Journal: iScience

Article Title: SARS-CoV-2 N protein mediates intercellular nucleic acid dispersion, a feature reduced in Omicron

doi: 10.1016/j.isci.2023.105995

Figure Lengend Snippet: N protein binds to and enters the cell through STEAP2 (A) Comparison of the cell-binding capacity of SARS-CoV-2 wild type (WT) N protein and Omicron N protein expressed in either E. coli or mammalian cells. 1 × 10 5 A549 cells were used to mixed with 1 μg WT N or Omicron N proteins. One hour after protein addition, allophycocyanin (APC) conjugated anti-His antibody was used to detect the cell binding capacity of WT N protein or Omicron N protein. The samples were analyzed by flow cytometry and data are shown as mean fluorescence intensity (MFI). (B) Antibody blocking assay. Aliquots of 10 μg of SARS-CoV-2 N protein were pre-mixed with 0, 1, 3, 10, 30, and 100 μg of normal mouse IgG or anti-N monoclonal antibody (NP-mAb-40) and incubated at 4°C overnight. The antibody/N protein complex was used for the A549 cell surface binding assay. The blocking capacity of anti-N antibody was normalized to N protein only control. (C) Membrane fractions of A549 and HPAEpiC cells were extracted and incubated with N protein conjugated beads for 3 h binding at 4°C, and pull-downed for LC-MS-MS analysis (upper panels). A549 and HPAEpiC cells were suspended and treated with N protein for 1 h on ice. After incubation, cells were crosslinked with 3 mM DTSSP for 1.5 h. Then, cells were lysed in RIPA lysis buffer, and N protein complex in the lysate was immunoprecipitated for LC-MS-MS analysis (lower panels). Y axis denotes −logP values while the X axis shows log2 fold change values. Orange dots highlight the statistically significant proteins, with p value < 0.05 (-Log p > 1.3) and fold change>2, and the enriched plasma membrane protein was labeled on the plot. Identified proteins were further sorted by HuMemProtDB. (D) To knock-down (KD) STEAP2 expression, HPAEpiC cells were infected with lentivirus carrying STEAP2 shRNA followed by puromycin selection for 14 days. The STEAP2 mRNA expression levels were assessed by qRT-PCR, and the relative KD efficiency of shSTEAP2 was compared to shLacZ control (left-hand side panel). N protein binding capabilities to HPAEpiC STEAP2 KD cells and shLacZ control KD cells were assessed by flow cytometry analysis, and data were shown as mean fluorescence intensity (MFI). (right-hand side panel). (E) Western blot analysis of STEAP2 in wild type (WT) and knock-out (KO) A549 cells were shown. N protein binding to A549 STEAP2 KO cells was assessed by flow cytometry analysis and shown as mean fluorescence intensity (MFI). Ccr (crotonyl-CoAcarboxylase/reductase, a bacterial protein) binding was used as a control. (F) SARS-CoV-2 N protein enters alveolar cells. HPAEpiC cells were treated with 10 μg SARS-CoV-2 N protein overnight and then stained with anti-N antibody. The localization of N protein (Red) was checked by fluorescence microscope and cell morphology was observed by dimensional interference contrast (DIC). Nuclei of cells were stained by DAPI (blue). (G) N protein entering cells by endocytosis and N protein co-localization with STEAP2. HPAEpiC alveolar cells were seeded on 8 well slides. Cells were pretreated with endocytosis inhibitors HCQ, or Dynasore. Then the cells were treated with N protein overnight. After treatment, the cells were stained by specific antibodies to detected N protein (red), endosome marker (EEA1) (green), and STEAP2 (yellow). Cells were observed under fluorescent microscopy (Invitrogen tech.). Scale bar: 50 μm. All data are shown as mean ± SEM. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗∗p < 0.0001; t test. See also Figure S5 .

Article Snippet: Human embryonic kidney 293T cells (American Type Culture Collection, CRL-3216), human cervical cancer HeLa cells (American Type Culture Collection, CCL-2) and mouse lung cancer LL2 cells (American Type Culture Collection, CRL-1642) were cultured in DMEM (Gibco, 11965-065) and human lung adenocarcinoma A549 cells (American Type Culture Collection, CCL-185), human colon adenocarcinoma HCT-8 cells (American Type Culture Collection, CCL-244) and mouse mammary gland epithelium 4T1 cells (American Type Culture Collection, CRL-2539) were cultured in RPMI 1640 Medium (Gibco, 22400-071), respectively.

