Journal: Nucleic Acids Research

Article Title: Dyskerin depletion increases VEGF mRNA internal ribosome entry site-mediated translation

doi: 10.1093/nar/gkt587

Figure Lengend Snippet: Dyskerin KD stimulates VEGF-IRES–mediated translation. Transient transfection of DKC1-specific siRNA strongly reduced DKC1 mRNA and protein level in MCF7 and MDA-MB231 cells ( A , left and B , left, respectively). [3H]-leucine incorporation indicates that the total protein synthesis is not compromised (A and B, center) after DKC1 KD. IRES-mediated translation was assessed by measuring the FLuc and RLuc activity in MCF-7 and MDA-MB231 (A, right, and B, right, respectively) cells 8 h after the transfection with the bicistronic mRNA transcribed from pRL-VEGF-IRES. siRNA transfection was performed 96 h before cell harvesting. Histograms represent means and SDs from at least three independent experiments. P < 0.05 are considered significant. NS = not significant.

Article Snippet: Sets of primers and fluorogenic probes specific for DKC1 (catalog number Hs00154737_m1), VEGF (Hs00900054_m1) and ®-Actin (Hs99999903_m1) mRNAs were purchased from Applied Biosystems.

Techniques: Transfection, Activity Assay, Cell Harvesting

Journal: Nucleic Acids Research

Article Title: Dyskerin depletion increases VEGF mRNA internal ribosome entry site-mediated translation

doi: 10.1093/nar/gkt587

Figure Lengend Snippet: Dyskerin KD drives VEGF mRNA translation in breast cancer cells. Total (left) and polysome-associated (right) VEGF mRNA levels assessed by real-time PCR after DKC1 KD in MCF7 ( A ) and MDA-MB231 cells ( C ). Representative polysomal profiles are shown. VEGF protein levels in supernatant and in whole cell extracts are also reported for MCF-7 ( B ) and MDA-MB231 cells ( D ). siRNA transfection was performed 96 h before cell harvesting. Histograms represent means and SDs from three independent experiments. P < 0.05 is considered significant.

Article Snippet: Sets of primers and fluorogenic probes specific for DKC1 (catalog number Hs00154737_m1), VEGF (Hs00900054_m1) and ®-Actin (Hs99999903_m1) mRNAs were purchased from Applied Biosystems.

Techniques: Real-time Polymerase Chain Reaction, Transfection, Cell Harvesting

Journal: Nucleic Acids Research

Article Title: Dyskerin depletion increases VEGF mRNA internal ribosome entry site-mediated translation

doi: 10.1093/nar/gkt587

Figure Lengend Snippet: The increased VEGF secretion in DKC1 KD cells is due to the up-regulation of VEGF mRNA IRES-mediated translation. ( A ) Histograms show the level of VEGF protein detected using an ELISA array. Experiments were performed in MCF-7 control (left) and DKC1 KD (right) cells treated with 15 nM PTC299 for 72 h. VEGF secretion of PTC299 treated cells is normalized on the correspondent untreated cells; for the effect of DKC1 KD on VEGF secretion on MCF7 cells see A ( B ) VEGF-IRES–mediated translation measured in MCF7 control and DKC1 KD cells after 72 h of 100 nM PTC299 treatment. siRNA transfection was performed 96 h before cell harvesting. ( C ) Representative image (left) and a summarizing graph (right) from clonogenic assays performed with control (empty vector) and shDKC1 MCF7 cells. Histograms represent means and SDs from three independent experiments. P < 0.05 is considered significant. NS = not significant.

Article Snippet: Sets of primers and fluorogenic probes specific for DKC1 (catalog number Hs00154737_m1), VEGF (Hs00900054_m1) and ®-Actin (Hs99999903_m1) mRNAs were purchased from Applied Biosystems.

Techniques: Enzyme-linked Immunosorbent Assay, Control, Transfection, Cell Harvesting, Plasmid Preparation

Journal: Nucleic Acids Research

Article Title: Dyskerin depletion increases VEGF mRNA internal ribosome entry site-mediated translation

doi: 10.1093/nar/gkt587

Figure Lengend Snippet: DKC1 KD increases VEGF-IRES recruitment to 48S preinitiation complex. ( A ) Left: representative profile at 260 nM O.D. obtained from MCF7 cytoplasmic extracts: fractions 18–21 were considered to correspond to the small ribosomal subunits. Right: Representative profile of a sucrose density gradient reporting the radioactive intensity per fraction in from DKC1 KD (circles) and control (SCR—triangles) cells extracts, respectively. Peaks of radioactivity coinciding with the identified fractions containing the 48S complexes was generated when MCF7 cytoplasmic extracts were incubated with a [ 32 P]VEGF IRES mRNA probe. ( B ) Histogram represent mean and SD of the radioactivity measured in the identified peaks for DKC1 KD and control (SCR) cells extracts. siRNA transfection was performed 96 h before cell harvesting. P < 0.05 is considered significant.

Article Snippet: Sets of primers and fluorogenic probes specific for DKC1 (catalog number Hs00154737_m1), VEGF (Hs00900054_m1) and ®-Actin (Hs99999903_m1) mRNAs were purchased from Applied Biosystems.

