hek293  (OriGene)

Journal: bioRxiv

Article Title: i 6 A-seq maps N 6 -isopentenyladenosine and uncovers its role as a regulator of mRNA stability through recruitment of DIS3L2

doi: 10.1101/2025.11.26.690722

Figure Lengend Snippet: (a) Relative normalized expression of Trit1 analysis using real-time qPCR for Trit1 KO validation, n =3, unpaired t -test * p < 0.05. (b) Venn diagram showing the decreased number of i 6 A sites found in mESCs Trit1 KO compared to control. (c) and (d) Analysis of TRIT1 OE after transfection with TRIT1 plasmid in HEK293 cells, using real-time qPCR (c), n =3, unpaired t -test, * p <0.05 and WB (d). (e) Venn diagram showing the overlap of i 6 A sites found in HEK293 TRIT1 OE and control in mRNA samples. (f-g) LC-MS/MS quantification of i 6 A/A ratio in HEK293 control and TRIT1 OE in small RNA (f) and mRNA (g), n =3, unpaired t -test *p < 0.05. (h) i 6 A sites in TRIT1 OE compared to control cells, represented according to their segment location in the transcript. (i) Sequence logo representing the deduced consensus of i 6 A sites in TRIT1 OE cells.

Article Snippet: Plasmids coding WT or mutant FLAG-tagged human TRIT1 (Origene, RC210476) were transfected into HEK293 and DMS273-shTRIT1 cells using TransIT-X2 reagent (Mirus Bio), according to manufacturer instructions, and harvested after 48 hours. siRNAs including scramble control or siRNA directed against DIS3L2 (Origene, SR315069) were transfected twice into DMS273-Scramble cells using RNAiMAX reagent (Invitrogen), according to manufacturer instructions, every 48 hours.

Techniques: Expressing, Biomarker Discovery, Control, Transfection, Plasmid Preparation, Liquid Chromatography with Mass Spectroscopy, Sequencing

Journal: bioRxiv

Article Title: i 6 A-seq maps N 6 -isopentenyladenosine and uncovers its role as a regulator of mRNA stability through recruitment of DIS3L2

doi: 10.1101/2025.11.26.690722

Figure Lengend Snippet: (a) Real-time qPCR showing TRIT1 OE effect on gene expression in highly isopentenylated genes in HEK293 cells, n =3, unpaired t -test, * p <0.05. (b) Real-time qPCR of the indicated genes in shTRIT1 cells compared to their control, before and after i 6 A-IP, n =3. (c-d) WB of the indicated cell lines, after TRIT1 OE/KD, displaying an inverse change in SLC38A10 (c) and AIG1 (d) in response to shifts in TRIT1 protein expression. (e) Immunofluorescence staining images of SLC38A10 and AIG1 in scramble and shTRIT1 DMS273 cells, revealing their increased expression under TRIT1 KD. (f) Analysis of TRIT1, SLC38A10 and AIG1 after TRIT1 WT or mutant rescue in shTRIT1 cells by real-time qPCR, n =3, unpaired t -test, * p <0.05. (g) WB of the indicated cell lines, representing similar trend for TRIT1 and SLC38A10 as in (f). (h) Real-time qPCR of SLC38A10 and AIG1 mRNA levels, in DMS273 scramble versus shTRIT1, monitored 6 hours after Actinomycin-D addition, compared to 0 hours, n =3.

Article Snippet: Plasmids coding WT or mutant FLAG-tagged human TRIT1 (Origene, RC210476) were transfected into HEK293 and DMS273-shTRIT1 cells using TransIT-X2 reagent (Mirus Bio), according to manufacturer instructions, and harvested after 48 hours. siRNAs including scramble control or siRNA directed against DIS3L2 (Origene, SR315069) were transfected twice into DMS273-Scramble cells using RNAiMAX reagent (Invitrogen), according to manufacturer instructions, every 48 hours.