Techniques: Comparison, Binding Assay, Flow Cytometry, Fluorescence, Antibody Blocking Assay, Incubation, Blocking Assay, Control, Membrane, Liquid Chromatography with Mass Spectroscopy, Lysis, Immunoprecipitation, Clinical Proteomics, Labeling, Knockdown, Expressing, Infection, shRNA, Selection, Quantitative RT-PCR, Protein Binding, Western Blot, Knock-Out, Staining, Microscopy, Marker

Journal: iScience

Article Title: SARS-CoV-2 N protein mediates intercellular nucleic acid dispersion, a feature reduced in Omicron

doi: 10.1016/j.isci.2023.105995

Figure Lengend Snippet: N protein delivers nucleic acids into cells (A) N protein-RNA complex binding to the cell surface. Aliquots of 10 μg SARS-CoV-2 N protein were incubated with 1 μg of indicated RNAs for 1 h at 4°C, and added to A549 or HPAEpiC cultures. SARS-CoV-2 N protein only without RNA was used as a control. The samples were analyzed by flow cytometry and data are shown as mean fluorescence intensity (MFI). Data are shown as mean ± SEM. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; t test. (B) The observation of N protein-RNA enters into cells. HPAEpiC were seeded onto 8-well glass slides (40,000 cells/well). SARS-CoV-2 N protein 10 μg and 40 μg RNA-FAM (green) were mixed for 1 h at 4°C. cells were treated with SARS-CoV-2 N-RNA-FAM mixture for 1 h. The groups of non-treated cells and RNA-FAM only were as controls. After treatment, N protein was detected by anti-N antibody (Red). The localization of RNA-FAM was green. DAPI (blue) indicates cell nuclei. Scale bar: 15 μm. (C) Lattice light sheet microscopy time lapse imaging of N protein-RNA complex entering into HPAEpiC cells. SARS-CoV-2 N protein 10 μg was mixed with 40 μg RNA-FAM (fluorescein) for 1 h at 4°C and then treated with ice-cooled alveolar cells. The signals of RNA-FAM and Hochest 33,342 were monitored by lattice light sheet microscopy at different time points.

Article Snippet: Human embryonic kidney 293T cells (American Type Culture Collection, CRL-3216), human cervical cancer HeLa cells (American Type Culture Collection, CCL-2) and mouse lung cancer LL2 cells (American Type Culture Collection, CRL-1642) were cultured in DMEM (Gibco, 11965-065) and human lung adenocarcinoma A549 cells (American Type Culture Collection, CCL-185), human colon adenocarcinoma HCT-8 cells (American Type Culture Collection, CCL-244) and mouse mammary gland epithelium 4T1 cells (American Type Culture Collection, CRL-2539) were cultured in RPMI 1640 Medium (Gibco, 22400-071), respectively.

Techniques: Binding Assay, Incubation, Control, Flow Cytometry, Fluorescence, Microscopy, Imaging

Journal: iScience

Article Title: SARS-CoV-2 N protein mediates intercellular nucleic acid dispersion, a feature reduced in Omicron

doi: 10.1016/j.isci.2023.105995

Figure Lengend Snippet: N protein-assisted nucleic acid dispersion and expression in the co-culture environment (A)The co-culture system consisted of A549 as recipient cells, and 293T pre-transfected with two plasmids, one expressing GFP and the other expressing SARS-CoV-2 N protein or the pcDNA3.1 empty vector. (B–E) After 24 h co-culture of the donor cells and recipient cells, cell pool was stained with cytokeratin 18 (an A549 marker) and SV40 large T antigen (a 293T marker). A549 cells in the cell pool were gated from cytokeratin 18 positive and large T antigen negative. A549 GFP positive percentage was further assessed by flow cytometry analysis. Effects of SARS-CoV-2 N variants (B), treatment with RANTES (C), the p38 inhibitor SB203580 (D), or anti-N neutralizing antibody (E) were accessed by adding these effectors to the medium. Experiments are performed in three to five biological replicates. ∗, p value <0.05 (paired two-tailed student’s t -test). (F) SARS-CoV-2 N protein promotes gene delivery by cell-free diffusion to neighboring cells. A549 cells were plated in the lower chamber, while 293T donor cells co-transfected with plasmids expressing EGFP and indicated N proteins in the upper chamber. After 3 days of co-culture, GFP positive A549 cells were observed and counted. See also Figures S10 and .

