Journal: Pathogens

Article Title: Evaluation of RNA Secondary Stem-Loop Structures in the UTRs of Mouse Hepatitis Virus as New Therapeutic Targets

doi: 10.3390/pathogens13060518

Figure Lengend Snippet: Inhibitory effects of shRNAs targeting RNA secondary SL structures in the 5′UTR on MHV-A59 replication. ( a ) RNA dot blot analysis for inhibitory effects of shRNA on MHV replication. DBT cells (DBT-5′shRNA cells) expressing shRNAs (5′shRNA) targeted for MHV-A59 5′UTR SL structures were established by the shRNA lentiviral system. MHV-A59 was infected at an M.O.I 10 −1 to 10 −4 into DBT-5′shRNA cells. RNA dot blot probes were made to detect the MHV N gene (NC_048217.1, nt 31,519-31,018). ( b ) As an internal control, the expression of the actin gene (NM_007393.5, nt 668-1180) in DBT-5′shRNA cells was analyzed by RNA dot blot assay. ( c ) The relative inhibitory effect of 5′shRNAs on MHV-A59 infection based on actin expression was quantified by ImageJ software ( https://imagej.net/ij/ , accessed on 1 January 2023) . One-way analysis of variance was performed, followed by Tukey’s multiple comparison test to verify statistical significance by 5′shRNAs. Statistically significant groups were labeled as A, B, and C to differentiate them. ( d ) Northern analysis for inhibitory effect of 5′shRNA on the MHV sgRNA synthesis. MHV-A59 at an M.O.I of 0.5 was infected into DBT-5′shRNA cells for 24 h. Mock (negative control) was performed with DBT-5′shRNA cells that were not infected with MHV-A59. Patterns of the MHV-A59 sgRNA production in DBT-5′shRNA cells were investigated with a DIG labelled N gene probe. ( e ) The effect of 5′shRNA inhibition on the MHV sgRNA synthesis was quantitatively analyzed with Image J software. ( f ) The one-step growth experiments of MHV-A59 infected DBT-5′shRNA cells. 5 × 10 4 DBT cells were seeded in 24-well plates and infected with MHV-A59 at an M.O.I of 3 for 0, 6, 12, 18, and 24 h the next day. After each infection time, MHV-A59 was harvested and the growth rate of the virus was analyzed. ( g ) Representative plaque shape formed by MHV-A59 infecting DBT-5′shRNA cells. ( h ) Average plaque size of MHV-A59 formed on DBT-5′shRNA cells. Statistical analysis was performed using a student’s t -test and a one-way analysis of variance (ANOVA). A post-hoc test was conducted using Tukey’s multiple comparisons test. ( i ) Analysis of the 5′UTR sequence of MHV-A59 infected DBT-5′shRNA cells. The MHV-A59 virus, harvested after infecting DBT-5′shRNA cells, was designated as P0. Subsequently, the harvested virus obtained after re-infecting cells was named P1, and the virus obtained after another round of infection and harvest was named P2. RNA was extracted from each of these virus samples, and sequence analysis was performed. * indicates a statistical difference in the t -test between the shCon-treated plaque size group and the shSL1/2 plaque size group; ** indicates a statistical difference in the t -test between the shCon-treated plaque size group and the shSL3/4 plaque size group; *** indicates a statistical difference in the t -test statistical difference between the shCon-treated plque size group and the siMIN-treated plque size group is significant.

Article Snippet: In the case of siRNA, the GenScript siRNA Design Tool ( https://www.genscript.com/tools/sirna-target-finder?page_no = 1&position_no = 2&sensors = googlesearch, accessed on 1 January 2022) was employed to design the siRNAs targeting the MHV-A59 5′UTR and 3′UTR.

Techniques: Dot Blot, shRNA, Expressing, Infection, Control, Software, Comparison, Labeling, Northern Blot, Negative Control, Inhibition, Virus, Sequencing

Journal: The Journal of Neuroscience

Article Title: Vesicular Glutamate Transporter Expression Level Affects Synaptic Vesicle Release Probability at Hippocampal Synapses in Culture

doi: 10.1523/JNEUROSCI.1444-14.2014

Figure Lengend Snippet: shRNA knockdown of VGLUT2 affects responses in VGLUT1 KO autapses. A, Example traces of EPSCs from VGLUT1 KO autapses expressing shRNA against VGLUT2 (red) or FoxP2 (green), or cytosolic green fluorescent protein (GFP; black). Black bars indicate 2 ms depolarization. Depolarization artifacts have been blanked for illustrative purposes. B, Average EPSC amplitude recorded from two cultures of groups described in A. C, Same traces as A scaled to the peak amplitude of the EPSC from pulse 1. D, Average paired-pulse ratios calculated from two EPSCs at a 25 ms interstimulus interval recorded from VGLUT1 KO autapses groups as described in A. For B and D, significance was determined by Kruskal–Wallis test with Dunn's post-test: ***p ≤ 0.001. For all graphs, error bars represent the SEM. n values (number of cells) are indicated in graphs.

