human embryo kidney  (ATCC)

Journal: PLOS Pathogens

Article Title: Signal peptidase complex mediates rotavirus VP7 processing and virion assembly

doi: 10.1371/journal.ppat.1013688

Figure Lengend Snippet: (A) WT, SPCS1 KO HEK293T, SPCS1 KO cells transfected with empty vector or FLAG-tagged SPCS1 were infected with RRV at an MOI of 3, and cells were collected for western blot analysis at 12 hpi using anti-FLAG and SPCS1 antibodies to confirm the protein level of SPCS1. (B) The lysates and supernatants were collected for FFU assays to determine the titers. (C) WT and SPCS1 KO HEK293T cells were infected with RRV, SA11 and UK strains at an MOI of 3. At 12 hpi, all the cell lysates and supernatants were collected for FFU assay to detect the titers. (D) WT and SPCS1 KO Huh7.5 cells were infected with RRV, SA11 and UK strains at an MOI of 3, respectively. At 12 hpi, all the cell lysates and supernatants were collected for FFU assay to detect the titers. (E) WT and SPCS1 KO HEK293T cells were transfected with siRNAs targeted against SPCS2 , SEC11A , SEC11C and a scrambled siRNA at the concentration of 20 nM. At 48 hpi, cells were infected with RRV at an MOI of 3. The cell lysates and supernatants were collected for FFU assay to detect the titers at 12 hpi. The results are the averages of data in three independent experiments and plotted as mean ± SD. Statistical significance was determined using two-way ANOVA with Sidak’s multiple comparisons test (**, P < 0.01; ***, P < 0.001, **** P < 0.0001).

Article Snippet: Human embryo kidney HEK293T cells (ATCC CRL-3216), were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, catalog number 11965118) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Avantor, catalog number 267820).

Techniques: Transfection, Plasmid Preparation, Infection, Western Blot, Concentration Assay

Journal: PLOS Pathogens

Article Title: Signal peptidase complex mediates rotavirus VP7 processing and virion assembly

doi: 10.1371/journal.ppat.1013688

Figure Lengend Snippet: (A) WT and SPCS1 KO HEK293T cells were infected with RRV at an MOI of 3. At 4, 8, 12 hpi, the cells were collected for qRT-PCR analysis of viral mRNA level by detecting NSP5. The NSP5 level was normalized to that of GAPDH. Results are the average of data in triplicates and plotted as mean ± SD. (B) WT and SPCS1 KO HEK293T cells were infected with RRV at an MOI of 3. At 4, 8, 12 hpi, the infected cells and mock cells were harvested for western blot analysis of viral protein level by detecting VP6 and VP7 protein. (C) WT and SPCS1 KO HEK293T cells were infected with RRV at an MOI of 3. At 12 hpi, an immunofluorescence assay was performed to detect the viroplasms in the infected cells. Cells were fixed and probed with in-house polyclonal antibodies against NSP2 and NSP5; the positive cells were shown as green fluorescence. Nuclei were counterstained with DAPI. Representative data from one of three independent experiments are shown. Scale bar, 1 μm. (D) The numbers of viroplasms in RV-infected WT and SPCS1 KO HEK293T cells were quantified using images from (C). (E) The sizes of viroplasms in RV-infected WT and SPCS1 KO HEK293T cells were quantified using images from (C) by Image J. The results are the averages of data in three different cells and plotted as mean ± SD. Statistical significance was determined using two-way ANOVA with Sidak’s multiple comparisons test.

Article Snippet: Human embryo kidney HEK293T cells (ATCC CRL-3216), were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, catalog number 11965118) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Avantor, catalog number 267820).