Article Snippet: Human embryonic kidney 293T cells (American Type Culture Collection, CRL-3216), human cervical cancer HeLa cells (American Type Culture Collection, CCL-2) and mouse lung cancer LL2 cells (American Type Culture Collection, CRL-1642) were cultured in DMEM (Gibco, 11965-065) and human lung adenocarcinoma A549 cells (American Type Culture Collection, CCL-185), human colon adenocarcinoma HCT-8 cells (American Type Culture Collection, CCL-244) and mouse mammary gland epithelium 4T1 cells (American Type Culture Collection, CRL-2539) were cultured in RPMI 1640 Medium (Gibco, 22400-071), respectively.

Techniques: Dispersion, Expressing, Co-Culture Assay, Transfection, Plasmid Preparation, Staining, Marker, Flow Cytometry, Two Tailed Test, Diffusion-based Assay

Journal: iScience

Article Title: SARS-CoV-2 N protein mediates intercellular nucleic acid dispersion, a feature reduced in Omicron

doi: 10.1016/j.isci.2023.105995

Figure Lengend Snippet:

Article Snippet: Human embryonic kidney 293T cells (American Type Culture Collection, CRL-3216), human cervical cancer HeLa cells (American Type Culture Collection, CCL-2) and mouse lung cancer LL2 cells (American Type Culture Collection, CRL-1642) were cultured in DMEM (Gibco, 11965-065) and human lung adenocarcinoma A549 cells (American Type Culture Collection, CCL-185), human colon adenocarcinoma HCT-8 cells (American Type Culture Collection, CCL-244) and mouse mammary gland epithelium 4T1 cells (American Type Culture Collection, CRL-2539) were cultured in RPMI 1640 Medium (Gibco, 22400-071), respectively.

Techniques: Bioprocessing, Recombinant, Magnetic Beads, Protease Inhibitor, Sequencing, Modification, SYBR Green Assay, shRNA

Journal: iScience

Article Title: Temporary serine protease inhibition and the role of SPINK2 in human bone marrow

doi: 10.1016/j.isci.2023.106949

Figure Lengend Snippet: SPINK2 is highly expressed in CD34 + bone marrow cells (A) Uniform manifold approximation and projection (UMAP) graph shows the CD34 + cell clusters within human bone marrow as annotated by Setty et al. (B) UMAP graph showing SPINK2 expression in CD34 + cells. (C) SPINK2 and SPINK9 expression values are reported as averaged normalized CPM (top graph), and percentage of SPINK2 or SPINK9 positive cells in different population of CD34 + cells within the bone marrow (bottom graph). A cell is considered positive if normalized CPM value is > 0. Numbers of analyzed cells (n) in each population are the following: HSC, hematopoietic stem cell (n = 4690); HMP, hematopoietic multipotent progenitor (n = 4306); CMP, common myeloid progenitor (n = 2328); GMP, granulocyte-monocyte progenitor (n = 3713); DP, dendritic progenitor (n = 2075); MP, megakaryocyte progenitor (n = 507); EP, erythroid progenitor (n = 3463); CLP, common lymphoid progenitor (n = 3237); (D) Averaged normalized CPM (left graph) and percentage of SPINK positive cells (right graph) in different populations of hematopoietic cells within the bone marrow of mouse C57BL/6. LT-HSC, Long Term-HSC (n = 216); HSCs/HMPs, hematopoietic stem and progenitor cells (n = 852); MP/EP/CMP/GMP, megakaryocyte progenitor/erythroid progenitor/common myeloid progenitor/granulocyte-monocyte progenitor (n = 851).

Article Snippet: Recombinant human SPINK2 , Sino Biological Inc. , Cat. n. 13636-H02H.

Techniques: Expressing

Journal: iScience

Article Title: Temporary serine protease inhibition and the role of SPINK2 in human bone marrow

doi: 10.1016/j.isci.2023.106949

Figure Lengend Snippet: Expression of putative tPRSS in human bone marrow Averages of normalized CPM values (top graph) and percentage of positive cells (bottom graph) in different CD34 + cell subpopulations for (A) PRSS1, PRSS2, and PRSS3; (B) PRSS27, PLAT, and DPP7; and (C) PRSS57, CTSG, ELANE, and PRTN3. (D) qRT-PCR analysis of PRSS1, PRSS2, and PRSS57 transcripts in purified mobilized CD34 + blood cells. CD34 − cells were used as calibrator for fold change calculation. (E) SPINK2/PRSS57 or SPINK2/PRSS2 ratios in different subpopulations of HSPCs. (F) Percentage of PRSS2+ and PRSS57+ cells in SPINK2+ cell population. (G) Percentage of SPINK2+ cells in PRSS2+ cell population and in PRSS57+ cell population.

