Journal: Clinical and Translational Medicine

Article Title: N 6 ‐methyladenosine‐mediated upregulation of LNCAROD confers radioresistance in esophageal squamous cell carcinoma through stabilizing PARP1

doi: 10.1002/ctm2.70039

Figure Lengend Snippet: METTL3‐mediated m6A modification enhances the stability of LNCAROD in a YTHDC1‐dependent manner. (A, B) RT‐qPCR analysis and Western blot analysis for the expression of METTL3 mRNA and protein in parental and radioresistant ESCC cell lines. (C) Total m6A levels in radioresistant and parental ESCC cell lines. (D) The Venn diagram shows that LNCAROD is the only lncRNA modified by METTL3‐mediated m6A and overlapping in both GSE53625 and TCGA independent ESCC cohorts. (E) RT‐PCR assay is employed to assess the expression of LNCAROD variants in ESCC cell lines and normal cells. (F) Expression levels of LNCAROD in ESCC cell lines and normal cells is examined by RT‐qPCR assay. (G) MeRIP assay results show the enrichment of m6A‐modified LNCAROD in the indicated cells. (H, I) MeRIP‐qPCR analysis of LNCAROD m6A modification in the indicated cells. (J) RT‐qPCR analysis of LNCAROD expression in the indicated cells. (K, L) The RNA stability of LNCAROD is measured by RT‐qPCR in the indicated cells. Actinomycin D (5 mg/mL) is added at time 0. (M) Cell nuclear/cytoplasmic fractionation and RT‐qPCR show the cellular distribution of LNCAROD in ESCC cells. GAPDH is used as a cytoplasmic marker and HOTAIR is used as a nuclear marker. (N) RIP‐qPCR assays show the relative enrichment of LNCAROD detected by YTHDC1 antibody. (O, P) RT–qPCR assays show the relative expression of YTHDC1 and LNCAROD in the indicated cells. (Q, R) The RNA stability of LNCAROD is measured by RT‐qPCR in the indicated cells. (S) Knockdown of YTHDC1 significantly abolished the upregulation of LNCAROD induced by ectopic METTL3 expression in KYSE‐150 cells. Data are shown as the mean ± SD from three independent experiments (* p < .05, ** p < .01, *** p < .001, **** p < .0001).

Article Snippet: Actinomycin D and proteasome inhibitor MG132 were acquired from MedChemExpress (New Jersey, USA), while cycloheximide (CHX), a protein synthesis inhibitor, was obtained from Selleck Chemicals (Houston, TX, USA).

Techniques: Modification, Quantitative RT-PCR, Western Blot, Expressing, Reverse Transcription Polymerase Chain Reaction, Fractionation, Marker, Knockdown

Journal: bioRxiv

Article Title: Gene transcription and chromatin packing domains form a self- organizing system

doi: 10.64898/2026.03.15.711889

Figure Lengend Snippet: Packing domain analysis following transcriptional inhibition of HCT116 cells treated with Actinomycin D (5 µg/mL) or treated with DMSO. Representative chromatin polymer chains from ( A ) a Self- Avoiding Walk model demonstrating a random walk with no forced returns (enzymatically driven loops) that does not form packing domains and ( B ) a Self-Returning-Excluded Volume model SR-EV demonstrating the formation of packing domains due to the contribution of loops and the excluded volume effect of monomers (nucleosomes). Color corresponds to monomer number in chain: Blue = 1, Red = 4000. ( C ) PWS following ActD treatment: average nuclear packing scaling of packing domains. Data was compiled from three independent biological replicates. (DMSO: N = 475; ActD: N=541). ( D-F ) ChromSTEM following treatment: packing scaling, domain radius, and chromatin volume concentration of individual domains, respectively (DMSO: N = 71; ActD: N=48). ( G ) ChromSTEM tomograms showing untreated chromatin state or domain swelling following treatment. XY axes are in nm. ( H ) Representative domain from ChromSTEM tomogram demonstrating radial density from center to periphery where values approaching 1 have high CVC and values approaching 0 have low CVC ( I ) Representative map of chromatin scaling values, D nucleus , generated using PWS from wildtype nuclei (Left) and ActD-treated nuclei (Right). For C–F : Significance was calculated by unpaired t test with Welch’s correction applied (**** < 0.0001).

Article Snippet: HCT116 “WT” cells were treated with either 5 μg/mL Actinomycin D (MP Biomedicals, #0210465810) or % v/v DMSO (Thermo Scientific, #J66650.AE) equal to the highest DMSO concentration in each experiment.

