Journal: PLOS Pathogens

Article Title: A novel ilarvirus protein CP-RT is expressed via stop codon readthrough and suppresses RDR6-dependent RNA silencing

doi: 10.1371/journal.ppat.1012034

Figure Lengend Snippet: (A) Schematic representation of sgRNA4 and its UGG and 2st mutant clones under a T7 promoter. (B) SDS-PAGE of proteins translated in wheatgerm extracts from in vitro transcripts of sgRNA4 and its two mutants. Mock–no RNA added. After drying the gel, proteins radioactively labelled with [ 35 S]Met were detected using a phosphorimager. (C) Schematic representation of the AV2 RNA3 with a tag (grey rectangle) appended to the 3′ end of the CP-RT gene. (D) Detection of CP and CP-RT-myc by western immunoblotting in plants infected with AV2 or AV2-myc. Samples were collected from the inoculated leaf and the 2nd upper non-inoculated leaf at 4 and 7 dpi, and the 3rd upper non-inoculated leaf (see ) at 14 dpi. (E) Detection of CP and CP-RT-myc by western blotting in extracts of plants infected with AV2, AV2-myc or AV2-2st-myc. Samples were collected from the 2nd upper non-inoculated leaf at 4 dpi (see ). Positions of CP and CP-RT-myc are indicated on the left. Sizes of molecular weight markers are indicated on the right in panels B, D and E. Ponceau red staining of the large Rubisco subunit (RbcL) was used as a loading control in panels D and E.

Article Snippet: The Cy5 probe was generated using the HighYield T7 Cy5 RNA Labelling Kit (Jena Bioscience) with a T7- gfp PCR product as template.

Techniques: Mutagenesis, Clone Assay, SDS Page, In Vitro, Western Blot, Infection, Molecular Weight, Staining, Control

Journal: PLOS Pathogens

Article Title: A novel ilarvirus protein CP-RT is expressed via stop codon readthrough and suppresses RDR6-dependent RNA silencing

doi: 10.1371/journal.ppat.1012034

Figure Lengend Snippet: (A) gfp-c3 was introduced by agroinfiltration into N . benthamiana leaves along with a plasmid encoding a candidate VSR (right half of leaf) or the empty plasmid (EP) (left half of leaf). (B) Agroinfiltrated N . benthamiana leaves were imaged under UV light at 4 dpi. The agroinfiltrated constructs are indicated above the images. (C) RT-qPCR analysis of gfp mRNA accumulation. Nb ACT-b (GI:380505031) was used as a reference gene. Values represent the mean +/− SD ( n = 4). (D) Northern blot analyses depicting the accumulation of gfp -specific siRNAs. A hydrolysed Cy5 labelled in vitro transcribed RNA fragment complementary to gfp was used as a probe. 5S ribosomal RNA was used as a loading control. (E–G) As for panels A–C except mgfp5 and dsGF were used instead of gfp-c3 .

Article Snippet: The Cy5 probe was generated using the HighYield T7 Cy5 RNA Labelling Kit (Jena Bioscience) with a T7- gfp PCR product as template.

Techniques: Plasmid Preparation, Construct, Quantitative RT-PCR, Northern Blot, In Vitro, Control

Journal: bioRxiv

Article Title: Restoring Shugoshin 1 reduces chromosome errors in human eggs

doi: 10.64898/2026.01.08.698387

Figure Lengend Snippet: (A) Representative immunofluorescence images showing the localization nascent transcripts (EU, green) inside the nucleus (blue) of early GV oocytes. Images show EU levels in negative control (no EU), control (with EU), upon treatment with α-amanitin, and triptolide. Scale bar: 10 µm. (B) Frequency of PSSC with α-amanitin measured by scoring the percentage of eggs with PSSC, similar to levels observed with triptolide . Plots show number of eggs analyzed on top. Statistical significance is measured by Fisher’s exact test, ns = not significant. (C) Representative time-lapse images showing the localization of MajSat RNA in GV and MI oocytes and MII eggs upon microinjection of GV oocytes with UTP-X-Cy3 labeled MajSat (top panel) and control (bottom panel) RNA. MajSat RNA localizes to the pericentromeres during metaphase I and metaphase II stages along with foci present in the cytoplasm. Cy3-labeled control RNA localizes only in the cytoplasm and not on chromosomes. MajSat and control RNA (green), chromosomes (H2B-SNAP, blue) are shown. Scale bars: 10 µm. (D) Co-localization of MajSat RNA and SGO1 observed on metaphase I chromosome spreads upon performing RNA FISH with immunofluorescence. Scale bar: 10 µm.

Article Snippet: The HighYield T7 Cy3 RNA Labelling Kit (Jena Bioscience, #RNT-101-CY3) was used to label amplified sequences with Cy3-labelled UTP (UTP-X-Cy3, 35%), followed by DNA removal using TurboTM DNAse (ThermoFisher).

Techniques: Immunofluorescence, Negative Control, Control, Microinjection, Labeling