Article Snippet: Recombinant human SPINK2 , Sino Biological Inc. , Cat. n. 13636-H02H.

Techniques: Expressing, Quantitative RT-PCR, Purification

Journal: iScience

Article Title: Temporary serine protease inhibition and the role of SPINK2 in human bone marrow

doi: 10.1016/j.isci.2023.106949

Figure Lengend Snippet: SPINK2 transcript and protein are highly expressed in CD34 + bone marrow cells (A) qRT-PCR by 2 −ΔΔCT method confirms high levels of SPINK2 in purified CD34 + cells. ELANE, typically expressed in differentiated neutrophil granulocytes and granulocyte-monocyte progenitors, has been evaluated as negative control. (B) SPINK2 and PRSS57 are highly expressed at protein level in CD34 + HSPCs. Protein intensity of SPINK2 and PRSS57 is expressed as normalized label-free (LF) score in CD34 + HSPC (Hennrich et al. ). Negative control results in bone marrow mesenchymal stromal cells (MSC) are also shown. (C) Western blot showing SPINK2 enrichment in CD34 + cells. GAPDH and ACTB were the loading control. (D) SPINK2 (P20155) and SPINK1 (P00995) structures have been downloaded from AlphaFold Protein Structure Database developed by DeepMind and EMBL-EBI ( https://alphafold.ebi.ac.uk/entry/P20155 ; https://alphafold.ebi.ac.uk/entry/P00995 ). , Kazal domain is highlighted in green square. (E) SPINK2 (NP_066937, 84 amino acids) and SPINK1 (NP_001366539) protein alignment was performed by Constrain-based Multiple Alignment tool (Cobalt, https://www.ncbi.nlm.nih.gov/tools/cobalt/cobalt.cgi ), and as showed in Figure 2 E the method highlights in red color highly conserved aminoacidic positions based on the relative entropy threshold of the residue, while blue color indicates lower conservation. Kazal domain is underlined (green) and is located among amino acids in position 36 and 84 and includes 6 typical cysteines forming three pairs of disulfide bonds that stabilize its conformation.

Article Snippet: Recombinant human SPINK2 , Sino Biological Inc. , Cat. n. 13636-H02H.

Techniques: Quantitative RT-PCR, Purification, Negative Control, Western Blot, Control, Residue

Journal: iScience

Article Title: Temporary serine protease inhibition and the role of SPINK2 in human bone marrow

doi: 10.1016/j.isci.2023.106949

Figure Lengend Snippet: SPINK2 expression in human tissues and cancer cell lines (A) SPINK2 expression in 1,293 cancer cell lines assembled for tissue of origin in 26 groups. Averages of TPM±SD and percentage of positive cell lines (>10 TPM) are shown in top and bottom histograms, respectively. (B) SPINK2 expression levels in AML cell lines (n = 42); (C) in ALL cell lines (n = 45); and (D) in Lymphoma cell lines (n = 86). (E) qRT-PCR confirmed SPINK2 expression in Jurkat cell line, a model of ALL, while other leukemia cell lines (HL-60, K562, KASUMI-1) did not express SPINK2. HCT116, a colon cancer cell line, was used as calibrator for fold change calculation.

Article Snippet: Recombinant human SPINK2 , Sino Biological Inc. , Cat. n. 13636-H02H.

Techniques: Expressing, Quantitative RT-PCR

Journal: iScience

Article Title: Temporary serine protease inhibition and the role of SPINK2 in human bone marrow

