Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Quantifying the mRNA epitranscriptome reveals epitranscriptome signatures and roles in cancer

doi: 10.1007/s00018-025-05805-7

Figure Lengend Snippet: siRNA decreases the mRNA and protein levels of m 1 A regulatory enzymes in HeLa cells. (A) Relative expression changes of mRNA for TRMT10C , ALKBH3 , TRMT6 , TRMT61A , and TRMT6-61 A were measured by RT-qPCR. The x-axis represents the treatment groups, while the y-axis indicates the mRNA expression changes of m 1 A regulatory enzymes relative to the reference gene (GAPDH). (B) Protein level changes of TRMT10C, ALKBH3, TRMT6, TRMT61A, and TRMT6-61 A were assessed by Western blotting. Protein bands were quantified using ImageJ software. The x-axis represents the treatment groups, and the y-axis shows the changes in m1A regulatory enzymes relative to the reference gene (GAPDH). Each group was conducted with three biological replicates. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: The protein samples were incubated with the corresponding primary antibodies: TRMT6 Polyclonal Antibody (Proteintech), TRMT10C Polyclonal Antibody (Proteintech), ALKBH3 Polyclonal Antibody (Proteintech), and TRMT61A Polyclonal Antibody (ORIGENE), followed by incubation with the respective secondary antibodies: Multi-rAb HRP-Goat Anti-Mouse Recombinant Secondary Antibody (Proteintech) and Multi-rAb HRP-Goat Anti-Rabbit Recombinant Secondary Antibody (Proteintech).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Software

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Quantifying the mRNA epitranscriptome reveals epitranscriptome signatures and roles in cancer

doi: 10.1007/s00018-025-05805-7

Figure Lengend Snippet: Effects of m 1 A regulatory enzyme knockdown on cell viability. (A) CCK8 assay evaluating changes in cell proliferation following knockdown of m 1 A regulatory enzymes TRMT10C , ALKBH3 , TRMT6 , TRMT61A , and TRMT6-61 A at 6 h, 24 h, 48 h, and 72 h. The x-axis represents treatment duration, and the y-axis indicates cell viability. Each group consists of three biological replicates. * p < 0.05, ** p < 0.01, *** p < 0.001. (B) Flow cytometry measurement of apoptosis rates after 72 h of knockdown of m 1 A regulatory enzymes TRMT10C , ALKBH3 , TRMT6 , TRMT61A , and TRMT6-61 A . Cell apoptosis percentages were quantified using GraphPad Prism software; the x-axis represents m 1 A regulatory enzymes, and the y-axis represents the percentage of apoptosis. Each group consists of three biological replicates. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: The protein samples were incubated with the corresponding primary antibodies: TRMT6 Polyclonal Antibody (Proteintech), TRMT10C Polyclonal Antibody (Proteintech), ALKBH3 Polyclonal Antibody (Proteintech), and TRMT61A Polyclonal Antibody (ORIGENE), followed by incubation with the respective secondary antibodies: Multi-rAb HRP-Goat Anti-Mouse Recombinant Secondary Antibody (Proteintech) and Multi-rAb HRP-Goat Anti-Rabbit Recombinant Secondary Antibody (Proteintech).

Techniques: Knockdown, CCK-8 Assay, Flow Cytometry, Software

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Quantifying the mRNA epitranscriptome reveals epitranscriptome signatures and roles in cancer

doi: 10.1007/s00018-025-05805-7

Figure Lengend Snippet: Transcriptomic and proteomic analysis of TRMT6-61 A treated HeLa cells: Transcriptome: (A) Top 20 KEGG enrichment bubble chart. Proteome: (B) Top 25 connectivity protein interaction network diagram. Combined transcriptomic and proteomic analysis: (C) Bar chart of the top 30 KEGG (GSEA) pathways shared across different omics

Article Snippet: The protein samples were incubated with the corresponding primary antibodies: TRMT6 Polyclonal Antibody (Proteintech), TRMT10C Polyclonal Antibody (Proteintech), ALKBH3 Polyclonal Antibody (Proteintech), and TRMT61A Polyclonal Antibody (ORIGENE), followed by incubation with the respective secondary antibodies: Multi-rAb HRP-Goat Anti-Mouse Recombinant Secondary Antibody (Proteintech) and Multi-rAb HRP-Goat Anti-Rabbit Recombinant Secondary Antibody (Proteintech).

