Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Validation of CRISPR activation system in Aedes cells using multicistronic plasmid vectors

doi: 10.3389/fbioe.2023.1142415

Figure Lengend Snippet: Efficiency of T2A peptides in Aedes cells. (A) Schematic diagram showing the design of dual fluorescent reporter cassettes to evaluate T2A (plasmid pJVG-T2A) and dT2A (plasmid pJVG-dT2A) activity. (B-D) Fluorescence micrograph of the cells transfected with plasmids as shown on the left of the panels. The untransfected cells (B-B”) were used to determine the baseline for imaging mCherry and eGFP channels, respectively. (E) Western blotting analysis of the extracts prepared from the transfected cells showing presence of processed mCherry and eGFP proteins. The blot was probed with anti-mCherry antibody (left panel) and anti-GFP antibody (right panel). Both blots were subsequently developed using anti-actin antibody. The arrow marks the position of the unprocessed mCherry-eGFP fusion protein.

Article Snippet: Post-transfer, the blot was cut into two and blocked using 5% BSA-TBST solution for 30 min at room temperature followed by incubation with rabbit anti-GFP antibody (1:1000, CST #2956S) and rabbit anti-mCherry antibody (1:1000, Millipore #AB356482) separately.

Techniques: Plasmid Preparation, Activity Assay, Fluorescence, Transfection, Imaging, Western Blot

Journal: Cell reports

Article Title: CARD-only proteins regulate in vivo inflammasome responses and ameliorate gout

doi: 10.1016/j.celrep.2023.112265

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit monoclonal anti-GFP, clone D5.1 , Cell Signaling Technology , Cat# 2956; RRID:AB_1196615.

Techniques: Purification, Recombinant, Protease Inhibitor, Staining, Magnetic Beads, Enzyme-linked Immunosorbent Assay, TaqMan Assay, LDH Cytotoxicity Assay, CyQUANT Assay, Proximity Ligation Assay, Western Blot, Software

Journal: Journal of Extracellular Vesicles

Article Title: Exosomal lipid PI4P regulates small extracellular vesicle secretion by modulating intraluminal vesicle formation

doi: 10.1002/jev2.12319

Figure Lengend Snippet: Profiling of phosphoinositide regioisomers in macrophage‐derived sEVs. (a) Schematic diagram of the PIP structure. Mono‐PIPs can be present as three isomers after the addition of a phosphate group at the −3, −4, and −5 sites, respectively. (b) Chromatograms of the three regioisomers of PIP Me 18:1(9Z)/18:1(9Z) standard (top panel) and separation of PIP Me 36:2 in RAW264.7‐cell‐derived sEVs (bottom panel). (c) MRM spectra of PI3P(purple) and PI4P(yellow) in RAW264.7‐cell‐derived sEVs. (d) Pie chart of the ratios of PI3P and PI4P in RAW264.7‐cell‐derived sEVs. (e) SIM imaging of isolated RAW264.7‐cell‐derived sEVs. Green represents sEVs carrying GFP or GFP fused PIP biosensor or CD63, and red represents PKH26 stained sEV particles. Scale bar, 2 μm. (f) Quantitative results from Figure . The bar shows the mean with SEM. The bar plot represents eight independent images from four biological replicates. Unpaired two‐tailed t ‐test ( *** P < 0.001; **** P < 0.0001; ns, not significant). (g) The TEM results of anti‐PI4P and anti‐GFP immunogold staining of RAW264.7‐cell‐derived sEVs. The negative control consists of only the secondary antibody. The white arrow indicates the immune gold particles. Scale bar, 200 nm.

Article Snippet: Anti‐EEA1 (#3288), anti‐GFP (#2956), anti‐GAPDH (#2118), and anti‐HSPA5 (#3177) aantibodies were purchased from Cell Signaling Technology.

