Journal: Cell Death & Disease

Article Title: Circular RNA TFRC/ SCD1 mRNA interaction regulates ferroptosis and metastasis in gastric cancer

doi: 10.1038/s41419-025-07759-x

Figure Lengend Snippet: A Volcano plot of differentially expressed circRNAs in three pairs of GC tissues and adjacent normal-appearing tissues. B Volcano plot of circRNA expression in plasma samples from three patients with GC and three healthy controls. Screening criteria: |log 2 FC| ≥ 1, P < 0.05. Red points indicate significantly upregulated circRNAs; blue points indicate significantly downregulated circRNAs. C Venn diagram displaying intersecting differentially expressed circRNAs between the two microarray datasets. D qRT–PCR analysis of circTFRC expression in 42 pairs of GC tissues and adjacent normal-appearing tissues. E qRT–PCR analysis of circTFRC expression in plasma from 40 patients with GC and 20 healthy volunteers. F qRT–PCR analysis comparing circTFRC expression in plasma from 24 GC individuals with small tumors (<5 cm) and 16 GC individuals with large tumors (≥5 cm). G qRT–PCR analysis comparing circTFRC expression in plasma from 30 GC individuals without distant metastases and 10 GC individuals with distant metastases. H ROC curve evaluating the diagnostic accuracy of circTFRC in plasma samples for distinguishing patients with GC from healthy controls. I ROC curve evaluating the diagnostic accuracy of circTFRC in plasma samples for distinguishing GC individuals with distant metastases from those without. J qRT–PCR analysis of circTFRC expression in the gastric normal epithelial cell line (GES-1) and various GC cell lines. Data are presented as mean ± SD. P values were calculated using a two-tailed paired ( D ) or unpaired Student’s t test ( E – G ) or one-way ANOVA ( J ); * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. See also Fig. .

Article Snippet: Two siRNAs targeting circTFRC BSJ regions and a negative control siRNA (si-NC) were synthesized by GenePharma.

Techniques: Expressing, Clinical Proteomics, Microarray, Quantitative RT-PCR, Diagnostic Assay, Two Tailed Test

Journal: Cell Death & Disease

Article Title: Circular RNA TFRC/ SCD1 mRNA interaction regulates ferroptosis and metastasis in gastric cancer

doi: 10.1038/s41419-025-07759-x

Figure Lengend Snippet: A Schematic illustration of circTFRC’s chromosomal location, exon structure, and back-splice junction. B , C qRT–PCR results showing circTFRC amplification from cDNA and gDNA of GC cells using divergent (DP) and convergent primers (CP). cDNA complementary DNA, gDNA genomic DNA. D , E Agarose gel electrophoresis of PCR products verifying circTFRC circularization in GC cells. F Sanger sequencing confirming the back-splice junction of circTFRC. G , H qRT–PCR analysis of circTFRC and TFRC mRNA expression in GC cells treated with Actinomycin D (5 µg/mL) at various time points. I , J qRT–PCR analysis of circTFRC and TFRC mRNA expression in GC cells following RNase R treatment (5 U/μg, 30 min). K RNA fluorescence in situ hybridization (FISH) depicting the subcellular localization of circTFRC in GC cells. Nuclei are counterstained with DAPI. Scale bar, 10 µm. Data are presented as mean ± SD. P -values were calculated using a two-tailed unpaired Student’s t test ( B , C , I , J ) or two-way ANOVA ( G , H ); *** P < 0.001, **** P < 0.0001. See also Fig. .

Article Snippet: Two siRNAs targeting circTFRC BSJ regions and a negative control siRNA (si-NC) were synthesized by GenePharma.