Techniques: Gene Expression, Control, Expressing, Immunofluorescence, Staining, Mutagenesis

Journal: bioRxiv

Article Title: i 6 A-seq maps N 6 -isopentenyladenosine and uncovers its role as a regulator of mRNA stability through recruitment of DIS3L2

doi: 10.1101/2025.11.26.690722

Figure Lengend Snippet: (a) Table of all i 6 A sites in tRNAs that were identified by i 6 A-seq in human and mouse. (b) IGV plots of tRNA Sec IP samples in iodine-treated (top) and untreated (bottom) samples, indicating the misincorporation profile and truncation pattern at the known i 6 A position. Presence of two additional modifications is indicated, along with their known locations. These additional modifications were not affected by iodine treatment. (c) HPLC graphs of an RNA oligonucleotide derived from the tRNA Sec before and after in vitro isopentenylation by TRIT1 enzyme. (d) IGV plots of tRNA Val before and after iodine labeling, in input and IP samples, indicating the mutation profile and truncation pattern at the identified i 6 A position and the location of the adjacent m 1 A modification. (e) HPLC graphs of an RNA oligonucleotide derived from the tRNA Val before and after in vitro isopentenylation by TRIT1 enzyme. (f) Sequencing of synthetic tRNA Val oligonucleotide with i 6 A after iodine treatment, showing elevated mutation rates at the modification position. (g) IGV plots of mitochondrial tRNA Ser in mouse liver tissue (right), HAP1 control cells (middle), and in HAP1 CDK5RAP1 KO cells (left), showing that the mutation profile of ms 2 i 6 A is independent of iodine treatment.

Article Snippet: TRIT1 mutations p.R323Q and p.E327K were introduced, separately, into a purchased human TRIT1 plasmid (Origene, RC210476) using the Q5 Site-Directed Mutagenesis Kit (NEB).

Techniques: Derivative Assay, In Vitro, Labeling, Mutagenesis, Modification, Sequencing, Control

Journal: bioRxiv

Article Title: i 6 A-seq maps N 6 -isopentenyladenosine and uncovers its role as a regulator of mRNA stability through recruitment of DIS3L2

doi: 10.1101/2025.11.26.690722

Figure Lengend Snippet: (a) Venn diagram showing the number of i 6 A sites in mRNA identified in the indicated human cell lines. (b) IGV plots of the NCKIPSD gene, illustrating the presence of an i 6 A site. (c) Metagene profiles before and after iodine treatment, depicting a drop in sequence coverage just before the i 6 A position. (d-e) Sequence logos representing the deduced consensus of i 6 A sites in human (d) and mouse (e). (f) Bar plot representing distribution of i 6 A sites within gene segments in human, mouse and common sites between the two species. (g) Bar plot representing amino acid distribution of i 6 A sites in human and mouse. (h) Venn diagram showing the number of i 6 A sites in mRNA identified in the two indicated mouse tissues and mouse ESCs. (i) and (k) HPLC graphs of RNA oligonucleotides derived from representative i 6 A sites in human ESRRA (i) and mouse Pcyox1 (k), before (left) and after (right) in vitro isopentenylation by TRIT1. (j) and (l) Sequencing of synthetic ESRRA (j) and Pcyox1 (k) RNA oligonucleotides with i 6 A after iodine treatment showing elevated mutation rates at the modification position.

Article Snippet: TRIT1 mutations p.R323Q and p.E327K were introduced, separately, into a purchased human TRIT1 plasmid (Origene, RC210476) using the Q5 Site-Directed Mutagenesis Kit (NEB).

