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: bioRxiv

Article Title: Microtubule poleward flux in human cells is driven by the coordinated action of four kinesins

doi: 10.1101/2020.06.16.155259

Figure Lengend Snippet: A . Representative spinning disk confocal live-cell imaging time series images of U2OS PA-GFP-α- tubulin cells conditionally co-expressing KIF4A shRNA and RNAi-resistant mCherry-Kif4A variants induced using doxycycline. Chromosomes were stained using SiR-DNA. Scale bars, 10 μm. Time, hour:min. B. Representative immunoblot of cell lysates obtained before and after doxycycline induction validating the efficiency of KIF4A shRNA construct and expression of the RNAi-resistant mCherry-KIF4A variants stained using anti-KIF4A antibody, with anti-vinculin used as a loading control. C. Quantification of MT-flux upon shRNA-mediated depletion of KIF4A alone or in combination with conditional expression of the RNAi-resistant mCherry-KIF4A variants. The bars in graph represent mean ± SD. N (number of cells, number of independent experiments): uninduced shKIF4A (48, 4), shKIF4A (60, 4), uninduced KIF4A WT (36, 5), shKIF4A + KIF4A WT (43, 5), uninduced KIF4A K64A (30, 4), shKIF4A + KIF4A K64A (32, 4), uninduced KIF4A ΔZip1 (32, 3), shKIF4A + ΔZip1 (30, 4). P-values were calculated using Student’s t -test. n.s. - not significant, **** P ≤ 0.0001. D. Model illustrating chromosome arms-localized KIF4A driving MT-flux.

Article Snippet: U2OS photoactivatable GFP (PA-GFP)-α-tubulin ( ) cell line expressing stable inducible short hairpin RNA (shKIF4A) targeting KIF4A sequence 5’-GCAAGATCCTGAAAGAGAT-3’ was generated using a multipurpose GATEWAY-based lentiviral tetracycline-regulated conditional RNAi system (GLTR) using pENTR-THT-III (Addgene plasmid #55791) and pGLTR-X-Puro (Addgene plasmid #58246) plasmids ( , ).

Techniques: Live Cell Imaging, Expressing, shRNA, Staining, Western Blot, Construct, Control

Journal: bioRxiv

Article Title: Microtubule poleward flux in human cells is driven by the coordinated action of four kinesins

doi: 10.1101/2020.06.16.155259

Figure Lengend Snippet: A . Representative spinning disk confocal live-cell image series of MT-flux in U2OS cells stably co-expressing PA-GFP-α- tubulin (cyan) and mCherry-α-tubulin (red) treated with indicated siRNAs. White arrowheads highlight poleward motion of the photoactivated regions due to MT-flux. Scale bars, 10 μm. Time, min:sec. B. Quantification of the impact of the MT-crosslinking proteins on MT-flux in U2OS PA-GFP/mCherry-α-tubulin cells transfected with respective siRNAs. Graph represent MT-flux values with mean ± SD. N (number of cells, number of independent experiments): siControl (49, 5), siPRC1 (45, 3), siNuMA (40, 3), siHSET (37, 3), siHSET + siNuMA (39, 3). P-values were calculated using one-way ANOVA. n.s., not significant, ** P ≤ 0.01, **** P ≤ 0.0001. C . Quantification of the impact of the motor activity of HSET on MT-flux in U2OS cells stably expressing mEOS-α-tubulin treated with control or HSET 3′UTR siRNAs in presence or absence of the respective RNAi-resistant GFP-HSET constructs. Graph represent MT-flux values with mean ± SD. N (number of cells, number of independent experiments): siControl (23, 3), siHSET 3′UTR (33, 3), siHSET 3′UTR + HSET WT (29, 5), siHSET 3′UTR + HSET N593K (30, 4). P-values were calculated using one-way ANOVA. n.s., not significant, **** P ≤ 0.0001. D. Representative kymographs of the photoactivated spindles from (B) displaying asynchronous flux motion (split of the two red dashed lines towards individual poles) upon RNAi-mediated depletion of MT-crosslinkers. E. Percentage of cells with asynchronous flux movements calculated from (B). F. Model illustrating the role of MT-crosslinking activities of NuMA and HSET in uniform distribution of poleward forces across the mitotic spindle.

Article Snippet: U2OS photoactivatable GFP (PA-GFP)-α-tubulin ( ) cell line expressing stable inducible short hairpin RNA (shKIF4A) targeting KIF4A sequence 5’-GCAAGATCCTGAAAGAGAT-3’ was generated using a multipurpose GATEWAY-based lentiviral tetracycline-regulated conditional RNAi system (GLTR) using pENTR-THT-III (Addgene plasmid #55791) and pGLTR-X-Puro (Addgene plasmid #58246) plasmids ( , ).

Techniques: Stable Transfection, Expressing, Transfection, Activity Assay, Control, Construct