Techniques:

Journal: Cell

Article Title: ALKBH1-Mediated tRNA Demethylation Regulates Translation

doi: 10.1016/j.cell.2016.09.038

Figure Lengend Snippet: (A) Western blot showing increased concentrations of glucose in the growth medium led to decreased ALKBH1 expression in HeLa cells; protein levels of the m1A58-transferase Trmt6/Trmt61 heterodimer remained mostly unchanged.

Article Snippet: Polyclonal rabbit anti-TRMT6 antibody (Sigma, SAB2107213) and polyclonal rabbit anti-TRMT61 antibody (Sigma SAB2700607) were both used with 1: 400 dilution.

Techniques: Western Blot, Expressing

Journal: Frontiers in Oncology

Article Title: Depletion of the m1A writer TRMT6/TRMT61A reduces proliferation and resistance against cellular stress in bladder cancer

doi: 10.3389/fonc.2023.1334112

Figure Lengend Snippet: Characterization of expression levels of m1A regulators in BLCA and control cell lines. (A) qRT-PCR was done to check the relative mRNA expression levels of TRMT6, TRMT61A, ALKBH1, and ALKBH3. GAPDH was used as endogenous control, and mRNA expression was normalized to SV-HUC1. qRT-PCR data represented as mean and SD, based on three independent experiments. Welch’s t-test, two tailed, was used to determine statistically significant differences compared to SV-HUC. TRMT6: T24 (** p = 0.003) TRMT61A: T24 (** p = 0.004), HT1376 (** p = 0.01), 5637 (p = * 0.038), SW780 (* p = 0.042). ALKBH1: T24 (**** p = <0.0001), 5637 (* p = 0.002) HT1197 (* p = 0.02, ALKBH3: HT1197 (** p= 0.002) (B) Protein expression of TRMT6, TRMT61A, ALKBH1, and ALKBH3 by western blot in BLCA cell lines versus SV-HUC1 control cell line, with GAPDH as loading control. Relative protein expression was normalized to GAPDH.

Article Snippet: Following blocking of the membranes for 1 h in 5% milk in PBS with 0.05% Tween-20 (PBST), the primary antibodies and corresponding dilutions were used in 5% milk in 0.05% PBST: anti-GAPDH (Abcam, #ab125247, mouse monoclonal, used at 1:3000), anti-TRMT6 (Abcam #ab235321, rabbit polyclonal, used at 1:1000), anti-TRMT61A (Biorbyt #orb411814, rabbit polyclonal, used at 1:500), anti-ALKBH1 (Abcam #ab128895, rabbit monoclonal, used at 1:3000), anti-ALKBH3 (Cell Signaling Technology #87620, rabbit monoclonal, used at 1:1000).

Techniques: Expressing, Quantitative RT-PCR, Two Tailed Test, Western Blot

Journal: Frontiers in Oncology

Article Title: Depletion of the m1A writer TRMT6/TRMT61A reduces proliferation and resistance against cellular stress in bladder cancer

doi: 10.3389/fonc.2023.1334112

Figure Lengend Snippet: Effect on TRMT6 and TRMT61A in BLCA cell proliferation in vitro . (A) qRT-PCR and western blot were used to determine the knockdown efficiency of TRMT6 and TRMT61A in 5637 and HT1197 cell lines. Bar graphs show relative mRNA levels represented as mean ± SD, based on three independent knockdown experiments. WB shows a representative experiment for each cell line. (B) Cell proliferation. Knockdown of TRMT6/TRMT61A reduced cell proliferation in 5637 and HT1197. ** p-value = 0.0143, Unpaired T-test, two-tailed.