Techniques: Quantitative RT-PCR, Amplification, Agarose Gel Electrophoresis, Sequencing, Expressing, Fluorescence, In Situ Hybridization, Two Tailed Test

Journal: Cell Death & Disease

Article Title: Circular RNA TFRC/ SCD1 mRNA interaction regulates ferroptosis and metastasis in gastric cancer

doi: 10.1038/s41419-025-07759-x

Figure Lengend Snippet: A, B qRT–PCR analysis confirming significant knockdown of circTFRC in AGS and HGC-27 cells following siRNA transfection. C , D) CCK-8 assay illustrating the proliferation of AGS and HGC-27 cells under control conditions (si-NC) or with circTFRC knockdown (si-circTFRC) at indicated time points (0, 24, 48, and 72 h). E , F , G Plate colony formation assay assessing colony formation in AGS and HGC-27 cells under control conditions (si-NC) or with circTFRC knockdown (si-circTFRC) over 7–10 days. H , I , J Transwell assay evaluating the migration ability of AGS and HGC-27 cells under control conditions (si-NC) or with circTFRC knockdown (si-circTFRC) over 24 h. Scale bar, 100 µm. Data are presented as mean ± SD. P- values were calculated using a two-tailed unpaired Student’s t test ( A , B , F , G , I , J ) or two-way ANOVA ( C , D ); ** P < 0.01, *** P < 0.001, **** P < 0.0001. See also Fig. .

Article Snippet: Two siRNAs targeting circTFRC BSJ regions and a negative control siRNA (si-NC) were synthesized by GenePharma.

Techniques: Quantitative RT-PCR, Knockdown, Transfection, CCK-8 Assay, Control, Colony Assay, Transwell Assay, Migration, Two Tailed Test

Journal: Cell Death & Disease

Article Title: Circular RNA TFRC/ SCD1 mRNA interaction regulates ferroptosis and metastasis in gastric cancer

doi: 10.1038/s41419-025-07759-x

Figure Lengend Snippet: A , B Propidium iodide (PI) staining showing the cell death rates of control (si-NC) and circTFRC knockdown (si-circTFRC) AGS and HGC-27 cells in the absence or presence of ferrostatin-1 (0.75 μM, 16 h). C , D Flow cytometry showing the lipid ROS levels (stained with C11 BODIPY 581/591) in control (si-NC) and circTFRC knockdown (si-circTFRC) AGS and HGC-27 cells in the absence or presence of ferrostatin-1 (0.75 μM, 16 h). ROS reactive oxygen species. E , F ELISA assays showing the relative MDA levels in control (si-NC) and circTFRC knockdown (si-circTFRC) AGS and HGC-27 cells in the absence or presence of ferrostatin-1 (0.75 μM, 16 h). MDA malondialdehyde. G , H Plate colony formation assay assessing colony formation in AGS cells under control conditions (sh-NC) or with circTFRC knockdown (sh-circTFRC) in the absence or presence of ferrostatin-1 (0.25 μM, 72 h). I , J Transwell assay evaluating the migration ability of AGS cells under control conditions (sh-NC) or with circTFRC knockdown (sh-circTFRC) in the absence or presence of ferrostatin-1 (0.75 μM, 16 h). K PI staining showing the cell death rates of control (sh-NC) and circTFRC knockdown (sh-circTFRC) AGS cells following treatment with RSL3 (1 μM) in the absence or presence of ferrostatin-1 (0.75 μM) for 16 h. L Flow cytometry showing the lipid ROS levels (stained with C11 BODIPY 581/591) in control (sh-NC) and circTFRC knockdown (sh-circTFRC) AGS cells following treatment with RSL3 (1 μM) in the absence or presence of ferrostatin-1 (0.75 μM) for 16 h. M Confocal microscopy showing the lipid ROS levels in control (sh-NC) and circTFRC knockdown (sh-circTFRC) AGS cells following treatment with RSL3 (1 μM) in the absence or presence of ferrostatin-1 (0.75 μM) for 16 h. Lipid ROS are stained with C11 BODIPY 581/591 probe (green). Nuclei are counterstained with DAPI (blue). Scale bar, 10 µm. Data are presented as mean ± SD. P -values were calculated using a two-tailed one-way ANOVA ( A – F ) or unpaired Student’s t test ( H , J – L ); ** P < 0.01, *** P < 0.001, **** P < 0.0001. See also Fig. .

Article Snippet: Two siRNAs targeting circTFRC BSJ regions and a negative control siRNA (si-NC) were synthesized by GenePharma.