Techniques: Derivative Assay, Imaging, Isolation, Staining, Two Tailed Test, Negative Control

Journal: Journal of Extracellular Vesicles

Article Title: Exosomal lipid PI4P regulates small extracellular vesicle secretion by modulating intraluminal vesicle formation

doi: 10.1002/jev2.12319

Figure Lengend Snippet: The PI4P content positively correlates with sEV release upon TLR4 activation. (a) TIRF imaging monitoring sEV release from the cell membrane surface. Each curve shows an MVB‐PM fusion event. (b) The instantaneous release rate of sEVs in RAW264.7 cells after LPS stimulation. The number of fluorescent flashes on the cell surface, as shown in Figure , was recorded. Each dot represents an MVB‐PM fusion event (fluorescent spots) over a time‐lapse recording of 3‐min onto a single cell. The separated scatter plot shows the mean with SEM, and represents three biological replicates. Unpaired two‐tailed t ‐test ( *** P < 0.001; **** P < 0.0001). (c) NTA determining the cumulative number of sEVs in RAW264.7 cells after different LPS stimulation periods. The bar plot shows the mean with SEM. Unpaired two‐tailed t ‐test ( *** P < 0.001; **** P < 0.0001). The bar plot represents three independent biological replicates. (d) The PIP content in sEVs detecting by LC‐MS/MS. The bar shows the mean with SEM. Unpaired two‐tailed t ‐test ( **** P < 0.0001). The bar plot represents three independent biological replicates. (e) sEVs derived from RAW264.7 cells stably expressing GFP‐tagged PI4P or PI3P sensor were observed by SIM imaging before and after LPS stimulation. Green indicates the PIP sensor‐GFP, and red indicates PKH26‐stained particles. Scale bar, 2 μm. (f) Quantitative results from Figure . The bar plot shows the mean with SEM. Unpaired two‐tailed t ‐test ( **** P < 0.0001). The bar plot represents four independent biological replicates. (g) The PIP species in RAW264.7‐cell‐derived sEVs before and after LPS stimulation. The concentration was equivalent to the internal standard. The bar plot shows the mean with SEM. Unpaired two‐tailed t ‐test ( **** P < 0.0001). The bar plot represents three independent biological replicates. (h) PIP species in RAW264.7 cells before and after LPS stimulation. The concentration was equivalent to the internal standard. The bar plot shows the mean with SEM. Unpaired two‐tailed t ‐test ( **** P < 0.0001). Bar plot represents three independent biological replicates. (i) WB detection of the distribution of PIP sensors in early endosomes and MVBs isolated from RAW264.7 cells by density gradient centrifugation. Left panel shows untreated cells, and right panel shows cells treated with LPS for 24 h. EEA1, early endosome marker; VPS16, MVB marker; GFP, PI4P sensor; FLOT1, cell membrane marker; P5, the pellets from 5000×g centrifugation; and P17, the pellets from 17,000×g centrifugation.

Article Snippet: Anti‐EEA1 (#3288), anti‐GFP (#2956), anti‐GAPDH (#2118), and anti‐HSPA5 (#3177) aantibodies were purchased from Cell Signaling Technology.

Techniques: Activation Assay, Imaging, Two Tailed Test, Liquid Chromatography with Mass Spectroscopy, Derivative Assay, Stable Transfection, Expressing, Staining, Concentration Assay, Isolation, Gradient Centrifugation, Marker, Centrifugation

Journal: bioRxiv

Article Title: Coronavirus endoribonuclease nsp15 induces host cellular protein synthesis shutoff

doi: 10.1101/2023.03.20.533404

Figure Lengend Snippet: (A) DF-1 cells were co-transfected with plasmids encoding Flag-tagged IBV nsp7, nsp8, nsp9, nsp12 or nsp15 and the plasmids encoding a constitutively active form of V5-chMDA5(N), or HA-chMAVS, or V5-chIRF7, or enhanced green fluorescent protein (EGFP). The empty PXJ40 vector was included as a control. After 24 h, cells were collected for western blot analysis. Protein signals were detected using the indicated antibodies, and β-actin was detected as loading control. The density of the protein bands was analysed with ImageJ, normalized by the density of β-actin, and the ratio was presented relative to the density detected in the corresponding PXJ40 sample. (B) PXJ40, or Flag-tagged IBV nsp7, nsp8, nsp9, nsp12 or nsp15 were co-transfected into DF-1 cells with V5-chMDA5(N) or HA-chMAVS. After 24 h, cells were collected and the RNA were extracted, subjected to quantitative RT-PCR, using primers spanning the tag (V5 or HA) sequence and the chMDA5(N) or chMAVS sequence. mRNA levels of V5-chMDA5(N) or HA-chMAVS were normalized relative to the β-actin housekeeping gene and presented relative to PXJ40 group. Values present results of one representative experiment, which was performed three times with comparable results. Error bars indicate standard deviation of triplicate values within one experiment. (C) DF-1 cells were transfected with plasmid encoding Flag-nsp15 or PXJ40 for 23 h and treated with puromycin (5 µg/ml) for 1 h to label de novo synthesized peptides. Indirect immunofluorescence was performed to detect nsp15 (magenta), puromycin (green), and nuclei (DAPI, blue). Fluorescence intensity of nsp15 and puromycin in individual cells along the white line (from a to b) is shown in the right panel (histogram plot).

