online shrna design tools  (Merck & Co)

Journal: Journal of Virology

Article Title: Tetraspanin CD81 serves as a functional entry factor for porcine circovirus type 2 infection

doi: 10.1128/jvi.01408-24

Figure Lengend Snippet: CD81 promotes PCV2 replication. ( A ) pCAGGS-CD81-Flag recombinant plasmids were transfected into PK-15 cells, followed by inoculation with 0.1 MOI PCV2. After 24 h of infection (hpi), Western blot was conducted to detect the expression levels of corresponding proteins. The protein bands were quantified using NIH ImageJ software. ( B ) TCID 50 assay was performed to determine the influence of overexpressing CD81 on the progeny of PCV2. ( C ) Indirect IFA was conducted to assess the infection status of PCV2 on PK-15 cells after overexpression of CD81. Scale bar, 50 µm. Quantification of CoraLite488 and DAPI (4',6-diamidino-2-phenylindole) fluorescence-positive cells was performed using the ImageJ plugin, respectively. The proportion of PCV2-positive cells relative to the total number of cells within the field of view was statistically analyzed. The vector in A–C is the pCAGGS control plasmid. ( D ) Western blot was conducted to detect the interference effect of CD81 siRNA. siNC (siRNA negative control) is the negative-control interference fragments. ( E–G ) RNA interference. PK-15 cells transfected with CD81 siRNA were infected with 0.1 MOI PCV2 for 36 h. Western blot was conducted to detect the expression levels of PCV2 Cap and endogenous CD81 ( F ). The protein bands were quantified using ImageJ software. TCID 50 assay was conducted to determine the influence of silencing CD81 gene on the progeny PCV2 ( E ). IFA was performed to evaluate the infection of PCV2 on PK-15 cells after silencing CD81 gene. Also, quantification of CoraLite488 and DAPI fluorescence-positive cells was performed using the ImageJ plugin, respectively. The proportion of PCV2-positive cells was statistically analyzed. ( G ). Scale bar, 50 µm. Data represent the mean ± SD of three independent replicate experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: Using the shRNA online design tool ( https://rnaidesigner.thermofisher.com ), shRNAs targeting porcine CD81 and RhoA genes were designed and the sequences were submitted to Nanjing GenScript Company for synthesis.

Techniques: Recombinant, Transfection, Infection, Western Blot, Expressing, Software, Over Expression, Fluorescence, Plasmid Preparation, Control, Negative Control

Journal: Journal of Virology

Article Title: Tetraspanin CD81 serves as a functional entry factor for porcine circovirus type 2 infection

doi: 10.1128/jvi.01408-24

Figure Lengend Snippet: CD81 is involved in PCV2 internalization into PK-15 cells. ( A–C ) CD81 knockdown (shCD81) and negative-control shRNA (shNC) PK-15 cells were infected with 0.1 MOI PCV2. After 36 h, RNA and protein samples were collected. qPCR was performed to detect the expression levels of CD81 mRNA ( A ) and PCV2 Cap mRNA ( B ). Western blot was conducted to detect the expression levels of PCV2 Cap protein and endogenous CD81. The protein bands were quantified using ImageJ software ( C ). TCID 50 assay was used to determine the viral titer of PCV2 ( D ). ( E ) The virus adsorption assay. CD81 knockdown and shNC PK-15 cells were inoculated with 5 MOI PCV2 and incubated for 1 h at 4°C. DNA samples were extracted, and qPCR was performed to detect the relative content of PCV2, with mtDNA (Mitochondrial DNA) as an internal control. The y-axis represents the fold change in viral DNA content between the experimental and the control groups, calculated using the 2 -ΔΔCT formula based on the CT values of the target gene Cap and the reference gene mtDNA (Mitochondrial DNA). ( F ) The virus internalization assay. CD81 knockdown and shNC PK-15 cells were inoculated with 5 MOI PCV2 and incubated for 1 h at 4°C, and then transferred to 37°C for an additional 2 h. DNA samples were extracted, and qPCR was performed to detect the relative content of PCV2, with mtDNA as an internal control. The y-axis represents the fold change in viral DNA content between the experimental and control groups, calculated using the 2 -△△CT formula based on the CT values of the target gene Cap and the reference gene mtDNA. Data represent the mean ± SD of three independent replicate experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: Using the shRNA online design tool ( https://rnaidesigner.thermofisher.com ), shRNAs targeting porcine CD81 and RhoA genes were designed and the sequences were submitted to Nanjing GenScript Company for synthesis.

