antibody. (D) Low-power immunofluorescence images of total and phosphorylated AQP2 in PKA dKO and control cells. Cells were transfected with AQP2 and grown on a permeable support without dDAVP for 4 d. Subsequently, cells were stimulated with 0.1 nM dDAVP for 30 min. (Scale bars, 50 μm.)

Article Snippet: Control and PKA dKO cells were transfected with a plasmid vector to express AQP2 (Sino Biological; MG57478-UT) and immediately seeded on permeable supports and grown to confluence in the absence of vasopressin (4 to 5 d). dDAVP (0.1 nM) was added to the basolateral medium before cells were prepared for immunofluorescence and Western blotting.

Techniques: Transfection, Western Blot, Immunofluorescence

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Systems-level identification of PKA-dependent signaling in epithelial cells

doi: 10.1073/pnas.1709123114

Figure Lengend Snippet: Phosphorylation of AQP2 in PKA single-KO cells. PKA single-KO cells were grown on membrane supports in the presence of dDAVP (0.1 nM) to assure high levels of endogenous AQP2. dDAVP was withdrawn for a 2-h incubation and then was readded at 0.1 nM dDAVP for 30 min. Loading volume was adjusted to be the same total AQP2 to allow direct comparison of phosphorylation. n = 3, mean ± SD. (A) PKA-Cα single-knockout cells. (B) PKA-Cβ single-knockout cells. Bars represent normalized band densities (normalized by mean value for control cells in the absence of dDAVP).

Article Snippet: Control and PKA dKO cells were transfected with a plasmid vector to express AQP2 (Sino Biological; MG57478-UT) and immediately seeded on permeable supports and grown to confluence in the absence of vasopressin (4 to 5 d). dDAVP (0.1 nM) was added to the basolateral medium before cells were prepared for immunofluorescence and Western blotting.

Techniques: Incubation, Knock-Out

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Systems-level identification of PKA-dependent signaling in epithelial cells

doi: 10.1073/pnas.1709123114

Figure Lengend Snippet: PKA signaling mapped to functional effects of vasopressin. (A) Direct PKA targets and their physiological and functional effects. (B–H) PKA-regulated signaling network in MAP kinase signaling (B), decreased apoptosis (C), Aqp2 gene transcription (D), actin dynamics (E), AQP2 phosphorylation (F), exocytosis (G), AQP2 endocytosis (H), and AQP2 protein stability (H). Data sources are given at https://hpcwebapps.cit.nih.gov/ESBL/PKANetwork/.

Article Snippet: Control and PKA dKO cells were transfected with a plasmid vector to express AQP2 (Sino Biological; MG57478-UT) and immediately seeded on permeable supports and grown to confluence in the absence of vasopressin (4 to 5 d). dDAVP (0.1 nM) was added to the basolateral medium before cells were prepared for immunofluorescence and Western blotting.

Techniques: Functional Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Systems-level identification of PKA-dependent signaling in epithelial cells

doi: 10.1073/pnas.1709123114

Figure Lengend Snippet: Role of PKA in nuclear translocation of transcriptional regulators, histone acetylation, actin polymerization, and apical membrane trafficking of AQP2. (A) Nuclear translocation of transcriptional regulators in response to vasopressin. Western blot of nuclear and cytoplasmic extracts of various transcriptional regulators (Left). Densitometric analysis showing mean and SD (Right). CE, cytoplasmic extract; NE, nuclear extract. (B) Distribution of ChIP-seq reads across gene bodies of selected genes for vehicle- or dDAVP-treated cells. Green boxes highlight two genomic regions in which increases were observable. (C) Confocal projection x–y (Top) and x–z (Bottom thin panels) images showing changes in actin polymerization in response to vasopressin. Alexa-594 phalloidin staining in cells treated with vehicle or dDAVP. (Scale bars, 10 μm.) (D) Vasopressin-dependent AQP2 trafficking to the apical plasma membrane in control and PKA dKO cells. Confocal x–y (Top) and x–z (Bottom thin panels) images of cells treated with vehicle or dDAVP using anti-AQP2 antibody (green). DAPI-stained nuclei, blue. (Scale bars, 10 μm.)

Article Snippet: Control and PKA dKO cells were transfected with a plasmid vector to express AQP2 (Sino Biological; MG57478-UT) and immediately seeded on permeable supports and grown to confluence in the absence of vasopressin (4 to 5 d). dDAVP (0.1 nM) was added to the basolateral medium before cells were prepared for immunofluorescence and Western blotting.

Techniques: Translocation Assay, Western Blot, ChIP-sequencing, Staining

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Systems-level identification of PKA-dependent signaling in epithelial cells

doi: 10.1073/pnas.1709123114

Figure Lengend Snippet: Materials

Article Snippet: Control and PKA dKO cells were transfected with a plasmid vector to express AQP2 (Sino Biological; MG57478-UT) and immediately seeded on permeable supports and grown to confluence in the absence of vasopressin (4 to 5 d). dDAVP (0.1 nM) was added to the basolateral medium before cells were prepared for immunofluorescence and Western blotting.

Techniques: Recombinant, Transfection, Electron Microscopy, Mass Spectrometry, Bicinchoninic Acid Protein Assay, Purification, Chromatin Immunoprecipitation, Magnetic Beads, Expressing, Plasmid Preparation, Sequencing, CRISPR, Software, Microscopy

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