Article Snippet: Mouse anti-V5 (Thermo fisher scientific, #R961-25, horseradish peroxidase HRP-conjugated), mouse anti-HA (MBL, #M180-7, HRP-conjugated), mouse anti-Flag (MBL, #M185-7, HRP-conjugated), were diluted by 1:2500 for Western Blot; mouse anti-β-actin (CST, #3700S), rabbit anti-GFP (CST, #2956), chicken anti-Flag (Gentaur, #AFLAG), rabbit anti-phosphorylated PKR (Abcam, #ab32036), rabbit anti-PKR (CST, #12297), rabbit anti-phosphorylated eIF2α (CST, #3398), rabbit anti-eIF2α (CST, #5324), anti-RPS6 rabbit mAb (CST, #2217) and eIF4E rabbit mAb (CST, #2067), were diluted by 1:1000 dilution for Western Blot; rabbit anti-IBV N (provided by Prof Dingxiang Liu, South China Agricultural University, China) was diluted by 1:2000 for Western blot; rabbit anti-human PABPC1 (Abcam, #Ab21060, cross-reacts against chicken PABPC1 in DF-1 cells), rabbit anti-IBV-N, anti-IBV nsp12 rabbit pAb (provided by Prof Dingxiang Liu, South China Agricultural University, China), anti-IBV nsp15 mouse mAb (provided by Dr. Min Liao’s lab, Zhejiang University, China), were diluted by 1:500 for immunofluorescence; mouse anti-puromycin (Sigma-Aldrich, #MABE343) was diluted by 1:25000 for Western blot and 1:10000 for immunofluorescence; anti-dsRNA mouse mAb J2 (Scicons, #10010200) was diluted by 1:1000 for dot blot analysis.

Techniques: Transfection, Plasmid Preparation, Western Blot, Quantitative RT-PCR, Sequencing, Standard Deviation, Synthesized, Immunofluorescence, Fluorescence

Journal: bioRxiv

Article Title: Coronavirus endoribonuclease nsp15 induces host cellular protein synthesis shutoff

doi: 10.1101/2023.03.20.533404

Figure Lengend Snippet: (A) Plasmid encoding Flag-tagged catalytic-deficient nsp15 H223A, H238A, oligomerization-deficient nsp15 D285A, D315A, wild type nsp15, and the vector PXJ40, were each co-transfected with plasmids encoding V5-chMDA5(N), HA-chMAVS, V5-IRF7, or EGFP into DF-1 cells. After 24 h, Western blot analysis was performed using corresponding antibodies. β-actin was detected as loading control. Density of the bands was analysed by Image J, normalized to the signal of β-actin, and the ratio was presented relative to the density detected in the corresponding PXJ40 transfected cells. (B) DF-1 (C) Vero and H1299 cells, were transfected with the plasmid encoding wild type or mutated nsp15 and treated with puromycin (5 µg/ml) for 1 h at 23 h post-transfection (h.p.t), to label the de novo synthesized peptides. Indirect immunofluorescence was performed to detect nsp15 (magenta), puromycin (green), and nuclei (DAPI, blue). Fluorescence intensity of nsp15 and puromycin signal along the white line (from a to b) is indicated in the right panel (histogram plot).