Techniques: Staining, Control, Knockdown, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Colony Assay, Transwell Assay, Migration, Confocal Microscopy, Two Tailed Test

Journal: Cell Death & Disease

Article Title: Circular RNA TFRC/ SCD1 mRNA interaction regulates ferroptosis and metastasis in gastric cancer

doi: 10.1038/s41419-025-07759-x

Figure Lengend Snippet: A Venn diagram showing the intersection of potential circTFRC-binding proteins predicted by ENCORI and RBPDB databases. B Representative Western blotting showing ELAVL1 enrichment upon circTFRC pulldown in GC cell lysates, with Actin as a negative control. C qRT–PCR analysis demonstrating circTFRC enrichment in an anti-ELAVL1 RIP assay in GC cells. IgG serves as a control. D Venn diagram displaying the intersection between ferroptosis-suppressor genes in the FerrDb database and ELAVL1-binding mRNAs predicted by ENCORI. E qRT–PCR analysis showing the expression of mRNAs in GC cells under control conditions (sh-NC) or circTFRC knockdown (sh-circTFRC). F qRT–PCR analysis showing the expression of mRNAs in GC cells transfected with control or ELAVL1 overexpression vector. G Western blotting showing SCD1 protein levels in GC cells under control conditions (sh-NC) or circTFRC knockdown (sh-circTFRC), with Actin as a loading control. H Western blotting of ELAVL1 and SCD1 in GC cells transfected with control vector, or ELAVL1 overexpression vector. I qRT–PCR analysis showing SCD1 3’ UTR enrichment in an anti-ELAVL1 RIP assay in GC cells, with CKAP5 mRNA as a positive control and GAPDH mRNA as a negative control. J qRT–PCR analysis of SCD1 mRNA stability in control, ELAVL1 -overexpressing, circTFRC knockdown, and ELAVL1- overexpressing + circTFRC knockdown cells following Actinomycin D treatment (5 µg/mL) at various time points (0, 1, 2, 4, and 8 h). K qRT–PCR demonstrating circTFRC and SCD1 3’ UTR enrichment upon circTFRC pulldown in GC cell lysates. GAPDH serves as a negative control. L qRT–PCR showing SCD1 3’ UTR enrichment after anti-ELAVL1 RIP in GC cells under control conditions (sh-NC) or circTFRC knockdown (sh-circTFRC). M Representative Western blot of ELAVL1 and SCD1 in GC cells after the transfection of the control vector or ELAVL1 overexpression vector or cotransfection of ELAVL1 + sh-circTFRC vectors. The data are shown as the mean ± SD. The P values were determined by a two-tailed unpaired Student’s t test ( C , E , F , I , K , L ) or two-tailed two-way ANOVA ( J ); * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. See also Figs. and .

Article Snippet: Two siRNAs targeting circTFRC BSJ regions and a negative control siRNA (si-NC) were synthesized by GenePharma.

Techniques: Binding Assay, Western Blot, Negative Control, Quantitative RT-PCR, Control, Expressing, Knockdown, Transfection, Over Expression, Plasmid Preparation, Positive Control, Cotransfection, Two Tailed Test

Journal: Cell Death & Disease

Article Title: Circular RNA TFRC/ SCD1 mRNA interaction regulates ferroptosis and metastasis in gastric cancer

doi: 10.1038/s41419-025-07759-x

Figure Lengend Snippet: A CCK-8 assay showing the proliferation of AGS cells under control conditions (sh-NC), or upon circTFRC knockdown (sh - circTFRC) or sh - circTFRC + SCD1 vector cotransfection at indicated time points (0, 24, 48, and 72 h). B , C Plate colony formation assay assessing colony formation in AGS cells under control conditions (sh-NC), or upon circTFRC knockdown (sh - circTFRC) or sh - circTFRC + SCD1 vector cotransfection over 10 days. D, E Transwell assay showing the migration of AGS cells under control conditions (sh-NC), or upon circTFRC knockdown (sh - circTFRC) or sh - circTFRC + SCD1 vector cotransfection over 24 h. Scale bar, 100 µm. F PI staining showing the cell death rates of AGS cells under control conditions (sh-NC), or upon circTFRC knockdown (sh - circTFRC) or sh - circTFRC + SCD1 vector cotransfection. G Flow cytometry showing the lipid ROS levels (stained with C11 BODIPY 581/591) of AGS cells under control conditions (sh-NC), or upon circTFRC knockdown (sh - circTFRC) or sh - circTFRC + SCD1 vector cotransfection. ROS reactive oxygen species. H ELISA assays showing the relative MDA levels of AGS cells under control conditions (sh-NC), or upon circTFRC knockdown (sh - circTFRC) or sh - circTFRC + SCD1 vector cotransfection. MAD malondialdehyde. The data are shown as the mean ± SD. The P values were determined by a two-tailed unpaired Student’s t test ( C , E – H ) or two-way ANOVA ( A ); ** P < 0.01, *** P < 0.001, **** P < 0.0001. See also Fig. .

