doxycycline inducible short hairpin rna shrna expression vector  (Addgene inc)

Journal: bioRxiv

Article Title: tRNA thiolation defects disrupt cellular proteostasis and tissue homeostasis in mammals

doi: 10.1101/2025.10.24.684405

Figure Lengend Snippet: (a) Western blot analysis of doxycycline (dox)-inducible CTU2 knockdown in HeLa cells over time. Duration of dox treatment is indicated in days. scrambled, non-targeting shRNA control. (b) APM-northern blot validation of tRNA thiolation loss in HeLa and RPE1 cells after 4 days of dox treatment. is shown representatively. (c) Viability analysis of CTU2 knockdown cell lines by Annexin V staining. +CTU2 res indicates shRNA-resistant CTU2 overexpression. One-way ANOVA, WT vs. CTU2 res p = 0.264. n = 3 technical replicates. (d) Western blot analysis of unfolded protein response (UPR) checkpoints during progressive tRNA hypothiolation in RPE1 cells. Time points indicate days of CTU2 depletion. (e) Western blot validation of selected proteins identified in DREAM-PL patient fibroblasts, analyzed under acute CTU2 knockdown conditions. (f) Ribosome profiling in 96 h CTU2 -depleted RPE1 cells compared with untreated controls. Normalized codon occupancy is shown for A- and G-ending codons of the indicated amino acids. Codons are divided into quartiles according to relative library abundance. n = 2 technical replicates. (g) Cumulative A-ending codon frequency (AAA, CAA, GAA, and AGA) in coding sequences (CDSs) of proteins quantified in CTU2 -depleted RPE1 cells after 96 hours. The dashed line indicates the average A-ending codon content (7.6%) across 19,085 analyzed human CDSs. Two-tailed unpaired t-test, p > 0.001 (down and up). (h) Gene Ontology (GO) enrichment analysis on the top 5% of human coding sequences ranked by A-ending codon content (AAA, CAA, GAA, AGA). Enriched pathways were clustered by GO Biological Process (2023). Cilium-associated terms are highlighted in orange. (i) Ribosome occupancy analysis of all mRNAs measured by ribosome profiling (f). Transcripts were ranked by A-ending codon frequency (AAA, CAA, GAA and AGA) and divided into ten equal bins (bin 1 = lowest, bin 10 = highest). A negative correlation was observed between A-ending codon frequency and ribosome abundance. ANOVA with post hoc test. Adjusted p-values are indicated. (j) Ciliogenesis assay in CTU2 -knockdown and wildtype RPE1 cells upon serum starvation. Representative immunofluorescence images show primary cilia (green) and nuclei (blue). The treatment scheme is shown above. Quantification of fractions of cilia-forming RPE1 cells under tRNA hypothiolation is provided. Two-tailed unpaired t-test, p-values are indicated. n = 3 technical replicates. Scale bars 10µm.

Article Snippet: For CTU2 knockdown lines we utilized the GLTR system, an all-in-one lentiviral, doxycycline inducible short hairpin RNA (shRNA) expression vector (Addgene 55790, 58246).

Techniques: Western Blot, Knockdown, shRNA, Control, Northern Blot, Biomarker Discovery, Staining, Over Expression, Two Tailed Test, Immunofluorescence

Journal: eLife

Article Title: SAFB regulates hippocampal stem cell fate by targeting Drosha to destabilize Nfib mRNA

doi: 10.7554/eLife.74940

Figure Lengend Snippet:

Article Snippet: Recombinant DNA reagent , pENTR-D-Topo- hTRIM9 (plasmid) , , RRID: Addgene_51032 , pENTR expression vector.

Techniques: Protease Inhibitor, Recombinant, Clone Assay, Bicinchoninic Acid Protein Assay, Mutagenesis, Transfection, Sequencing, esiRNA, Blocking Assay, Plasmid Preparation, Expressing, Software