Article Snippet: Mouse anti-V5 (Thermo fisher scientific, #R961-25, horseradish peroxidase HRP-conjugated), mouse anti-HA (MBL, #M180-7, HRP-conjugated), mouse anti-Flag (MBL, #M185-7, HRP-conjugated), were diluted by 1:2500 for Western Blot; mouse anti-β-actin (CST, #3700S), rabbit anti-GFP (CST, #2956), chicken anti-Flag (Gentaur, #AFLAG), rabbit anti-phosphorylated PKR (Abcam, #ab32036), rabbit anti-PKR (CST, #12297), rabbit anti-phosphorylated eIF2α (CST, #3398), rabbit anti-eIF2α (CST, #5324), anti-RPS6 rabbit mAb (CST, #2217) and eIF4E rabbit mAb (CST, #2067), were diluted by 1:1000 dilution for Western Blot; rabbit anti-IBV N (provided by Prof Dingxiang Liu, South China Agricultural University, China) was diluted by 1:2000 for Western blot; rabbit anti-human PABPC1 (Abcam, #Ab21060, cross-reacts against chicken PABPC1 in DF-1 cells), rabbit anti-IBV-N, anti-IBV nsp12 rabbit pAb (provided by Prof Dingxiang Liu, South China Agricultural University, China), anti-IBV nsp15 mouse mAb (provided by Dr. Min Liao’s lab, Zhejiang University, China), were diluted by 1:500 for immunofluorescence; mouse anti-puromycin (Sigma-Aldrich, #MABE343) was diluted by 1:25000 for Western blot and 1:10000 for immunofluorescence; anti-dsRNA mouse mAb J2 (Scicons, #10010200) was diluted by 1:1000 for dot blot analysis.

Techniques: Plasmid Preparation, Transfection, Western Blot, Synthesized, Immunofluorescence, Fluorescence

Journal: bioRxiv

Article Title: Coronavirus endoribonuclease nsp15 induces host cellular protein synthesis shutoff

doi: 10.1101/2023.03.20.533404

Figure Lengend Snippet: The plasmid encoding wild type or catalytic-deficient nsp15 from the indicated coronaviruses was co-transfected with the plasmid encoding EGFP or IBV N into Vero cells. After 24 h, Western blot analysis was performed using corresponding antibodies. β-actin was detected as loading control. Density of the bands of EGFP or IBV N were analysed by Image J, normalized to the signal of β-actin and presented relative to the PXJ40 group.

Article Snippet: Mouse anti-V5 (Thermo fisher scientific, #R961-25, horseradish peroxidase HRP-conjugated), mouse anti-HA (MBL, #M180-7, HRP-conjugated), mouse anti-Flag (MBL, #M185-7, HRP-conjugated), were diluted by 1:2500 for Western Blot; mouse anti-β-actin (CST, #3700S), rabbit anti-GFP (CST, #2956), chicken anti-Flag (Gentaur, #AFLAG), rabbit anti-phosphorylated PKR (Abcam, #ab32036), rabbit anti-PKR (CST, #12297), rabbit anti-phosphorylated eIF2α (CST, #3398), rabbit anti-eIF2α (CST, #5324), anti-RPS6 rabbit mAb (CST, #2217) and eIF4E rabbit mAb (CST, #2067), were diluted by 1:1000 dilution for Western Blot; rabbit anti-IBV N (provided by Prof Dingxiang Liu, South China Agricultural University, China) was diluted by 1:2000 for Western blot; rabbit anti-human PABPC1 (Abcam, #Ab21060, cross-reacts against chicken PABPC1 in DF-1 cells), rabbit anti-IBV-N, anti-IBV nsp12 rabbit pAb (provided by Prof Dingxiang Liu, South China Agricultural University, China), anti-IBV nsp15 mouse mAb (provided by Dr. Min Liao’s lab, Zhejiang University, China), were diluted by 1:500 for immunofluorescence; mouse anti-puromycin (Sigma-Aldrich, #MABE343) was diluted by 1:25000 for Western blot and 1:10000 for immunofluorescence; anti-dsRNA mouse mAb J2 (Scicons, #10010200) was diluted by 1:1000 for dot blot analysis.

