Journal: Developmental Cell

Article Title: S-acylation controls SARS-CoV-2 membrane lipid organization and enhances infectivity

doi: 10.1016/j.devcel.2021.09.016

Figure Lengend Snippet: S-Acylation controls the lipid environment of spike See also Figure S4 . (A) Coarse-grained (CG) model of spike’s transmembrane (TM) helix + C-terminal region (residues 1,201-end in UniProt P0DTC2 ) in trimeric form, inserted into a DPPC-(yellow)-DLiPC-(red)-cholesterol-(green) membrane model. Left, starting conditions: random lipid positions and stretched cytoplasmic tail; center, representative end state of same view after 3.75 μs of CG MD simulation of the S-palmitoylated protein depicts phase separation and spike partitioning. Right, side view of the same model after 2 μs of atomistic MD simulation in the S-palmitoylated form in a POPC membrane. (B) Quantification of contacts at equilibrium between the full TM helix, the luminal half, or the cytosolic half, and each lipid phase. Results are means ± SD (n = 10). (C) Vero E6 expressing spike-HA WT surface labeled with biotin and processed for fractionation. Western blot (WB) of biotinylated proteins (Surface-Strp. Pull-down) precipitated from each fraction were compared to unbound fractions (intracellular-unbound). (D) Quantification of spike-HA (all forms) in each fraction relative to the total signal in all 6 fractions. Results are mean ± SD, n = 3. (E and F) High-throughput automated microscopy of Vero E6 expressing spike-HA (WT, or 10C-A mutant) for 24 h immunolabeled for HA and stained with Filipin. (E) Total levels of filipin staining within HA-positive cells (or per cell—mock sample) quantified per number of nuclei. The data were averaged for 49 frames over 8 wells per condition. Each dot represents one independent well, and results are mean ± SEM. Equivalent results were obtained for three independent assays. (G) Vero E6 infected with SARS-CoV-2 24 h, MOI ≈ 0.1 were incubated with beta-methylcyclodextrin (βMCD) or not (Ctrl) for 30 min. Cell extracts were fractionated as in (C). DRM marker Caveolin1 (Cav1)-positive control, transferrin receptor (TfR)-negative control. (H and I) Quantification of spike forms (H) and caveolin 1 (I) signals as in (C). Results are mean ± SD, n = 3. (J) Vero E6 transfected with indicated siRNA (siCtrl or siMixT pool), infected and fractionated as in (G). (K–M) Quantification of (K) and (L). Spike (all forms) and (M) ERGIC53 in each fraction as in (C). Results are mean ± SD, n = 3. All p values were obtained by two-way (except for E, one-way) ANOVA.

Article Snippet: ERGIC53 , ProSci , RRID: AB_796913 Cat#PSC-PM-7213.

Techniques: Membrane, Expressing, Labeling, Fractionation, Western Blot, High Throughput Screening Assay, Microscopy, Mutagenesis, Immunolabeling, Staining, Infection, Incubation, Marker, Positive Control, Negative Control, Transfection

Journal: Developmental Cell

Article Title: S-acylation controls SARS-CoV-2 membrane lipid organization and enhances infectivity

doi: 10.1016/j.devcel.2021.09.016

Figure Lengend Snippet: Spike S-acylation controls lipid organization of VLPs See also Figure S5 . (A) Untyped (Bald) or spike-HA (WT or 10C-A) pseudotyped VLPs purified from supernatants of HEK293T (see ). Normalized VLP released (mean ± SEM) was determined by quantification of Nluc-bioluminescence from 5 independent preparations. (B–D) Western blot (WB) of cell extracts and VLPs: non-typed (bald) or VLPs pseudotyped with spike-HA (WT or 10C-A mutant). GAPDH used as cellular-control. (C) Spike-HA levels in VLPs normalized against N or M and set to 1 for WT. (D) Spike cleavage in VLPs quantified as the ratio between S2 and S2 + S full length. Results are mean ± SEM, n = 3. (E) Acylrac capture assay in purified VLPs and correspondent cell extracts isolated as in (B). Total cell extracts (input, In), S-palmitoylated proteins (+NH 2 OH), and control fractions (−NH 2 OH) analyzed by WB against HA (spike). (F) WB of fractions from concentrated VLPs processed for fractionation and DRM isolation. (G and H) Quantification of (G). Spike-HA (all forms) or (H) M in each fraction divided by the total signal in the 6 fractions. Results are mean ± SD, n = 3. (I and J) Same as in (F) from lysates of VLP-producing HEK293T. Fractions were analyzed by WB and ERGIC53 levels in each fraction quantified as in (G) and (H). Results are mean ± SD, n = 3. p values were obtained by two-way ANOVA.

Article Snippet: ERGIC53 , ProSci , RRID: AB_796913 Cat#PSC-PM-7213.

Techniques: Purification, Western Blot, Mutagenesis, Control, Isolation, Fractionation

Journal: Developmental Cell

Article Title: S-acylation controls SARS-CoV-2 membrane lipid organization and enhances infectivity

doi: 10.1016/j.devcel.2021.09.016

Figure Lengend Snippet:

Article Snippet: ERGIC53 , ProSci , RRID: AB_796913 Cat#PSC-PM-7213.