Techniques: Knockdown, Negative Control, shRNA, Infection, Expressing, Western Blot, Software, Virus, Adsorption, Incubation, Control

Journal: Journal of Virology

Article Title: Tetraspanin CD81 serves as a functional entry factor for porcine circovirus type 2 infection

doi: 10.1128/jvi.01408-24

Figure Lengend Snippet: Syndecan-1 collaborates with CD81 to facilitate PCV2 infection. ( A ) The interaction between CD81 and Syndecan-1 was detected with Co-IP. ( B ) The interaction between PCV2 Cap protein and Syndecan-1 was detected with Co-IP. ( C ) The endogenous CD81 and Syndecan-1 were detected in PK-15 cells with Co-IP. PK-15 cells were infected with PCV2 for 36 h, lysed, and immunoprecipitated with anti-Syndecan-1 antibody. The whole-cell lysates and immunoprecipitated products were analyzed by Western blot. ( D ) Immunofluorescence confocal microscopy was used to detect co-localization of CD81 and Syndecan-1 at the cell membrane. Scale bar, 5 µm. ( E ) Western blot was conducted to detect the interference effect of three siRNAs targeting Syndecan-1. ( F–J ) PK-15 cells transfected with the siRNA targeting CD81 and Syndecan-1 were infected with 0.1 MOI PCV2 or 5 MOI PCV2. Western blot was performed to detect the expression levels of PCV2 Cap protein, and the protein bands were quantified using ImageJ software ( F ). qPCR was performed to detect the expression levels of CD81 mRNA ( G ), Syndecan-1 mRNA ( H ), PCV2 Cap mRNA ( I ), and the relative content of adsorbed PCV2, with mtDNA (Mitochondrial DNA) as an internal control ( J ). The y-axis represents the fold change in viral DNA content between the experimental and control groups, calculated using the 2 -ΔΔCT formula based on the CT values of the target gene Cap and the reference gene mtDNA (Mitochondrial DNA). Data represent the mean ± SD of three independent experiments. *, P < 0.05, **, P < 0.01, ***, P < 0.001.

Article Snippet: Using the shRNA online design tool ( https://rnaidesigner.thermofisher.com ), shRNAs targeting porcine CD81 and RhoA genes were designed and the sequences were submitted to Nanjing GenScript Company for synthesis.

Techniques: Infection, Co-Immunoprecipitation Assay, Immunoprecipitation, Western Blot, Immunofluorescence, Confocal Microscopy, Membrane, Transfection, Expressing, Software, Control

Journal: Virology Journal

Article Title: Intracellular delivery of antiviral shRNA using penetratin-based complexes effectively inhibits respiratory syncytial virus replication and host cell apoptosis

doi: 10.1186/s12985-024-02519-3

Figure Lengend Snippet: Anti-viral activity of penetratin-shRNA, ribavirin, and their combination. ( A ) Protection of HEp-2 cells against RSV infection, visualized under a microscope. HEp-2 cells were infected with RSV (MOI = 0.1) and treated with penetratin-shRNA (20:1), penetratin-scramble (20:1), ribavirin (35 µM), or a combination of penetratin-shRNA and ribavirin. RSV infection CPE was observed using a 10x objective lens. ( B ) Relative inhibition rate of ribavirin in HEp-2 cells against RSV. ( C ) Real-time analysis of the F gene in different treatments of HEp-2 cells infected with RSV. Viral RNA was extracted 24 h post-transfection, and the F gene was quantified. Samples were analyzed in triplicate, and RSV F gene copy number is expressed as mean ± SD. ( D ) Plaque formation by RSV in HEp-2 cells in the presence of 0.3% agarose. HEp-2 cells were stained 5 days post-infection, following the established plaque-forming assay. (E) Plaque-forming units (PFU/ml) were counted 5 days after incubation. Experiments were repeated three times, and the virus titer is expressed as mean ± SD

Article Snippet: The Biosettia online shRNA Designer tool ( https://biosettia.com/shrna/ ) was used to design shRNA targeting the RSV-F gene to enhance shRNA functionality, reduce off-target effects, and stabilize the shRNA molecules.