Techniques: Sequencing, Derivative Assay, In Vitro, Mutagenesis, Modification

Journal: bioRxiv

Article Title: i 6 A-seq maps N 6 -isopentenyladenosine and uncovers its role as a regulator of mRNA stability through recruitment of DIS3L2

doi: 10.1101/2025.11.26.690722

Figure Lengend Snippet: (a) Relative normalized expression of Trit1 analysis using real-time qPCR for Trit1 KO validation, n =3, unpaired t -test * p < 0.05. (b) Venn diagram showing the decreased number of i 6 A sites found in mESCs Trit1 KO compared to control. (c) and (d) Analysis of TRIT1 OE after transfection with TRIT1 plasmid in HEK293 cells, using real-time qPCR (c), n =3, unpaired t -test, * p <0.05 and WB (d). (e) Venn diagram showing the overlap of i 6 A sites found in HEK293 TRIT1 OE and control in mRNA samples. (f-g) LC-MS/MS quantification of i 6 A/A ratio in HEK293 control and TRIT1 OE in small RNA (f) and mRNA (g), n =3, unpaired t -test *p < 0.05. (h) i 6 A sites in TRIT1 OE compared to control cells, represented according to their segment location in the transcript. (i) Sequence logo representing the deduced consensus of i 6 A sites in TRIT1 OE cells.

Article Snippet: TRIT1 mutations p.R323Q and p.E327K were introduced, separately, into a purchased human TRIT1 plasmid (Origene, RC210476) using the Q5 Site-Directed Mutagenesis Kit (NEB).

Techniques: Expressing, Biomarker Discovery, Control, Transfection, Plasmid Preparation, Liquid Chromatography with Mass Spectroscopy, Sequencing

Journal: bioRxiv

Article Title: i 6 A-seq maps N 6 -isopentenyladenosine and uncovers its role as a regulator of mRNA stability through recruitment of DIS3L2

doi: 10.1101/2025.11.26.690722

Figure Lengend Snippet: (a) Real-time qPCR showing TRIT1 OE effect on gene expression in highly isopentenylated genes in HEK293 cells, n =3, unpaired t -test, * p <0.05. (b) Real-time qPCR of the indicated genes in shTRIT1 cells compared to their control, before and after i 6 A-IP, n =3. (c-d) WB of the indicated cell lines, after TRIT1 OE/KD, displaying an inverse change in SLC38A10 (c) and AIG1 (d) in response to shifts in TRIT1 protein expression. (e) Immunofluorescence staining images of SLC38A10 and AIG1 in scramble and shTRIT1 DMS273 cells, revealing their increased expression under TRIT1 KD. (f) Analysis of TRIT1, SLC38A10 and AIG1 after TRIT1 WT or mutant rescue in shTRIT1 cells by real-time qPCR, n =3, unpaired t -test, * p <0.05. (g) WB of the indicated cell lines, representing similar trend for TRIT1 and SLC38A10 as in (f). (h) Real-time qPCR of SLC38A10 and AIG1 mRNA levels, in DMS273 scramble versus shTRIT1, monitored 6 hours after Actinomycin-D addition, compared to 0 hours, n =3.

Article Snippet: TRIT1 mutations p.R323Q and p.E327K were introduced, separately, into a purchased human TRIT1 plasmid (Origene, RC210476) using the Q5 Site-Directed Mutagenesis Kit (NEB).

Techniques: Gene Expression, Control, Expressing, Immunofluorescence, Staining, Mutagenesis

Journal: Cancers

Article Title: Gene Amplification-Associated Overexpression of the Selenoprotein tRNA Enzyme TRIT1 Confers Sensitivity to Arsenic Trioxide in Small-Cell Lung Cancer