Techniques: Plasmid Preparation, Transfection, Western Blot

Journal: bioRxiv

Article Title: Coronavirus endoribonuclease nsp15 induces host cellular protein synthesis shutoff

doi: 10.1101/2023.03.20.533404

Figure Lengend Snippet: Plasmid encoding wild type or catalytic-deficient IBV nsp15 and reporter plasmid encoding IBV N or IBV M, or luciferase DNA, were co-incubated with Rabbit Reticulocyte Lysate for 1 h followed by Western blot analysis or luciferase assay. Density of the bands corresponding to the reporter proteins was normalized to the signal of β-actin and presented relative to the sample transfected with the empty vector PXJ40.

Article Snippet: Mouse anti-V5 (Thermo fisher scientific, #R961-25, horseradish peroxidase HRP-conjugated), mouse anti-HA (MBL, #M180-7, HRP-conjugated), mouse anti-Flag (MBL, #M185-7, HRP-conjugated), were diluted by 1:2500 for Western Blot; mouse anti-β-actin (CST, #3700S), rabbit anti-GFP (CST, #2956), chicken anti-Flag (Gentaur, #AFLAG), rabbit anti-phosphorylated PKR (Abcam, #ab32036), rabbit anti-PKR (CST, #12297), rabbit anti-phosphorylated eIF2α (CST, #3398), rabbit anti-eIF2α (CST, #5324), anti-RPS6 rabbit mAb (CST, #2217) and eIF4E rabbit mAb (CST, #2067), were diluted by 1:1000 dilution for Western Blot; rabbit anti-IBV N (provided by Prof Dingxiang Liu, South China Agricultural University, China) was diluted by 1:2000 for Western blot; rabbit anti-human PABPC1 (Abcam, #Ab21060, cross-reacts against chicken PABPC1 in DF-1 cells), rabbit anti-IBV-N, anti-IBV nsp12 rabbit pAb (provided by Prof Dingxiang Liu, South China Agricultural University, China), anti-IBV nsp15 mouse mAb (provided by Dr. Min Liao’s lab, Zhejiang University, China), were diluted by 1:500 for immunofluorescence; mouse anti-puromycin (Sigma-Aldrich, #MABE343) was diluted by 1:25000 for Western blot and 1:10000 for immunofluorescence; anti-dsRNA mouse mAb J2 (Scicons, #10010200) was diluted by 1:1000 for dot blot analysis.

Techniques: Plasmid Preparation, Luciferase, Incubation, Western Blot, Transfection

Journal: bioRxiv

Article Title: Coronavirus endoribonuclease nsp15 induces host cellular protein synthesis shutoff

doi: 10.1101/2023.03.20.533404

Figure Lengend Snippet: (A) DF-1 cells or (B) H1299 cells were infected with IBV-WT or rIBV-nsp15H1238A at an MOI of 1. At 6, 12, 24 h.p.i., cells were treated with puromycin (5 µg/ml) for 1 h, followed by western blot analysis to detect puromycin-labelled de novo peptides, IBV-N protein, and β-actin. Density of the puromycin labelled proteins was normalized to the signal of β-actin. Ratio of the puromycin–labelled de novo peptides of the infected cells (+) to that of the uninfected cells (-) at the same time h.p.i. is shown. (B) H1299 cells were infected as described above followed by dot blot analysis to detects dsRNA and western blot analysis to detect p-PKR, PKR, p-eIF2α, eIF2α.