Techniques: Inhibition, Polymer, Concentration Assay, Generated

Journal: bioRxiv

Article Title: Gene transcription and chromatin packing domains form a self- organizing system

doi: 10.64898/2026.03.15.711889

Figure Lengend Snippet: RNA synthesis is downregulated but gene transcription persists in nucleoplasm despite Pol-II depletion. POLR2A-AID2 Degron line was treated with auxin, DMSO, or Actinomycin D treated (5 µg/mL) for 6 hours and imaged following labeling of nascent RNA with 1 mM 5-ethynyl-uridine for 1 hour. ( A ) Representative image of each condition captured using widefield fluorescent microscopy. Scale bar = 10 µm. ( B ) Cell total corrected fluorescence of widefield fluorescent images for each condition. Significance was calculated by unpaired t test with Welch’s correction applied (**** < 0.0001) (POLR2A: N = 142; 6hrActD: N=20; 1hrActD: N=28; DMSO: N= 204). ( C-D ) Representative images from SMLM of EU-labeled RNA for each condition. ( E-G ) Quantification of SMLM distributions for each condition generated using DBscan: fluorophore blink density, cluster density, and cluster size (POLR2A: N = 18; DMSO: N= 28). Significance was calculated by unpaired t test with Welch’s correction applied. P-values are listed. ( H-I ) MA plot for POLR2A degraded cells and ActD treated cells showing log2 expression as a function of log fold change using a LFC cutoff of (abs > 1) and signficance level of Padj < 0.05. X axis = Log2 Mean Expression (averaged over all samples). ( J ) Scatterplot of log fold chance values for each gene in one condition compared to log fold change values for the same gene in another condition. Heatmap color scale corresponds to gene density. Marginal distribution shows DEGs with same cutoffs as MA plots. For B, E– G : Significance was calculated by unpaired t test with Welch’s correction applied (**** < 0.0001).

Article Snippet: HCT116 “WT” cells were treated with either 5 μg/mL Actinomycin D (MP Biomedicals, #0210465810) or % v/v DMSO (Thermo Scientific, #J66650.AE) equal to the highest DMSO concentration in each experiment.

Techniques: Labeling, Microscopy, Fluorescence, Generated, Expressing

Journal: bioRxiv

Article Title: Gene transcription and chromatin packing domains form a self- organizing system

doi: 10.64898/2026.03.15.711889

Figure Lengend Snippet: Genome connectivity and in situ packing domains are perturbed by Pol-II loss. POLR2A-AID2 degron line was treated with auxin for 6 hours, DMSO for 6 hours, or Actinomycin D treated (5 µg/mL) for 1 hour and Hi-C was generated for each condition. ( A-C ) Scatterplot of log10 loop strength against log10 size for each loop. Loops called using HICCUPS. Heatmap shows loop density (POLR2A Loops: N =56,420; DMSO Loops: N=18,123; ActD Loops: N=39,484). ( D ) Top: difference plot showing change relative to WT. Scale bar: normalized mean loss or gain of contact strength. Bottom: Pileup plots of loop insulation strength for each. Scale bar: normalized mean contact strength. Insulation plots for loop strength were generated in GENOVA . ( E ) TAD insulation plots showing no change to TADs between conditions in POLR2A depleted cells. TADs called using Arrowhead and plotted with GENOVA ( F ) Contact map highlighting loop loss upon POLR2A depletion. Red arrows mark loops that have decreased focal enrichment. ( G ) Contact map from reanalyzed Micro-C data showing distal loop gain and proximal loop loss following POLR2A depletion. Red arrows mark loops that have increased focal enrichment. Yellow arrows mark loops that have decreased focal enrichment. ( H) Compartment pile-up plot showing no change to compartments (both TAD and compartment pileup plots were generated using GENOVA. See methods for more details). ( I) Contact scaling for individual genes binned and ranked by E/I score (E/I = Exon Length/(Gene Length – Exon Length) ). Accompanying schematic shows how E/I scales with domain size. Low E/I = large domains, average E/I = small domains, and high E/I = chained aggregates that have are less likely to form domains on their own. (J) Schematic demonstrating how the ratio of exons to introns (E/I ratio) within genes organizes domain geometry. Genes with a low E/I ratio are more likely to form domains, wherein heterochromatic introns form sticky cores that exclude euchromatic exons in a transcriptionally poised superficial ideal zone for coherent gene transcription. For I : Significance was calculated by unpaired t test with Welch’s correction applied (**** < 0.0001).

Article Snippet: HCT116 “WT” cells were treated with either 5 μg/mL Actinomycin D (MP Biomedicals, #0210465810) or % v/v DMSO (Thermo Scientific, #J66650.AE) equal to the highest DMSO concentration in each experiment.

Techniques: In Situ, Hi-C, Generated, Insulation