Article Snippet: Following blocking of the membranes for 1 h in 5% milk in PBS with 0.05% Tween-20 (PBST), the primary antibodies and corresponding dilutions were used in 5% milk in 0.05% PBST: anti-GAPDH (Abcam, #ab125247, mouse monoclonal, used at 1:3000), anti-TRMT6 (Abcam #ab235321, rabbit polyclonal, used at 1:1000), anti-TRMT61A (Biorbyt #orb411814, rabbit polyclonal, used at 1:500), anti-ALKBH1 (Abcam #ab128895, rabbit monoclonal, used at 1:3000), anti-ALKBH3 (Cell Signaling Technology #87620, rabbit monoclonal, used at 1:1000).

Techniques: In Vitro, Quantitative RT-PCR, Western Blot, Two Tailed Test

Journal: Frontiers in Oncology

Article Title: Depletion of the m1A writer TRMT6/TRMT61A reduces proliferation and resistance against cellular stress in bladder cancer

doi: 10.3389/fonc.2023.1334112

Figure Lengend Snippet: Cell migration and displacement of 5637 are inhibited by the knockdown of TRMT6/TRMT61A. (A) Wound-healing assay. Bars represent a relative quantitation of migration distance (%) at 7 hours after the scratch was made. (B) Cell displacement analysis. Average displacement speed was calculated based on particle tracking data. The boxplot represents the average cell displacement speed over a time period of 4 hours, n = 9 separate microscopic fields of view. *** p-value = 8.56·10 -9 .

Article Snippet: Following blocking of the membranes for 1 h in 5% milk in PBS with 0.05% Tween-20 (PBST), the primary antibodies and corresponding dilutions were used in 5% milk in 0.05% PBST: anti-GAPDH (Abcam, #ab125247, mouse monoclonal, used at 1:3000), anti-TRMT6 (Abcam #ab235321, rabbit polyclonal, used at 1:1000), anti-TRMT61A (Biorbyt #orb411814, rabbit polyclonal, used at 1:500), anti-ALKBH1 (Abcam #ab128895, rabbit monoclonal, used at 1:3000), anti-ALKBH3 (Cell Signaling Technology #87620, rabbit monoclonal, used at 1:1000).

Techniques: Migration, Wound Healing Assay, Quantitation Assay

Journal: Frontiers in Oncology

Article Title: Depletion of the m1A writer TRMT6/TRMT61A reduces proliferation and resistance against cellular stress in bladder cancer

doi: 10.3389/fonc.2023.1334112

Figure Lengend Snippet: The Unfolded Protein Response and cell survival. (A) 5637 and HT1197 cells were untreated (T=0) or treated with tunicamycin (5 ug/mL) for 8h (T=8) before qRT-PCR was used to investigate mRNA expression levels of S1P, ATF6, and CREB3L2. β-actin was used as endogenous control, and mRNA expression was normalized to siCtrl T=0. Bar graphs show relative mRNA levels represented as mean ± SD, based on three independent knockdown experiments. * p value = 0.04, Unpaired T-test, two-tailed. (B) Cell survival after treatment with increasing concentration of tunicamycin. Measurements were done 72 h after drug treatment. Nonlinear regression was performed to determine the IC50 for the cell lines depleted of TRMT6/61A compared to the control. Regression line is marked as a solid line on the graph. IC50 *** p-value=0.0003.Unpaired T-test, two-tailed.

Article Snippet: Following blocking of the membranes for 1 h in 5% milk in PBS with 0.05% Tween-20 (PBST), the primary antibodies and corresponding dilutions were used in 5% milk in 0.05% PBST: anti-GAPDH (Abcam, #ab125247, mouse monoclonal, used at 1:3000), anti-TRMT6 (Abcam #ab235321, rabbit polyclonal, used at 1:1000), anti-TRMT61A (Biorbyt #orb411814, rabbit polyclonal, used at 1:500), anti-ALKBH1 (Abcam #ab128895, rabbit monoclonal, used at 1:3000), anti-ALKBH3 (Cell Signaling Technology #87620, rabbit monoclonal, used at 1:1000).