Article Snippet: Two siRNAs targeting circTFRC BSJ regions and a negative control siRNA (si-NC) were synthesized by GenePharma.

Techniques: CCK-8 Assay, Control, Knockdown, Plasmid Preparation, Cotransfection, Colony Assay, Transwell Assay, Migration, Staining, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Cell Death & Disease

Article Title: Circular RNA TFRC/ SCD1 mRNA interaction regulates ferroptosis and metastasis in gastric cancer

doi: 10.1038/s41419-025-07759-x

Figure Lengend Snippet: A Images of tumors in mice received intratumoral injections of control siRNA or in vivo-optimized circTFRC siRNA ( n = 7 per group). B Tumor growth curves in mice in each group. C Tumor weights in mice in each group. D qRT‒PCR showing the expression levels of circTFRC and SCD1 mRNA in control tumors (Ctrl siRNA) or circTFRC knockdown tumors (CircTFRC siRNA). E , F Immunohistochemistry (IHC) staining showing the expression levels of SCD1 protein in control tumors (Ctrl siRNA) or circTFRC knockdown tumors (CircTFRC siRNA). Scale bar, 100 µm (left), 20 µm (right). G ELISA assays showing the relative intratumoral levels of MDA in control tumors (Ctrl siRNA) or circTFRC knockdown tumors (CircTFRC siRNA). MAD malondialdehyde. H , I The spread of GC cells in control group (Ctrl siRNA) or circTFRC knockdown group (CircTFRC siRNA) was presented through bioluminescence imaging (BLI) using the IVIS® Spectrum imaging system ( n = 7 per group). Bioluminescent signals were detected by administering 150 mg/kg of the luciferase substrate D-luciferin via intraperitoneal injection 10 min before imaging. J Images of lung metastasis nodules in mice received intravenous injections of control siRNA or in vivo-optimized circTFRC siRNA. Scale bar, 200 µm. K The number of lung metastasis nodules in mice in control group (Ctrl siRNA) or circTFRC knockdown group (CircTFRC siRNA). The data are shown as the mean ± SD. The P values were determined by a two-tailed unpaired Student’s t test ( C , D , F , G , I , K ) or two-way ANOVA ( B ); ** P < 0.01, *** P < 0.001.

Article Snippet: Two siRNAs targeting circTFRC BSJ regions and a negative control siRNA (si-NC) were synthesized by GenePharma.

Techniques: Control, In Vivo, Expressing, Knockdown, Immunohistochemistry, Enzyme-linked Immunosorbent Assay, Imaging, Luciferase, Injection, Two Tailed Test

Journal: Journal of Advanced Research

Article Title: Histone deacetylases facilitate Th17-cell differentiation and pathogenicity in autoimmune uveitis via CDK6/ID2 axis