Article Snippet: Mouse anti-V5 (Thermo fisher scientific, #R961-25, horseradish peroxidase HRP-conjugated), mouse anti-HA (MBL, #M180-7, HRP-conjugated), mouse anti-Flag (MBL, #M185-7, HRP-conjugated), were diluted by 1:2500 for Western Blot; mouse anti-β-actin (CST, #3700S), rabbit anti-GFP (CST, #2956), chicken anti-Flag (Gentaur, #AFLAG), rabbit anti-phosphorylated PKR (Abcam, #ab32036), rabbit anti-PKR (CST, #12297), rabbit anti-phosphorylated eIF2α (CST, #3398), rabbit anti-eIF2α (CST, #5324), anti-RPS6 rabbit mAb (CST, #2217) and eIF4E rabbit mAb (CST, #2067), were diluted by 1:1000 dilution for Western Blot; rabbit anti-IBV N (provided by Prof Dingxiang Liu, South China Agricultural University, China) was diluted by 1:2000 for Western blot; rabbit anti-human PABPC1 (Abcam, #Ab21060, cross-reacts against chicken PABPC1 in DF-1 cells), rabbit anti-IBV-N, anti-IBV nsp12 rabbit pAb (provided by Prof Dingxiang Liu, South China Agricultural University, China), anti-IBV nsp15 mouse mAb (provided by Dr. Min Liao’s lab, Zhejiang University, China), were diluted by 1:500 for immunofluorescence; mouse anti-puromycin (Sigma-Aldrich, #MABE343) was diluted by 1:25000 for Western blot and 1:10000 for immunofluorescence; anti-dsRNA mouse mAb J2 (Scicons, #10010200) was diluted by 1:1000 for dot blot analysis.

Techniques: Infection, Western Blot, Dot Blot

Journal: bioRxiv

Article Title: Coronavirus endoribonuclease nsp15 induces host cellular protein synthesis shutoff

doi: 10.1101/2023.03.20.533404

Figure Lengend Snippet: (A) DF-1 cells were transfected with a plasmid coding Flag-tagged nsp15 (PXJ40F-nsp15). At 24 h.p.t, indirect immunofluorescence was performed with a chicken anti-Flag-tag antibody (red). Nuclei were stained with DAPI (blue). (B) Vero cells were infected with IBV-WT or rIBV-nsp15-H238A at an MOI=1. At 18 h.p.i, indirect immunofluorescence was performed with a mouse anti-IBV-nsp15 monoclonal antibody (red), a rabbit anti-IBV-nsp12 polyclonal antibody (green) and the nuclei were stained with DAPI (blue). (C) Vero cells were infected with IBV or rIBV-nsp15-H238A with an MOI of 1. At 18 h.p.i., cells were treated with 100 μg/mL cycloheximide (CHX) for 15 min at 37°C and subjected to 7–47% sucrose density gradient ultracentrifugation (38,000 rpm for 3 h), and the fractions were analysed by Western blot to detect nsp15, Rsp6, eIF4E, and β-actin (left panel).

Article Snippet: Mouse anti-V5 (Thermo fisher scientific, #R961-25, horseradish peroxidase HRP-conjugated), mouse anti-HA (MBL, #M180-7, HRP-conjugated), mouse anti-Flag (MBL, #M185-7, HRP-conjugated), were diluted by 1:2500 for Western Blot; mouse anti-β-actin (CST, #3700S), rabbit anti-GFP (CST, #2956), chicken anti-Flag (Gentaur, #AFLAG), rabbit anti-phosphorylated PKR (Abcam, #ab32036), rabbit anti-PKR (CST, #12297), rabbit anti-phosphorylated eIF2α (CST, #3398), rabbit anti-eIF2α (CST, #5324), anti-RPS6 rabbit mAb (CST, #2217) and eIF4E rabbit mAb (CST, #2067), were diluted by 1:1000 dilution for Western Blot; rabbit anti-IBV N (provided by Prof Dingxiang Liu, South China Agricultural University, China) was diluted by 1:2000 for Western blot; rabbit anti-human PABPC1 (Abcam, #Ab21060, cross-reacts against chicken PABPC1 in DF-1 cells), rabbit anti-IBV-N, anti-IBV nsp12 rabbit pAb (provided by Prof Dingxiang Liu, South China Agricultural University, China), anti-IBV nsp15 mouse mAb (provided by Dr. Min Liao’s lab, Zhejiang University, China), were diluted by 1:500 for immunofluorescence; mouse anti-puromycin (Sigma-Aldrich, #MABE343) was diluted by 1:25000 for Western blot and 1:10000 for immunofluorescence; anti-dsRNA mouse mAb J2 (Scicons, #10010200) was diluted by 1:1000 for dot blot analysis.

Techniques: Transfection, Plasmid Preparation, Immunofluorescence, FLAG-tag, Staining, Infection, Western Blot