doi: 10.3390/cancers13081869

Figure Lengend Snippet: Determination of TRIT1 gene amplification and RNA and protein overexpression in small-cell lung cancer cell lines. ( A ) Schematic representation of adenosine derivatives synthesis at position A37 of human tRNA. The enzymes catalyzing these reactions are shown in blue. Abbreviations are: A, adenosine; i 6 A, N 6 -isopentenyladenosine; ms 2 i 6 A, 2-methylthio-N 6 -isopentenyladenosine. ( B ) Frequency of TRIT1 gene amplification in the panel of cancer cell lines. ( C ) TRIT1 gene amplification was significantly associated with high levels of the TRIT1 transcript in the small-cell lung cancer cell lines for which expression patterns were available ( n = 36). Non-Amp, non-amplified; Amp, amplified. TPM, transcripts per million. p -value obtained by Wilcoxon rank sum test. *** p < 0.001. ( D ) Fluorescence in situ hybridization for the TRIT1 gene. The UCSC genome browser was used to select the bacterial artificial chromosome (BAC) clone spanning the 1p34.2 region for the TRIT1 gene: RP11-613D14. The BAC was obtained from the BACPAC Resource Center at the Children’s Hospital Oakland Research Institute (Oakland, CA, USA). TRIT1 probe was labeled with Red dUTP (Abbott, Wiesbaden, Germany), using a CGH Nick Translation Reagent Kit (Abbott Molecular Inc., Des Plaines, IL, USA). The samples were counterstained with DAPI and analyzed under a fluorescent microscope (NIKON, Eclipse E400). Gene amplification was found in the interphases of DMS-273 and HCC-33. The D-5099-100-OG probe (1p32.3, MetaSystems) was used as control. TRIT1/Control ratios are shown. ( E ) Multiplex ligation-dependent probe amplification (MLPA) assay. Probe mixes contained two probes for exons 4 and 9 of the TRIT1 gene (in orange). Six reference probes were also included (in grey). Values greater than 2 (two copies, corresponding to MLPA ratio of 1) were considered to indicate the presence of extra copies. DMS-273 and HCC-33 cell lines showed TRIT1 gene amplification, whilst NCI-H82 is shown as an example of TRIT1 two copy number cells. ( F ) TRIT1 expression levels in gene-unamplified (NCI-H82) and amplified (DMS-273 and HCC-33) cancer cell lines determined by Western blot analysis. Actin is shown as the loading control. The uncropped Western blots have been shown in .

Article Snippet: Lentiviral plasmids for TRIT1 human shRNA (TL300819-C, Origene, Rockville, MD, USA) and scrambled shRNA (TR30021, Origene, Rockville, MD, USA), both cloned in pGFP-C-shLenti vector, were used.

Techniques: Amplification, Over Expression, Expressing, Fluorescence, In Situ Hybridization, Labeling, Nick Translation, Microscopy, Control, Multiplex Assay, Ligation, Western Blot

Journal: Cancers

Article Title: Gene Amplification-Associated Overexpression of the Selenoprotein tRNA Enzyme TRIT1 Confers Sensitivity to Arsenic Trioxide in Small-Cell Lung Cancer

doi: 10.3390/cancers13081869

Figure Lengend Snippet: Effect of TRIT1 depletion on tumor growth and the RNA transcriptome of small-cell lung cancer. ( A ) Stable downregulation of the TRIT1 gene by short hairpin RNA in the small-cell lung cancer cell line DMS-273 (shTRIT1) determined by western blot analysis. SCR, scramble shRNA. ( B ) Effect of TRIT1 shRNA-mediated depletion on the growth of subcutaneous tumors in nude mice derived from DMS-273 cells (amplified and overexpressing TRIT1). There was a significant reduction in tumor volume in the TRIT1 shRNA-depleted cells. Data are summarized as the mean and standard deviation ( n = 9). Student’s t test, ** p < 0.01. ( C ) Nucleoside analysis of tRNAs by LC/MS showing that shRNA-mediated depletion of TRIT1 in DMS-273 cells induces the depletion of the i 6 A-modified nucleoside. Student’s t test, *** p = 0.0002. ( D ) Volcano plot summarizing the results of the RNA-seq experiment to find mRNAs differentially expressed in TRIT1 shRNA-depleted DMS-273 cells compared with scramble-shRNA DMS-273 cells. ( E ) Gene ontology (GO) analysis of Biological Process categories in the transcripts downregulated on TRIT1 depletion in DMS-273 cells shows the GO Biological Process category “regulation of cell differentiation” to be the most highly enriched.