doi: 10.1016/j.jare.2024.07.029

Figure Lengend Snippet: CDK6 regulated a positive feedback loop with ID2 to promote Th17 pathogenicity and EAU development. A. Correlation heatmap showing the relative levels between downregulated rescue-TFs and CDK6. B. Venn diagram showing the intersection of downregulated rescue-TFs and downregulated rescue-DEGs. C. Venn diagram showing the intersection of ID2′s target genes and Top 10 Downregulated rescue-DEGs in PPI. D. The flow cytometry histograms (left) and column charts (right) showing the level of CDK6 in CD4 + IL-17A + Th17 cells among IRBP 1-20 , IRBP 1-20 + CDK6 siRNA, and IRBP 1-20 + CDK6i groups in vitro ( n = 5/group). E. The flow cytometry histograms (left) and column charts (right) showing the level of ID2 in CD4 + IL-17A + Th17 cells among IRBP 1-20 and IRBP 1-20 + ID2i groups in vitro ( n = 5/group). F. The flow cytometry histograms (left) and column charts (right) showing the percentage of the level of CDK6 in CD4 + IL-17A + Th17 cells among IRBP 1-20 , IRBP 1-20 + ID2i groups in vitro ( n = 5/group). G-H. The flow cytometry histograms (left) and column charts (right) showing the percentage of CD4 + IL-17A + Th17 cells ( G ), the level of PIM1 ( H ) in CD4 + IL-17A + Th17 cells among IRBP 1-20 , IRBP 1-20 + CDK6 siRNA, and IRBP 1-20 + CDK6i groups in vitro ( n = 5/group). I. The representative fundus examination images of EAU mice, CDK6i-treated EAU mice, and ID2i-treated EAU mice after immunization at day 14 (left). The white arrows indicate retinal inflammatory exudates. The column charts showing the clinical scores of the three groups ( n = 5/group, right). J. The representative H&E-stained images of EAU mice, CDK6i-treated EAU mice, and ID2i-treated EAU mice after immunization at day 14 (left). Scale bars: 50 μm. The black arrows indicate retinal folding. The column charts showing the pathology scores between the three groups ( n = 5/group, right). K-L. The flow cytometry histograms (left) and column charts (right) showing the percentage of retinal CD4 + cells ( K ) and CD4 + IL-17A + (Th17) ( L ) of EAU, CDK6i, and ID2i groups ( n = 5/group). Significance in D-E, G-L was calculated using one-way ANOVA test; significance in F was calculated using Mann-Whitney U test; *P<0.05, **P<0.01, ****P<0.0001.

Article Snippet: Isolated PBMCs were incubated with anti-human CD3/CD28 magnetic beads alone or with beads plus HDACi Belinostat (0.3 μM) with or without CDK6i BSJ-03–123 (3 μM, MedChem Express, cat. no. HY-111556) and ID2i helichrysetin (50 μM, TargetMol, cat. no. TN1727) , .

Techniques: Flow Cytometry, In Vitro, Staining, MANN-WHITNEY

Journal: Journal of Advanced Research

Article Title: Histone deacetylases facilitate Th17-cell differentiation and pathogenicity in autoimmune uveitis via CDK6/ID2 axis

doi: 10.1016/j.jare.2024.07.029

Figure Lengend Snippet: The HDACs/CDK6/ID2 axis is associated with AU progression and regulates the pathogenicity of human Th17 cells. A. The column charts showing the levels of HDAC1, pHDAC3, HDAC6, CDK6 and ID2 in human Th17 cells from HC, initial AU, relapsed AU, and recovery AU patients in flow cytometry analysis ( n = 6/group). B. The flow cytometry histograms (left) and column charts (right) showing the level of CDK6 in human CD3 + CD8 - T cells among Beads and Beads + CDK6i groups in vitro ( n = 5/group). C. The flow cytometry histograms (left) and column charts (right) showing the level of ID2 in human CD3 + CD8 - T cells among Beads and Beads + ID2i groups in vitro ( n = 5/group). D-G. The flow cytometry histograms (left) and column charts (right) showing the percentage of human Th17 cells ( D ), the level of GM-CSF ( E ), CDK6 ( F ), and ID2 ( G ) in human Th17 cells among Blank, Beads, Beads + HDACi, Beads + HDACi + CDK6i, and Beads + HDACi + ID2i groups in vitro ( n = 5/group). H-I. The flow cytometry histograms (left) and column charts (right) showing the level of Acetyl-H3 ( H ) and p53 ( I ) in human Th17 cells among Blank, Beads, Beads + HDACi groups in vitro ( n = 5/group). Significance in B-C was calculated using one-way ANOVA test; significance in D-I was calculated using Mann-Whitney U test; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Article Snippet: Isolated PBMCs were incubated with anti-human CD3/CD28 magnetic beads alone or with beads plus HDACi Belinostat (0.3 μM) with or without CDK6i BSJ-03–123 (3 μM, MedChem Express, cat. no. HY-111556) and ID2i helichrysetin (50 μM, TargetMol, cat. no. TN1727) , .

Techniques: Flow Cytometry, In Vitro, MANN-WHITNEY