Techniques: Activity Assay, shRNA, Infection, Microscopy, Inhibition, Transfection, Staining, Incubation, Virus

Journal: Virology Journal

Article Title: Intracellular delivery of antiviral shRNA using penetratin-based complexes effectively inhibits respiratory syncytial virus replication and host cell apoptosis

doi: 10.1186/s12985-024-02519-3

Figure Lengend Snippet: ( A ) Fluorescence microscopy at 48 h post-transfection in A549 cells using a 10x objective lens. ( B ) DNA fragmentation in RSV-infected (MOI = 0.1) A549 cells. The hallmark of apoptosis, DNA fragmentation, was observed via GelRed staining. ( C ) Topological changes in nuclear particles in A549 cells. PI staining revealed the nuclear morphology in different groups of A549 cells under fluorescence microscopy. ( D ) Nuclear fragmentation and the presence of apoptotic bodies in A549 cells were observed as indicators of apoptosis. PI staining of uninfected A549 cells revealed normal nuclei with minimal debris, while infected-treated cells with penetratin-shRNA (20:1) and a combination of penetratin-shRNA with ribavirin showed decreased apoptotic signs compared to infected-untreated cells. ( E ) Caspase-3/7 activity in A549 cells. Caspase-3/7 activity in infected and non-infected A549 cells with various treatments was evaluated. The Caspase-3/7 activity in uninfected control groups was set to 1. At least two independent experiments were performed, and ratios are presented as mean ± SD

Article Snippet: The Biosettia online shRNA Designer tool ( https://biosettia.com/shrna/ ) was used to design shRNA targeting the RSV-F gene to enhance shRNA functionality, reduce off-target effects, and stabilize the shRNA molecules.

Techniques: Fluorescence, Microscopy, Transfection, Infection, Staining, shRNA, Activity Assay, Control

Journal: eLife

Article Title: RAPSYN-mediated neddylation of BCR-ABL alternatively determines the fate of Philadelphia chromosome-positive leukemia

doi: 10.7554/eLife.88375

Figure Lengend Snippet: ( A ) Analyses of RAPSN mRNA levels in peripheral blood mononuclear cells (PBMCs) of healthy donors and non-leukemic patients compared to those of patients with chronic myeloid leukemia (CML) from GSE13204, GSE13159, GSE138883, and GSE140385 datasets. ( B ) Quantification of RAPSN mRNA levels in PBMCs of healthy donors and patients with Ph + leukemia from the cohort of primary samples using reverse transcription-PCR (RT-PCR). ( C ) Quantification of RAPSN mRNA levels in Ph + leukemia cells (K562, KU812, MEG-01, and Jurkat) compared to normal bone marrow stromal cells (HS-5) using RT-PCR (n=4). ( D ) Quantification of RAPSN mRNA levels in K562 cells transduced with shNC or three independent shRNAs targeting RAPSN using RT-PCR (n=5). ( E ) Immunoblotting of RAPSYN in K562 cells transduced with shNC or three different shRNAs targeting RAPSN . ( F ) Cytotoxicity induced by shRNA-mediated RAPSN knockdown in KU812 cells. Representative results from at least three independent experiments are shown. ( G ) Analysis of SNARF-1 labeling intensity in K562 cells transducted with shNC or sh RAPSN #3. ( H ) Representative Fluorescence-activated cell sorting (FACS) cell cycle profiles of K562 cells transduced with shNC or sh RAPSN #3 (n=3). ( I ) Representative FACS blots of apoptosis analysis of K562 cells transduced with shNC or sh RAPSN #3 (n=3). ( J ) Individual growth curves of subcutaneous xenograft tumors were measured every 2 days from the third day after tumor inoculation for 19 days. ( K ) Quantification of RAPSYN and BCR-ABL expression in mouse xenograft tumor biopsies from K562 cells transduced with sh RAPSN #3 or shNC (n=5). ( L ) Verification of RAPSN KO in K562 cells. The red dotted line indicates deleted sequences. RAPSN mRNA levels were normalized to that of ACTIN ( B ) or GAPDH ( C, D ); error bars, mean ± standard deviation (SD); *p < 0.05, **p < 0.01, ****p < 0.0001, n.s., not significant; unpaired Student’s t -test (A, B, J, K and I) or one-way ANOVA test ( C, D ). Figure 1—figure supplement 1—source data 1. Original file for the Western blot analysis in (anti-RAPSYN, anti-GAPDH). Figure 1—figure supplement 1—source data 2. PDF containing and original scan of the relevant Western blot analysis (anti-RAPSYN, anti-GAPDH) with highlighted bands and sample labels.

Article Snippet: All shRNAs ( ) were designed using online shRNA design tools ( https://rnaidesigner.thermofisher.com and https://portals.broadinstitute.org ).

Techniques: Reverse Transcription, Reverse Transcription Polymerase Chain Reaction, Transduction, Western Blot, shRNA, Knockdown, Labeling, Fluorescence, FACS, Expressing, Standard Deviation