Article Snippet: Two VGLUT2-specific siRNA (1: 5′-TCA CTT GGA TAT TGC TCC A-3′; 2: 5′-CAG CAC TAT CCA CCG CGG A-3′) and one FoxP2-specific siRNA as control (5′-GGA ACG CGA ACG TCT TCA A-3′) were obtained using GenScript siRNA Target Finder (GenScript) and cloned as shRNA in a U6 promoter expression cassette within a lentiviral shuttle vector.

Techniques: shRNA, Expressing

Journal: The Journal of Neuroscience

Article Title: Vesicular Glutamate Transporter Expression Level Affects Synaptic Vesicle Release Probability at Hippocampal Synapses in Culture

doi: 10.1523/JNEUROSCI.1444-14.2014

Figure Lengend Snippet: The transport-deficient VGLUT2 protein does not rescue the distorted morphology of empty vesicles in standard EM. A–C, Example electron micrographs of synapses from VGLUT1 KO, VGLUT2 knock-down (KD) hippocampal cultures rescued with shRNA-resistant VGLUT2 WT (A) or VGLUT2 TPM (B) proteins, or cultures without rescue (C). Scale bars: full images, 100 nm; insets, 40 nm (insets are indicated by a dashed box in the full image). D, Quantification of vesicle circularity measurement averaged per culture and normalized to the VGLUT2 rescue condition. Total number of vesicles measured: VGLUT2 WT: n = 1202, three cultures; VGLUT2 TPM: n = 1245, three cultures; no rescue: n = 656, two cultures. Significance was determined by Kruskal–Wallis test with Dunn's post-test: *p ≤ 0.05. Error bars represent the SEM. E, Ratiometric immunofluorescence signals for VGLUT2 over synaptophysin 1 measured for the same cultures as measured in D. VGLUT2/Syp1 signal normalized to the VGLUT2 WT rescue per culture plotted as a box-and-whisker plot with median (horizontal bar), mean (cross), 25th to 75th percentiles (box), and minimum to maximum values (whiskers). For each group, n = 19 images over three cultures. Significance was determined by Kruskal–Wallis test with Dunn's post test: ***p ≤ 0.001.

Article Snippet: Two VGLUT2-specific siRNA (1: 5′-TCA CTT GGA TAT TGC TCC A-3′; 2: 5′-CAG CAC TAT CCA CCG CGG A-3′) and one FoxP2-specific siRNA as control (5′-GGA ACG CGA ACG TCT TCA A-3′) were obtained using GenScript siRNA Target Finder (GenScript) and cloned as shRNA in a U6 promoter expression cassette within a lentiviral shuttle vector.

Techniques: shRNA, Immunofluorescence, Whisker Assay

Journal:

Article Title: PSD-93 Deficiency Protects Cultured Cortical Neurons from NMDA Receptor-triggered Neurotoxicity

doi: 10.1016/j.neuroscience.2010.01.030

Figure Lengend Snippet: Efficacy of pCMV-U6-siRNA constructs. (A)RT-PCR analysis showed that a 495 bp product from PSD-93 was inhibited significantly by siP3, GAPDH mRNA was used as a loading Control. siP3 significantly decreased the mRNA of PSD93 by 80.2% of control.(B) Confirmation by western blot analysis of silencing PSD93 expression, The blots were reprobed with anti-GAPDH antibody to verify protein loading. (C) Relative protein level of PSD-93 after siRNA transfection, PSD93 decreased by 79.5% compared to control group when transfected with siP3.

Article Snippet: Small RNA (siRNA) Target Finder software (from Genescript Corporation, http://www.genscript.com ) was used to design siRNA and construct small hairpin RNA against PSD93 (shRNA). shRNAs were synthesized and subsequently cloned into pCMV-U6 vector using Bbsl and BglII (Genscript, USA).

Techniques: Construct, Reverse Transcription Polymerase Chain Reaction, Control, Western Blot, Expressing, Transfection