Techniques: Virus, Recombinant, Amplex Red Cholesterol Assay, Luciferase, Electron Microscopy, Protease Inhibitor, Enzyme-linked Immunosorbent Assay, Neutralization, SYBR Green Assay, Lysis, Generated

Journal: BMC Cancer

Article Title: SNHG17 drives malignant behaviors in astrocytoma by targeting miR-876-5p/ERLIN2 axis

doi: 10.1186/s12885-020-07280-8

Figure Lengend Snippet: SNHG17 was up-regulated in astrocytoma cell lines and promoted the process of astrocytoma cells ( a ) SNHG17 expression was detected in astrocytoma cell lines (LN-215, ADF, U138 and A-382) and human astrocytes (NHA). b SNHG17 expression was examined in cells transfected with sh-SNHG17#1/2. c - d EdU assays and CCK8 assessed proliferation in sh-SNHG17#1/2 and sh-NC group. e - f Flow cytometry analysis and TUNEL appraised apoptosis in different groups. g - h Sphere formation efficiency was assessed by sphere formation assay and the expression of stemness-associated markers was measured by RT-qPCR. i - j Transwell assays evaluated cell migration and invasion in different groups. * P < 0.05, ** P < 0.01

Article Snippet: Cells were suspended in serum-free DMEM and inoculated into Transwell upper compartment (Corning, Corning, NY) with matrigel for cell invasion, without matrigel for cell migration.

Techniques: Expressing, Transfection, Flow Cytometry, TUNEL Assay, Tube Formation Assay, Quantitative RT-PCR, Migration

Journal: BMC Cancer

Article Title: SNHG17 drives malignant behaviors in astrocytoma by targeting miR-876-5p/ERLIN2 axis

doi: 10.1186/s12885-020-07280-8

Figure Lengend Snippet: MiR-876-5p suppressed proliferation and motility in astrocytoma cells ( a - b ) Nuclear cytoplasm fractionation and FISH were built to judge the subcellular place of SNHG17 in U138 and LN-215 cells. c RNA pull down was used to select out miRNAs could bind to SNHG17. d MiR-876-5p expression was tested in astrocytoma cell lines. e The binding sites between SNHG17 and miR-876-5p were predicted by starBase. f RNA pull down manifested that miR-876-5p could bind to SNHG17. g RIP verified that SNHG17 could bind to Ago2. h MiR-876-5p expression was detected in astrocytoma cells. i Luciferase reporter assays illustrated that SNHG17 could bind to miR-876-5p in both LN-215 and U138 cells. j - k Proliferative capacities were examined in EdU and CCK8 in cells with NC mimics and miR-876-5p mimics. l - m Cell apoptosis was evaluated in different groups by TUNEL assay and flow cytometry analysis. n - o Stemness of cells in different groups was probed via sphere formation assay and RT-qPCR analysis of related genes. p - q Transwell assay examined migration and invasion. ** P < 0.01

Article Snippet: Cells were suspended in serum-free DMEM and inoculated into Transwell upper compartment (Corning, Corning, NY) with matrigel for cell invasion, without matrigel for cell migration.

Techniques: Fractionation, Expressing, Binding Assay, Luciferase, TUNEL Assay, Flow Cytometry, Tube Formation Assay, Quantitative RT-PCR, Transwell Assay, Migration

Journal: Neural Regeneration Research

Article Title: Substance P promotes epidural fibrosis via induction of type 2 macrophages

doi: 10.4103/1673-5374.369120

Figure Lengend Snippet: SP promotes neutrophil infiltration and NET formation. (A) Quantification of CXCL1 in SP-treated macrophages by ELISA. (B) SP-treated macrophages increased neutrophil migration in a Transwell assay. As the image shows, macrophages treated with SP attracted greater numbers of neutrophils. (C) Immunofluorescence staining for MPO (green, Alexa Fluor® 488) and citH3 (red, Alexa Fluor® 555) in wound tissues 3 days after spinal surgery. More NETs were observed in the operated group. (D) Western blot showing citH3 expression in SP-treated neutrophils. (E) Immunofluorescence staining for citH3 (yellow, Alexa Fluor® 555) in SP-treated neutrophils. Greater citH3 expression was observed after treatment with SP. (F) Western blot showing that the NK1-R inhibitor RP67580 blocked the effects of SP on citH3 in SP-treated neutrophils. (G) Immunofluorescence staining for MPO (green, Alexa Fluor® 488) and citH3 (red, Alexa Fluor® 555) in wound tissues 3 days after spinal surgery in mice treated with an NK1-R inhibitor (RP67580) or left untreated. As the images show, citH3 expression was decreased after treatment with RP67580. Scale bars: 20 μm. (H) An NADPH inhibitor (apocynin) blocked the effects of SP on citH3 in SP-treated neutrophils. (I) Western blot showing Arg-1/iNOS expression in macrophages treated with NETs. (J) Western blot showing Arg-1/iNOS expression in macrophages treated with NETs with or without elastase inhibitor. All data are presented as the mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 (one-way analysis of variance with post hoc Tukey’s honest significant difference test). Arg-1: Arginase-1; CXCL1: C-X-C motif chemokine ligand 1; ELISA: enzyme-linked immunosorbent assay; iNOS: inducible nitric oxide synthase; MPO: myeloperoxidase; NADPH: nicotinamide adenine dinucleotide phosphate; NETs: neutrophil extracellular traps; NK1-R: neurokinin receptor 1; SP: substance P.

Article Snippet: Neutrophils (5 × 10 5 ) were added to the Transwell compartment (Corning, Beijing, China), and macrophages (5 × 10 5 ) or SP were added to the lower compartment of the 24-well plate.

Techniques: Enzyme-linked Immunosorbent Assay, Migration, Transwell Assay, Immunofluorescence, Staining, Western Blot, Expressing