Techniques: Quantitative RT-PCR, Expressing, Two Tailed Test, Concentration Assay

Journal: Frontiers in Oncology

Article Title: Depletion of the m1A writer TRMT6/TRMT61A reduces proliferation and resistance against cellular stress in bladder cancer

doi: 10.3389/fonc.2023.1334112

Figure Lengend Snippet: m1A mismatch rate is decreased by TRMT6/61A knock-down but not altered by tunicamycin treatment. (A) Heatmap representation of mismatch index of selected m1A sites from tRFs. Mismatch index is scaled across samples (rows). As expected, m1A mismatch rate is relatively decreased upon siTRMT6/61A. (B-D) Box plots showing m1A mismatch rate on tRFs from selected amino acid groups. Mismatch indexes from small RNA-seq are shown as box plots. One-way ANOVA was used to determine any global difference in mismatch indexes by the conditions (p value shown). Post-hoc Tukey test (confidence level 0.95) was used to examine pair-wise comparisons. Each pair with significant difference (p < 0.01) are labeled with “a” and “b”.

Article Snippet: Following blocking of the membranes for 1 h in 5% milk in PBS with 0.05% Tween-20 (PBST), the primary antibodies and corresponding dilutions were used in 5% milk in 0.05% PBST: anti-GAPDH (Abcam, #ab125247, mouse monoclonal, used at 1:3000), anti-TRMT6 (Abcam #ab235321, rabbit polyclonal, used at 1:1000), anti-TRMT61A (Biorbyt #orb411814, rabbit polyclonal, used at 1:500), anti-ALKBH1 (Abcam #ab128895, rabbit monoclonal, used at 1:3000), anti-ALKBH3 (Cell Signaling Technology #87620, rabbit monoclonal, used at 1:1000).

Techniques: RNA Sequencing Assay, Labeling

Journal: bioRxiv

Article Title: Active role of free RNA during the early phase of proteostasis stress

doi: 10.1101/564799

Figure Lengend Snippet: (A) Enrichment values of the subunits of eIF2 in individual experiments (black symbols) and the enrichment averages (red symbols). (B) Left panels, amounts of total eIF2α and its serine 51-phosporylated form (p-eIF2α) as determined by western blotting. Right panels, enrichment of non-phosphorylated eIF2α on RNA upon heat shock. *p<0.05, two-tailed t-test; N=3 independent experiments (mean + SD). (C) Enrichment values of the subunits of the m A methyltransferase TRMT6/61A in individual experiments (black symbols) and the enrichment averages (red symbols). (D) Upper panel, amounts of TRMT6 as determined by western blotting. Lower panel, enrichment of TRMT6 on RNA upon heat shock. ***p<0.001, two-tailed t-test; N=3 independent experiments (mean + SD). (E) Increased association of TRMT6 with mRNAs during heat shock as determined by western blot analysis of PolyT pulldowns. ***p<0.001, two-tailed t-test, N=3 independent experiments (mean + SD).

Article Snippet: The following antibodies were used: anti-TRMT6 (A303-008A-M) from Bethyl (Montgomery, TX); anti-TRMT61A (sc-107105) and anti-S6 (sc-74459) from Santa Cruz Biotechnology (Dallas, TX); anti-eIF2α (9722), anti-eIF2α (phosphor-Ser51) (3398), anti-eIF4E (9742), HRP-conjugated anti-rabbit-IgG (7074) and Alexa Fluor 647-conjugated anti-rabbit IgG (4414) from Cell Signaling (Danvers, MA); anti-Flag (F1804) and HRP-conjugated anti-mouse IgG (A9044) from Sigma-Aldrich.

Techniques: Western Blot, Two Tailed Test

Journal: bioRxiv

Article Title: Active role of free RNA during the early phase of proteostasis stress

doi: 10.1101/564799

Figure Lengend Snippet: (A) Increased association of eIF2α with TRMT61A-containing complexes during heat shock as determined by immunoprecipitation (IP) of Flag-tagged TRMT6/61A. *p<0.05, two-tailed t-test; N=3 independent experiments (mean + SD). (B) RNA hydrolysis (+nuclease) abolishes association of eIF2α with the TRMT61A-containing complexes. One representative out of three independent experiments is shown.

Article Snippet: The following antibodies were used: anti-TRMT6 (A303-008A-M) from Bethyl (Montgomery, TX); anti-TRMT61A (sc-107105) and anti-S6 (sc-74459) from Santa Cruz Biotechnology (Dallas, TX); anti-eIF2α (9722), anti-eIF2α (phosphor-Ser51) (3398), anti-eIF4E (9742), HRP-conjugated anti-rabbit-IgG (7074) and Alexa Fluor 647-conjugated anti-rabbit IgG (4414) from Cell Signaling (Danvers, MA); anti-Flag (F1804) and HRP-conjugated anti-mouse IgG (A9044) from Sigma-Aldrich.

Techniques: Immunoprecipitation, Two Tailed Test

Journal: bioRxiv

Article Title: Active role of free RNA during the early phase of proteostasis stress

doi: 10.1101/564799

Figure Lengend Snippet: (A) Left panel, Melting analysis of TRMT6/61A without and with tRNA. First derivatives of the fluorescence change are plotted and indicate two melting points (T m’ and T m ) upon increase of the temperature. Right panel, T m’ is significantly lower than T m . ***p<0.001, two-tailed t-test; N=3 independent experiments (mean + SD). (B) Proteinase K sensitivity indicates partial unfolding of the RNA-binding TRMT6 subunit at 45°C. Right panel, the amount of protein at timepoint 0 was set as 1. **p<0.01, two-tailed t-test; N=3 independent experiments (mean + SD).

Article Snippet: The following antibodies were used: anti-TRMT6 (A303-008A-M) from Bethyl (Montgomery, TX); anti-TRMT61A (sc-107105) and anti-S6 (sc-74459) from Santa Cruz Biotechnology (Dallas, TX); anti-eIF2α (9722), anti-eIF2α (phosphor-Ser51) (3398), anti-eIF4E (9742), HRP-conjugated anti-rabbit-IgG (7074) and Alexa Fluor 647-conjugated anti-rabbit IgG (4414) from Cell Signaling (Danvers, MA); anti-Flag (F1804) and HRP-conjugated anti-mouse IgG (A9044) from Sigma-Aldrich.

Techniques: Fluorescence, Two Tailed Test, RNA Binding Assay

Journal: bioRxiv

Article Title: Active role of free RNA during the early phase of proteostasis stress

doi: 10.1101/564799

Figure Lengend Snippet: (A and B) Comparison of TRMT6/61A binding to 1 μM endogenous tRNA (A) or 1 μM in vitro transcribed tRNA (B) at physiological and febrile temperature. #, not significant difference; two-tailed t-test; N=3 independent experiments (mean + SD). (C) Direct association of RNA and TRMT6/61A in vitro . The efficiency of TRMT6/61A pulldowns by RNA-coated beads (+/-1 μM tRNA) was estimated by western blotting. In vitro transcribed tRNA (Unmod. tRNA) or endogenous tRNA (Mod. tRNA) were used. Control, beads without RNA. #, not significant difference; two-tailed t-test; N=3 (mean+SD).

Article Snippet: The following antibodies were used: anti-TRMT6 (A303-008A-M) from Bethyl (Montgomery, TX); anti-TRMT61A (sc-107105) and anti-S6 (sc-74459) from Santa Cruz Biotechnology (Dallas, TX); anti-eIF2α (9722), anti-eIF2α (phosphor-Ser51) (3398), anti-eIF4E (9742), HRP-conjugated anti-rabbit-IgG (7074) and Alexa Fluor 647-conjugated anti-rabbit IgG (4414) from Cell Signaling (Danvers, MA); anti-Flag (F1804) and HRP-conjugated anti-mouse IgG (A9044) from Sigma-Aldrich.

Techniques: Binding Assay, In Vitro, Two Tailed Test, Western Blot

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Quantifying the mRNA epitranscriptome reveals epitranscriptome signatures and roles in cancer

doi: 10.1007/s00018-025-05805-7

Figure Lengend Snippet: siRNA decreases the mRNA and protein levels of m 1 A regulatory enzymes in HeLa cells. (A) Relative expression changes of mRNA for TRMT10C , ALKBH3 , TRMT6 , TRMT61A , and TRMT6-61 A were measured by RT-qPCR. The x-axis represents the treatment groups, while the y-axis indicates the mRNA expression changes of m 1 A regulatory enzymes relative to the reference gene (GAPDH). (B) Protein level changes of TRMT10C, ALKBH3, TRMT6, TRMT61A, and TRMT6-61 A were assessed by Western blotting. Protein bands were quantified using ImageJ software. The x-axis represents the treatment groups, and the y-axis shows the changes in m1A regulatory enzymes relative to the reference gene (GAPDH). Each group was conducted with three biological replicates. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: The protein samples were incubated with the corresponding primary antibodies: TRMT6 Polyclonal Antibody (Proteintech), TRMT10C Polyclonal Antibody (Proteintech), ALKBH3 Polyclonal Antibody (Proteintech), and TRMT61A Polyclonal Antibody (ORIGENE), followed by incubation with the respective secondary antibodies: Multi-rAb HRP-Goat Anti-Mouse Recombinant Secondary Antibody (Proteintech) and Multi-rAb HRP-Goat Anti-Rabbit Recombinant Secondary Antibody (Proteintech).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Software

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Quantifying the mRNA epitranscriptome reveals epitranscriptome signatures and roles in cancer

doi: 10.1007/s00018-025-05805-7

Figure Lengend Snippet: Effects of m 1 A regulatory enzyme knockdown on cell viability. (A) CCK8 assay evaluating changes in cell proliferation following knockdown of m 1 A regulatory enzymes TRMT10C , ALKBH3 , TRMT6 , TRMT61A , and TRMT6-61 A at 6 h, 24 h, 48 h, and 72 h. The x-axis represents treatment duration, and the y-axis indicates cell viability. Each group consists of three biological replicates. * p < 0.05, ** p < 0.01, *** p < 0.001. (B) Flow cytometry measurement of apoptosis rates after 72 h of knockdown of m 1 A regulatory enzymes TRMT10C , ALKBH3 , TRMT6 , TRMT61A , and TRMT6-61 A . Cell apoptosis percentages were quantified using GraphPad Prism software; the x-axis represents m 1 A regulatory enzymes, and the y-axis represents the percentage of apoptosis. Each group consists of three biological replicates. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: The protein samples were incubated with the corresponding primary antibodies: TRMT6 Polyclonal Antibody (Proteintech), TRMT10C Polyclonal Antibody (Proteintech), ALKBH3 Polyclonal Antibody (Proteintech), and TRMT61A Polyclonal Antibody (ORIGENE), followed by incubation with the respective secondary antibodies: Multi-rAb HRP-Goat Anti-Mouse Recombinant Secondary Antibody (Proteintech) and Multi-rAb HRP-Goat Anti-Rabbit Recombinant Secondary Antibody (Proteintech).

Techniques: Knockdown, CCK-8 Assay, Flow Cytometry, Software

Journal: BMC Gastroenterology

Article Title: Hypoxia-induced exosomal circNRIP1 activates cancer-associated fibroblasts to promote esophageal squamous cell carcinoma migration and invasion

doi: 10.1186/s12876-025-03978-w

Figure Lengend Snippet: Hypoxia enhances exosome release. A Observation of exosomes using TEM. B Western blot detected the protein expression of exosome marker ALIX, TSG101, and CD81 in KYSE-150 cells and the exosomes using equal total protein. C Western blot detected the protein expression of exosome marker ALIX, TSG101, and CD81 in TE-10 cells and the exosomes using equal total protein. D BCA assay detected the total protein of normoxic exosomes (Nor-exo) and hypoxic exosomes (Hypo-exo) from equal KYSE-150 cells. E BCA assay detected the total protein of normoxic exosomes and hypoxic exosomes from equal TE-10 cells. Data represent mean ± S.D. of three independent experiments. ** P < 0.01

Article Snippet: Membranes were then treated with primary antibodies against ALIX (Proteintech, 12,422–1-AP, USA), TSG101 (Abcam, ab125011, USA), CD81 (Proteintech, 27,855–1-AP, USA), α-SMA (Proteintech, 14,395–1-AP, USA), COL1A1 (Proteintech,14,695–1-AP, USA), COL3A1 (Proteintech, 21,898–1-AP, USA), TRMT6 (Proteintech,16,727–1-AP,1:6000), GAPDH (Proteintech, 60,004–1-Ig, USA) at 4°C overnight.

Techniques: Western Blot, Expressing, Marker, BIA-KA