Article Snippet: Lentiviral plasmids for TRIT1 human shRNA (TL300819-C, Origene, Rockville, MD, USA) and scrambled shRNA (TR30021, Origene, Rockville, MD, USA), both cloned in pGFP-C-shLenti vector, were used.

Techniques: shRNA, Western Blot, Derivative Assay, Amplification, Standard Deviation, Liquid Chromatography with Mass Spectroscopy, Modification, RNA Sequencing, Cell Differentiation

Journal: Cancers

Article Title: Gene Amplification-Associated Overexpression of the Selenoprotein tRNA Enzyme TRIT1 Confers Sensitivity to Arsenic Trioxide in Small-Cell Lung Cancer

doi: 10.3390/cancers13081869

Figure Lengend Snippet: Clinicopathological features of the studied small cell lung cancer patients according to TRIT1 gene amplification status.

Article Snippet: Lentiviral plasmids for TRIT1 human shRNA (TL300819-C, Origene, Rockville, MD, USA) and scrambled shRNA (TR30021, Origene, Rockville, MD, USA), both cloned in pGFP-C-shLenti vector, were used.

Techniques: Amplification

Journal: Cancers

Article Title: Gene Amplification-Associated Overexpression of the Selenoprotein tRNA Enzyme TRIT1 Confers Sensitivity to Arsenic Trioxide in Small-Cell Lung Cancer

doi: 10.3390/cancers13081869

Figure Lengend Snippet: TRIT1 gene amplification in primary small-cell lung cancer patients and response to arsenic trioxide. ( A ) MLPA assay of primary small-cell lung cancer samples. Probe mixes contained two probes for exons 4 and 9 of the TRIT1 gene (in orange). Six reference probes were also included (in grey). Values greater than 2 (two copies, corresponding to MLPA ratio of 1) were considered to indicate the presence of extra copies. Patient 1241 is shown as example of a TRIT1 two copy number case, whilst patient 391 shows TRIT1 gene amplification. ( B ) TRIT1 RNA expression levels derived from Affymetrix U133Plus2.0 microarray data in six primary small-cell lung cancer samples where TRIT1 copy number was determined. The only patient that exhibited TRIT1 gene amplification (#012) showed the highest TRIT1 expression level. Two copies of TRIT1 were observed in the 006, 010, 011, 014 and 018 samples. ( C ) IC50 determination by MTT assay. TRIT1 shRNA-depleted DMS-273 cells were significantly less sensitive to the antiproliferative effect of arsenic trioxide than were the shRNA scramble-transfected cells harboring TRIT1 gene amplification-associated overexpression. TRIT1 shRNA-mediated depletion did not affect sensitivity to cisplatin. Student’s t test, ** p < 0.01; ns, non-significant. ( D ) shRNA scramble (SCR, left panel) and TRIT1 shRNA-depleted (shTRIT1, right panel) DMS-273 cells were injected into the flanks of nude mice to form subcutaneous tumors. Tumor volume over time according to treatment conditions, vehicle (black lines) vs. arsenic trioxide-treated group (red lines) are shown. Black arrow indicates the time at which the mice were randomized and started to be treated with arsenic trioxide or vehicle. P values are those corresponding to Student’s t tests. Means and standard deviations (bars) are illustrated. Tumors derived from shRNA scramble-transfected DMS-273 cells were sensitive to the growth inhibition effect of arsenic trioxide (left panel), whilst TRIT1 shRNA-mediated depletion eliminates the enhanced sensitivity to arsenic trioxide, the tumor size reduction effect being similar to that obtained with the vehicle treatment (right panel). * p < 0.05; ns, non-significant.

Article Snippet: Lentiviral plasmids for TRIT1 human shRNA (TL300819-C, Origene, Rockville, MD, USA) and scrambled shRNA (TR30021, Origene, Rockville, MD, USA), both cloned in pGFP-C-shLenti vector, were used.

Techniques: Amplification, RNA Expression, Derivative Assay, Microarray, Expressing, MTT Assay, shRNA, Transfection, Over Expression, Injection, Inhibition