Techniques: Control, Radioactivity, Generated, Incubation, Transfection, Cell Harvesting

Journal: Nucleic Acids Research

Article Title: Dyskerin depletion increases VEGF mRNA internal ribosome entry site-mediated translation

doi: 10.1093/nar/gkt587

Figure Lengend Snippet: Reduction of dyskerin levels differentially affects IRES-mediated translation of viral and cellular IRESs. ( A ) IRES-mediated translation assessed by measuring the FLuc and RLuc activity in MCF-7 DKC1 KD cells, 8 h after transfection with a bicistronic mRNA transcribed from viral pR-CrPV-IRES-F (left), pR-HCV-IRES-F (center) and pF-EMCV-IRES-R (right). ( B ) IRES-mediated translation assessed by measuring the FLuc and RLuc activity in MCF-7 DKC1 KD cells 8 h after transfection with a bicistronic mRNA transcribed from cellular pR-HSP70-IRES-F (left), pR-c-MYC-IRES-F (center) and pR-p53IRES-F (right). siRNA transfection was performed 96 h before cell harvesting. Histograms represent means and SDs from three independent experiments. P < 0.05 is considered significant. NS = not significant.

Article Snippet: Sets of primers and fluorogenic probes specific for DKC1 (catalog number Hs00154737_m1), VEGF (Hs00900054_m1) and ®-Actin (Hs99999903_m1) mRNAs were purchased from Applied Biosystems.

Techniques: Activity Assay, Transfection, Cell Harvesting

Journal: Nature Cell Biology

Article Title: A comprehensive tRNA pseudouridine map uncovers targets dependent on human stand-alone pseudouridine synthases

doi: 10.1038/s41556-025-01803-w

Figure Lengend Snippet: a , Comparison of the modification levels of Ψ sites at selected positions of human tRNAs upon PUS1 depletion. Box plots visualize all Ψ sites at each position; boxes represent the 25th to 75th percentiles with a line at the median; whiskers correspond to 1.5 times the interquartile range (cy-tRNA: Ψ27/28, n = 126 Ψ sites; mt-tRNA: Ψ27/28, n = 21 Ψ sites; Ψ66/67/68, n = 4 Ψ sites; Ψ20, n = 1 Ψ site; Ψ25, n = 1 Ψ site). Results are adapted from ref. . b , Sequence motifs of PUS1-dependent Ψ sites in human cy-tRNAs (top) and mt-tRNAs (bottom). Results are adapted from ref. . c , Comparison of the modification levels of Ψ sites at selected positions of human tRNAs upon PUS3 depletion. Box plots visualize all Ψ sites at each position; boxes represent the 25th to 75th percentiles with a line at the median; whiskers correspond to 1.5 times the interquartile range (cy-tRNA: Ψ38, n = 28 Ψ sites; Ψ39, n = 86 Ψ sites; Ψ40, n = 12 Ψ sites). d , Comparison of Ψ38–40 modification levels in human cy-tRNAs and mt-tRNAs following PUS3 or PUSL1 depletion. Box plots visualize all Ψ sites at each position; boxes represent the 25th to 75th percentiles with a line at the median; whiskers correspond to 1.5 times the interquartile range (cy-tRNA: Ψ38/39/40, n = 126 Ψ sites; mt-tRNA: Ψ38/39/40, n = 16 Ψ sites). e , Comparison of the modification levels of Ψ sites at selected positions of human tRNAs upon PUSL1 depletion. Box plots visualize all Ψ sites at each position; boxes represent the 25th to 75th percentiles with a line at the median; whiskers correspond to 1.5 times the interquartile range (mt-tRNA: Ψ38, n = 2 Ψ sites; Ψ39, n = 9 Ψ sites; Ψ40, n = 5 Ψ sites). f , Sequence motif of PUS3-dependent Ψ sites in human tRNAs. g , Sequence motif of PUSL1-dependent Ψ sites in human tRNAs.

Article Snippet: The following primary antibodies were used: RPUSD1 antibody (Invitrogen, PA5-59448, lot R37969 ; 1:1,000 dilution), RPUSD2 antibody (Proteintech, 25707-1-AP, lot 00057317; 1:1,000 dilution), RPUSD3 antibody (Santa Cruz, sc-393209, lot 10413; 1:500 dilution), RPUSD4 antibody (Sigma, HPA039689, lot A118277; 1:1,000 dilution), PUS10 antibody (Abcam, ab313622, lot 1059517-4; 1:1,000 dilution), TRUB2 antibody (Proteintech, 19891-1-AP, lot 00076382; 1:1,000 dilution), PUS3 antibody (Proteintech, 17248-1-AP, lot 00099351; 1:1,000 dilution), PUSL1 antibody (Sigma, HPA032057, lot R32031 ; 1:1,000 dilution), and anti-vinculin antibody (Invitrogen, 700062, lot 2616511, clone number 42H89L44; 1:3,000 dilution).

Techniques: Comparison, Modification, Sequencing

Journal: Nature Cell Biology

Article Title: A comprehensive tRNA pseudouridine map uncovers targets dependent on human stand-alone pseudouridine synthases

doi: 10.1038/s41556-025-01803-w

Figure Lengend Snippet: (a) Sanger sequencing of PUS3-KO and PUSL1-KO cells, illustrating CRISPR-induced indels in HCT116 cells. (b) Representative immunoblots from two independent experiments depict the efficiency of two distinct monoclonal KOs in HCT116 cells. Anti-Vinculin served as a loading control.

Article Snippet: The following primary antibodies were used: RPUSD1 antibody (Invitrogen, PA5-59448, lot R37969 ; 1:1,000 dilution), RPUSD2 antibody (Proteintech, 25707-1-AP, lot 00057317; 1:1,000 dilution), RPUSD3 antibody (Santa Cruz, sc-393209, lot 10413; 1:500 dilution), RPUSD4 antibody (Sigma, HPA039689, lot A118277; 1:1,000 dilution), PUS10 antibody (Abcam, ab313622, lot 1059517-4; 1:1,000 dilution), TRUB2 antibody (Proteintech, 19891-1-AP, lot 00076382; 1:1,000 dilution), PUS3 antibody (Proteintech, 17248-1-AP, lot 00099351; 1:1,000 dilution), PUSL1 antibody (Sigma, HPA032057, lot R32031 ; 1:1,000 dilution), and anti-vinculin antibody (Invitrogen, 700062, lot 2616511, clone number 42H89L44; 1:3,000 dilution).

Techniques: Sequencing, CRISPR, Western Blot, Control

Journal: Med (New York, N.y.)

Article Title: Evolution of long-term vaccine-induced and hybrid immunity in healthcare workers after different COVID-19 vaccine regimens

doi: 10.1016/j.medj.2023.02.004

Figure Lengend Snippet:

Article Snippet: SARS-CoV-2 (B.1.617.2; AY.1, AY.2, AY.3) Stabilized Spike Glycoprotein, His-Strep-Tag (HEK293) , The Native Antigen Company , Cat#REC31975-100.

Techniques: Purification, Virus, Recombinant, Modification, Synthesized, Saline, Capsules, Staining, Activation Assay, Control, Enzyme-linked Immunospot, Software, Flow Cytometry

Journal: Nature Methods

Article Title: Absolute quantitative and base-resolution sequencing reveals comprehensive landscape of pseudouridine across the human transcriptome

doi: 10.1038/s41592-024-02439-8

Figure Lengend Snippet: a , Integrated view of the PUS-dependent Ψ profiles of HeLa cy-tRNAs and mt-tRNAs. Ψ 55 in HeLa cy-tRNAs is partially dependent on TRUB1, which is labeled by the dashed line. b , Comparison of the modification levels of Ψ 55 in HeLa cy-tRNAs and mt-tRNAs upon TRUB1 depletion. Box plots visualize all Ψ sites at each position; boxes represent the 25th to 75th percentiles with a line at the median; whiskers correspond to 1.5 times the interquartile range (cy-tRNA Ψ 55 , n = 180; mt-tRNA Ψ 55 , n = 6). c , Scatterplot illustrating all TRUB1-dependant Ψ sites across the HeLa transcriptome. d , Sequence motifs of TRUB1-dependent Ψ sites in mt-tRNAs and poly-A-tailed RNA. e , Comparison of the modification levels of Ψ sites at selected positions of HeLa cy-tRNAs upon PUS7 depletion. Box plots visualize all Ψ sites at each position; boxes represent the 25th to 75th percentiles with a line at the median; whiskers correspond to 1.5 times the interquartile range (cy-tRNA: Ψ 13 , n = 43; Ψ 20B , n = 12; Ψ 35 , n = 7; Ψ 36 , n = 4; Ψ 50 , n = 3). f , Comparison of the modification levels of Ψ 50 in mt-tRNA Met between wild-type (WT) and PUS7-KO cell lines. g , Sequence motifs of PUS7-dependent Ψ sites in tRNAs and poly-A-tailed RNA. h , Comparison of the modification levels of Ψ sites at selected positions of HeLa cy-tRNAs and mt-tRNAs upon PUS1 depletion. Box plots visualize all Ψ sites at each position; boxes represent the 25th to 75th percentiles with a line at the median; whiskers correspond to 1.5 times the interquartile range (cy-tRNA: Ψ 27/28 , n = 130; mt-tRNA: Ψ 27/28 , n = 21; Ψ 66/67/68 , n = 4). i , Scatterplot illustrating all PUS1-dependant Ψ sites in HeLa mt-mRNAs. j , Sequence motifs of PUS1-dependent Ψ sites in cy-tRNAs and mt-tRNAs.

Article Snippet: Finally, clones were expanded and picked for western blot validation with TRUB1 antibody (Proteintech, 12520-1-AP; 1:1,000 dilution), PUS7 antibody (Abcam, ab226257; 1:10,000 dilution) and PUS1 antibody (Proteintech, 11512-1-AP; 1:1,000 dilution).

Techniques: Labeling, Comparison, Modification, Sequencing