doi: 10.1016/j.isci.2023.106949

Figure Lengend Snippet: Kinetic properties of trypsin inhibition by SPINK2 (A) Graph showing experimental initial reaction rates at different concentrations of SPINK2 (range 3.75–120 nM) and fixed concentration of total enzyme (0.0032 μM) and initial substrate (125 μM); K m was constrained at 2,450 μM. Ki was determined by fitting experimental data to Morrison’s equation by GraphPad Prism 8.0.2. Results are expressed as averages ±SEM of two independent experiments. (B) Initial reaction rates expressed as % of uninhibited reaction rate versus Log 10 concentration of SPINK2. Blue lines and points are calculated values with a Ki = 0.011 μM; red points are experimental values. (C) Experimental “product vs. time” curves; enzymatic reactions were followed for 17 h by monitoring spectrophotometrically the conversion of the substrate FVR-NA in p -nitroaniline at 410 nm. Trypsin (3.2 nM) was incubated with two different SPINK2 concentrations (3.75 and 60 nM) corresponding to an inhibitor/enzyme ratio of 1.2 and 19, respectively. (D) Calculated data obtained with TI-Model, showing the different “product vs. time” curves obtained with temporary and persistent inhibition; (E) Graph showing the “% of initial SPINK2 concentration” vs. Time obtained by TI-Model with Ki = 3 nM and increasing concentration of SPINK2. A SPINK2 k cat of 1.2 x 10 −3 sec −1 and a fixed concentration of 3.2 nM trypsin were used in the simulation. (F) In vitro degradation of recombinant SPINK2 by trypsin at different times. Each incubation was performed in duplicate. Averages of densitometric areas of western blotting bands (shown in the inset) are expressed as percentages of value at time zero (t0). (G) Kinetic reaction scheme for enzyme activity, competitive enzyme inhibition, and inhibitor degradation. Differential rate equations and abbreviations for kinetic constants (k0-k5) and chemical species are also reported in the figure.

Article Snippet: Recombinant human SPINK2 , Sino Biological Inc. , Cat. n. 13636-H02H.

Techniques: Inhibition, Concentration Assay, Incubation, In Vitro, Recombinant, Western Blot, Activity Assay, Enzyme Inhibition Assay

Journal: iScience

Article Title: Temporary serine protease inhibition and the role of SPINK2 in human bone marrow

doi: 10.1016/j.isci.2023.106949

Figure Lengend Snippet: Schematic drawing illustrating the TI-D theory for SPINK2 role in hematopoietic stem cell niche

Article Snippet: Recombinant human SPINK2 , Sino Biological Inc. , Cat. n. 13636-H02H.

Techniques:

Journal: iScience

Article Title: Temporary serine protease inhibition and the role of SPINK2 in human bone marrow

doi: 10.1016/j.isci.2023.106949

Figure Lengend Snippet: The temporary inhibition-diffusion model for SPINK2 in hematopoietic stem cell niche (A) The concentration of the complex tPRSS SPINK2, indicated as [EI] and shown in top graphs, and the velocity of conversion of protein substrate ( S v), shown in bottom graphs, were calculated at different distances from the source cell using different values of D (μ 2 /sec), as indicated above each plot. Results obtained for temporary or persistent inhibitor are shown in each plot. A SPINK2/tPRSS ratio = 2 is used in these simulations. (B) The velocity of conversion of protein substrate ( S v) per unit volume was calculated at different distances from the source cell using different ratios [I]/[E] at the source boundary, as indicated above each plot. Results obtained for a temporary inhibitor or a persistent one are compared in each plot. A D value = 5 μ 2 /sec is used in these simulations. (C) The concentration of the complex [EI] is shown in top panels, and the velocity of conversion of protein substrate (Sv) per unit volume in bottom panels. A fixed total concentration of [E] is used in left panels, and an inverse concentration gradient in the right panels. Results obtained for a temporary inhibitor or a persistent one are compared in each plot. A SPINK2/tPRSS ratio = 2 at the boundary of inhibitor source and a D value = 5 μ 2 /sec are used in these simulations.

Article Snippet: Recombinant human SPINK2 , Sino Biological Inc. , Cat. n. 13636-H02H.

Techniques: Inhibition, Diffusion-based Assay, Concentration Assay

Journal: iScience

Article Title: Temporary serine protease inhibition and the role of SPINK2 in human bone marrow

doi: 10.1016/j.isci.2023.106949

Figure Lengend Snippet:

Article Snippet: Recombinant human SPINK2 , Sino Biological Inc. , Cat. n. 13636-H02H.

Techniques: Recombinant, Sequencing, Modification, Isolation, SYBR Green Assay, Mass Spectrometry, Extraction, Synthesized, Software

Journal: iScience

Article Title: Temporary serine protease inhibition and the role of SPINK2 in human bone marrow

doi: 10.1016/j.isci.2023.106949

Figure Lengend Snippet: Primers details used for qRT-PCR

Article Snippet: Recombinant human SPINK2 , Sino Biological Inc. , Cat. n. 13636-H02H.

Techniques: