Journal: iScience

Article Title: m6A-dependent regulation of DGUOK-AS1 by RBM15 and HNRNPH1 in lung adenocarcinoma

doi: 10.1016/j.isci.2026.117078

Figure Lengend Snippet: DGUOK-AS1 201 and miR-2467-5p regulated PRMT5 expression (A) Predicted binding sites of miR-2467-5p on wild-type and mutant DGUOK-AS1 201 sequences. (B) Dual-luciferase reporter assay in A549 cells co-transfected with wild-type (WT) or mutant (MUT) DGUOK-AS1 201 and miR-2467-5p mimics ( n = 3, ∗∗ p < 0.01; ns, not significant). (C) RIP-qPCR analysis of DGUOK-AS1 enrichment on Ago2 relative to IgG in A549 cells ( n = 3, ∗∗∗ p < 0.001). (D) miR-2467-5p expression in BEAS-2B, A549, and H1975 cells was determined by RT-qPCR ( n = 3, ∗∗∗ p < 0.001). (E and F) miR-2467-5p levels in A549 and H1975 cells following DGUOK-AS1 knockdown or DGUOK-AS1 201 overexpression ( n = 3, ∗ p < 0.05, ∗∗ p < 0.01). (G–J) Colony formation and Transwell assays assessing proliferation and migration of A549 cells co-transfected with DGUOK-AS1 201 overexpression vector and miR-2467-5p mimics (G and H: n = 3; I and J: scale bars, 100 μm; n = 5; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001). (K) Schematic of the predicted binding sites between miR-2467-5p and the wild-type or mutant 3′ UTR of PRMT5 mRNA. (L) Dual-luciferase reporter assay validating the interaction between miR-2467-5p and the PRMT5 3′ UTR in A549 cells ( n = 3, ∗∗∗ p < 0.001; ns, not significant). (M) RIP-qPCR analysis of PRMT5 mRNA enrichment using anti-Ago2 antibody or IgG control ( n = 3, ∗∗∗ p < 0.001). (N) PRMT5 protein expression in A549 and H1975 cells transfected with NC mimic or miR-2467-5p mimics. (O) PRMT5 protein levels in A549 and H1975 cells overexpressing DGUOK-AS1 201. (P and Q) CCK-8 and Transwell assays of proliferation and migration in A549 cells co-transfected with PRMT5 overexpression vector and DGUOK-AS1 siRNA (P: n = 3; Q: scale bars, 100 μm; n = 5; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001). The data are shown as the mean ± SEM based on three independent trials. For (A–Q), n represents the number of independent biological replicates, unless otherwise specified. Statistical analysis was performed using a paired t test (C, E, F, and M), a one-way ANOVA (D, H, J, and Q), or two-way ANOVA (B and L), followed by Tukey’s multiple comparison test.

Article Snippet: Membranes were blocked using a solution of 5% nonfat dry milk and incubated with primary antibodies PRMT5 (1:800, A19533, ABclonal), RBM15 (1:1000, 60386, CST), HNRNPH1 (1:1000, 14774-1-AP, Proteintech), E-cadherin (1:1000, 20874-1-AP, Proteintech), Vimentin (1:1000, 10366-1-AP, Proteintech) and GAPDH (1:6000, AP0063, Bioworld), followed by reaction with HRP-conjugated secondary antibodies (1:4000, Proteintech).

Techniques: Expressing, Binding Assay, Mutagenesis, Luciferase, Reporter Assay, Transfection, Quantitative RT-PCR, Knockdown, Over Expression, Migration, Plasmid Preparation, Control, CCK-8 Assay, Comparison

Journal: iScience

Article Title: m6A-dependent regulation of DGUOK-AS1 by RBM15 and HNRNPH1 in lung adenocarcinoma

doi: 10.1016/j.isci.2026.117078

Figure Lengend Snippet: RBM15 upregulates the expression of DGUOK-AS1 201 and promotes the proliferation and migration of LUAD cells (A–C) Tumor volume and weight in nude mice injected with A549 cells stably transfected with sh-RBM15 or sh-control ( n = 5 mice per group; ∗∗∗ p < 0.001). (D) Immunohistochemistry (IHC) staining of Ki67 in subcutaneous xenograft tumors (scale bars, 100 μm). (E and F) RT-qPCR analysis of DGUOK-AS1 expression in A549 and H1975 cells upon RBM15 knockdown or overexpression ( n = 3, ∗ p < 0.05, ∗∗∗ p < 0.001; ns, not significant). (G) Relative DGUOK-AS1 RNA levels measured by RT-qPCR following actinomycin D treatment ( n = 3, ∗ p < 0.05, ∗∗∗ p < 0.001). (H) Colony formation assay of proliferation in A549 cells co-transfected with RBM15 knockdown lentivirus and DGUOK-AS1 201 overexpression vector ( n = 3, ∗∗∗ p < 0.001). (I) Transwell migration assay in RBM15-silenced A549 cells overexpressing DGUOK-AS1 201 (scale bars, 100 μm; n = 5, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001). (J) RT-qPCR analysis of miR-2467-5p expression in A549 and H1975 cells upon RBM15 overexpression or knockdown ( n = 3, ∗ p < 0.05, ∗∗∗ p < 0.001). (K) Protein levels of RBM15 and PRMT5 in A549 cells with RBM15 overexpression or knockdown. Data are presented as mean ± SEM from three independent experiments. For (A–K), n represents the number of independent biological replicates, unless otherwise specified (mice: n = number of animals). Statistical analysis was performed using a paired t test (C and J), a one-way ANOVA (H and I), or a two-way ANOVA (E, F, and G) followed by Tukey’s multiple comparison test.

Article Snippet: Membranes were blocked using a solution of 5% nonfat dry milk and incubated with primary antibodies PRMT5 (1:800, A19533, ABclonal), RBM15 (1:1000, 60386, CST), HNRNPH1 (1:1000, 14774-1-AP, Proteintech), E-cadherin (1:1000, 20874-1-AP, Proteintech), Vimentin (1:1000, 10366-1-AP, Proteintech) and GAPDH (1:6000, AP0063, Bioworld), followed by reaction with HRP-conjugated secondary antibodies (1:4000, Proteintech).

Techniques: Expressing, Migration, Injection, Stable Transfection, Transfection, Control, Immunohistochemistry, Quantitative RT-PCR, Knockdown, Over Expression, Colony Assay, Plasmid Preparation, Transwell Migration Assay, Comparison

Journal: iScience

Article Title: m6A-dependent regulation of DGUOK-AS1 by RBM15 and HNRNPH1 in lung adenocarcinoma

doi: 10.1016/j.isci.2026.117078

Figure Lengend Snippet: HNRNPH1 regulates the expression of DGUOK-AS1 (A and B) RT-qPCR assay measuring the expression of DGUOK-AS1 following knockdown or overexpression of HNRNPH1 ( n = 3, ∗∗ p < 0.01, ∗∗∗ p < 0.001). (C and D) The RNA stability of DGUOK-AS1 in A549 and H1975 cells was assessed after HNRNPH1 overexpression ( n = 3, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001). (E) The migration ability was measured using Transwell assay when DGUOK-AS1 201 was overexpressed in HNRNPH1-overexpression cells (scale bars, 100 μm; n = 4, ∗ p < 0.05). (F) CCK-8 assay was used to assess the proliferation of A549 cells co-transfected with HNRNPH1 and DGUOK-AS1 201 overexpression vectors ( n = 3, ∗∗∗ p < 0.001). (G) The expression of miR-2467-5p in A549 cells after overexpressing or knocking down HNRNPH1 by RT-qPCR analysis. (H) The protein levels of PRMT5 in A549 cells with overexpressed or knocked down HNRNPH1 ( n = 3, ∗ p < 0.05, ∗∗ p < 0.01). (I) Molecular mechanism diagram of DGUOK-AS1 promoting cancer progression in LUAD. Each data point represents the mean ± SEM based on three independent trials. For (A–I), n represents the number of independent biological replicates, unless otherwise specified. Statistical analysis was performed using a paired t test (G), one-way ANOVA (E), or two-way ANOVA (A–D) and (F) followed by Tukey’s multiple comparison test.

Article Snippet: Membranes were blocked using a solution of 5% nonfat dry milk and incubated with primary antibodies PRMT5 (1:800, A19533, ABclonal), RBM15 (1:1000, 60386, CST), HNRNPH1 (1:1000, 14774-1-AP, Proteintech), E-cadherin (1:1000, 20874-1-AP, Proteintech), Vimentin (1:1000, 10366-1-AP, Proteintech) and GAPDH (1:6000, AP0063, Bioworld), followed by reaction with HRP-conjugated secondary antibodies (1:4000, Proteintech).

Techniques: Expressing, Quantitative RT-PCR, Knockdown, Over Expression, Migration, Transwell Assay, CCK-8 Assay, Transfection, Comparison

Journal: iScience

Article Title: m6A-dependent regulation of DGUOK-AS1 by RBM15 and HNRNPH1 in lung adenocarcinoma

doi: 10.1016/j.isci.2026.117078

Figure Lengend Snippet:

Article Snippet: Membranes were blocked using a solution of 5% nonfat dry milk and incubated with primary antibodies PRMT5 (1:800, A19533, ABclonal), RBM15 (1:1000, 60386, CST), HNRNPH1 (1:1000, 14774-1-AP, Proteintech), E-cadherin (1:1000, 20874-1-AP, Proteintech), Vimentin (1:1000, 10366-1-AP, Proteintech) and GAPDH (1:6000, AP0063, Bioworld), followed by reaction with HRP-conjugated secondary antibodies (1:4000, Proteintech).

Techniques: Virus, Recombinant, Transfection, RNA Extraction, Transwell Assay, Magnetic Beads, Lysis, Cell Counting, RNA Immunoprecipitation, Labeling, Silver Staining, Methylation, Immunoprecipitation, Immunohistochemistry, Luciferase, Reporter Assay, Mass Spectrometry, Software, RNA sequencing

Journal: Research

Article Title: Dysregulation of the Cant1/β-Catenin/TCF4–CHSY1 Axis Underpins Impaired ECM Biosynthesis in Skeletal Disorders

doi: 10.34133/research.1227

Figure Lengend Snippet: Mechanism of ECM biosynthesis disorders and abnormal Wnt/β-Catenin signaling pathway transduction in TD models. (A) mRNA levels of ECM biosynthesis factors and canonical Wnt/β-Catenin signaling pathway components. (B) mRNA levels of Cant1. (C) Quantitative analysis of GAG content. (D) Protein blots. (E) Protein levels of ACAN, Col2α1, β-Catenin, and Cant1. (F) Immunofluorescence digital images of CON tibia and TD tibia. (G and H) Relative fluorescence intensity of Cant1 and β-Catenin in ROI of CON tibia and TD tibia.

Article Snippet: Protein bands were blocked in tris–borate–sodium Tween-20 (TBST) with 5% skim milk and then incubated with primary antibodies against Cant1 (no. A6341, ABclonal, China), β-Catenin (no. 66379-1-Ig, Proteintech, China), phospho-β-Catenin (no. AP1076, ABclonal, China), Col2α1 (no. A1520, ABclonal, China), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (no. 60004-1-1g, Proteintech, China), and ACAN (no. A12045, ABclonal, China).

Techniques: Transduction, Immunofluorescence, Fluorescence

Journal: Research

Article Title: Dysregulation of the Cant1/β-Catenin/TCF4–CHSY1 Axis Underpins Impaired ECM Biosynthesis in Skeletal Disorders

doi: 10.34133/research.1227

Figure Lengend Snippet: Regulation of Cant1 on ECM biosynthesis and canonical Wnt/β-Catenin signaling pathway activity in chondrocytes. (A) Illustration of Cant1 inhibition/overexpression and canonical Wnt/β-Catenin signaling pathway activation/inhibition in chondrocytes. (B) mRNA levels of Cant1. (C) mRNA levels of ECM biosynthesis factors. (D) mRNA levels of Wnt/β-Catenin signaling pathway components. (E) Protein blots. (F) Protein levels of ACAN, Col2α1, Cant1, and β-Catenin. (G) Quantitative analysis of GAG content. (H) Wnt/β-Catenin transcriptional activity (TOP/FOPFLASH ratio). (I and J) Relative β-Catenin fluorescence intensity in CON and TD chondrocytes.

Article Snippet: Protein bands were blocked in tris–borate–sodium Tween-20 (TBST) with 5% skim milk and then incubated with primary antibodies against Cant1 (no. A6341, ABclonal, China), β-Catenin (no. 66379-1-Ig, Proteintech, China), phospho-β-Catenin (no. AP1076, ABclonal, China), Col2α1 (no. A1520, ABclonal, China), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (no. 60004-1-1g, Proteintech, China), and ACAN (no. A12045, ABclonal, China).

Techniques: Activity Assay, Inhibition, Over Expression, Activation Assay, Fluorescence

Journal: Research

Article Title: Dysregulation of the Cant1/β-Catenin/TCF4–CHSY1 Axis Underpins Impaired ECM Biosynthesis in Skeletal Disorders

doi: 10.34133/research.1227

Figure Lengend Snippet: The inhibition of the canonical Wnt/β-Catenin signaling pathway down-regulates ECM biosynthesis promoted by Cant1 overexpression. (A) Illustration of the cell model for Cant1 overexpression combined with activation or inhibition of the canonical Wnt/β-Catenin signaling pathway. (B) Quantitative analysis of GAG content. (C) mRNA levels of ECM biosynthesis factors. (D) mRNA levels of Cant1. (E) Protein blots. (F) Protein levels of ACAN, Col2α1, Cant1, and β-Catenin. (G) Wnt/β-Catenin transcriptional activity (TOP/FOPFLASH ratio).

Article Snippet: Protein bands were blocked in tris–borate–sodium Tween-20 (TBST) with 5% skim milk and then incubated with primary antibodies against Cant1 (no. A6341, ABclonal, China), β-Catenin (no. 66379-1-Ig, Proteintech, China), phospho-β-Catenin (no. AP1076, ABclonal, China), Col2α1 (no. A1520, ABclonal, China), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (no. 60004-1-1g, Proteintech, China), and ACAN (no. A12045, ABclonal, China).

Techniques: Inhibition, Over Expression, Activation Assay, Activity Assay