Techniques: Infection, Quantitative RT-PCR, Western Blot, Immunofluorescence, Fluorescence

Journal: PLOS Pathogens

Article Title: Signal peptidase complex mediates rotavirus VP7 processing and virion assembly

doi: 10.1371/journal.ppat.1013688

Figure Lengend Snippet: (A) WT and SPCS1 KO HEK293T cells were infected with RRV at an MOI of 3 for 16 hours, and the supernatants were subjected to sucrose cushion followed by CsCl gradient ultracentrifugation. (B) Western blot validation of sucrose cushion products by detecting DLPs and TLPs using guinea pig polyclonal serum against TLP. (C) Electron micrograph (low magnification) of RRV-infected HEK293T WT cells. ‘V’ indicates the viroplasm. (D) Enlarged view of the area boxed in red in panel (C). Blue arrows indicate TLPs. (E) Electron micrograph (low magnification) of RRV-infected SPCS1 KO cells. ‘V’ indicates the viroplasm. (F) Enlarged view of the area boxed in red in panel (E). Magenta arrows indicate abnormal morphology of TLPs. Scale bars: low magnification, 50 nm; high magnification, 100 nm.

Article Snippet: Human embryo kidney HEK293T cells (ATCC CRL-3216), were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, catalog number 11965118) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Avantor, catalog number 267820).

Techniques: Infection, Western Blot, Biomarker Discovery

Journal: PLOS Pathogens

Article Title: Signal peptidase complex mediates rotavirus VP7 processing and virion assembly

doi: 10.1371/journal.ppat.1013688

Figure Lengend Snippet: (A) Schematic diagram of rotavirus VP7, cleavage site was indicated by an arrow. (B) WT and SPCS1 KO HEK293T cells were transfected with plasmids expressing -EV, -WT VP7, -A50V mutant VP7, -non-structural proteins NSP4, respectively. Those plasmids were all tagged with GFP. At 24 hours post-transfection, cells were harvested for western blot analysis by probing GFP. (C) Quantification of the cleavage ratio of WT and A50V mutant VP7 proteins in WT and SPCS1 KO HEK293T cells. The results are the averages of data in three independent experiments and plotted as mean ± SD. Statistical significance was determined by two-way ANOVA with Sidak’s multiple comparisons test (***, P < 0.001, **** P < 0.0001).

Article Snippet: Human embryo kidney HEK293T cells (ATCC CRL-3216), were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, catalog number 11965118) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Avantor, catalog number 267820).

Techniques: Transfection, Expressing, Mutagenesis, Western Blot

Journal: PLOS Pathogens

Article Title: Signal peptidase complex mediates rotavirus VP7 processing and virion assembly

doi: 10.1371/journal.ppat.1013688

Figure Lengend Snippet: (A) Alpha-Fold 3 prediction of VP7 with SPC complex (SPCS1, SPCS2, SPCS3, and SEC11A). (B) The magnification of the interaction site of VP7 and SPCS3 from (A). (C) Co-IP of SPCS1 with VP7 mutants. HEK293 cells were co-transfected with plasmids expressing SPCS1-FLAG with GFP-WT VP7, -E256R mutant VP7, -NSP4 and -EV, respectively. Cell lysates of transfected cells were immunoprecipitated with anti-GFP antibody or its corresponding isotype IgG control. The resulting precipitates and whole-cell lysates used for immunoprecipitation were examined by immunoblot using anti-GFP and anti-FLAG antibodies. (D) WT and SPCS1 KO HEK293T cells were infected with rSA11-WT or rSA11-E256R at an MOI of 3. At 12 hpi, all the cell lysates and supernatants were collected for FFU assay to detect the titers. Statistical significance was determined by two-way ANOVA with Sidak’s multiple comparisons test (***, P ＜0.001, **** P ＜0.0001). (E) Samples were collected from WT HEK293T cells infected with rSA11-VP7-WT and rSA11-VP7-E256R in three independent experiments. The purified PCR products targeting the VP7 gene were subjected to Sanger sequencing, and the results are shown in the chart (right panel). Direct sequencing of VP7 from rSA11-VP7-WT and rSA11-VP7-E256R viruses served as controls. The red box indicates the codon of mutated amino acids.

Article Snippet: Human embryo kidney HEK293T cells (ATCC CRL-3216), were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, catalog number 11965118) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Avantor, catalog number 267820).

Techniques: Co-Immunoprecipitation Assay, Transfection, Expressing, Mutagenesis, Immunoprecipitation, Control, Western Blot, Infection, Purification, Sequencing