Journal: bioRxiv

Article Title: Kinetic proofreading as a mechanism for transcriptional specificity in living human cells

doi: 10.64898/2026.03.17.711757

Figure Lengend Snippet: (A) Screen readout using smRNA-FISH with ERRFI1 -intronic and MYH9 -3’UTR probes to detect nascent transcription sites in fixed cells. (B) Screening workflow: sgRNAs targeting ∼1,100 genes are transfected in arrayed 384-well plates, followed by 72-hr knockdown, hormone depletion, dexamethasone treatment, and fixation for smFISH. Plates are imaged by spinning confocal microscopy (60X, 9 fields of view/well), and nuclear spots are quantified per cell via automated DAPI segmentation and spot detection. (C) 2D scatter plot of averaged Z-scores (2 replicates) for ERRFI1 (x-axis) and MYH9 (y-axis) nuclear spots, zeroed to the mean of negative controls (sgNEG, sgOR10A5). Hits classified into four categories: “common up” (n=8), “common down” (n=111), “MYH9-specific” (n=16), and “ERRFI1-specific” (n=72). (D) Representative smFISH images for each hit category alongside negative (sgNEG) and positive (sgNR3C1) controls. (E) STRING interaction network of “ERRFI1-specific” hits (left) and representative smFISH images for selected hits (right) (F) RT-qPCR measurement of nascent transcription (intron 1 primers) after 30-min pretreatment with triptolide (TPL; TFIIH inhibitor), BRM-014 (BRM/BRG1 inhibitor), or Pevonedistat (MLN-4924; NEDD8-activating enzyme inhibitor), followed by 2-hr vehicle (left) or 100nM Dex (right) treatment.

Article Snippet: For inhibitor perturbations , hormone-depleted cells were pre-treated for 30 min with triptolide (TPL; 50 nM or 100 nM) (Sigma-Aldrich, T3652), BRM-014 (10 μM) (MedChemExpress, HY-119374), or MLN-4924 (500 nM or 1 μM) (Cell Signaling, #85923), followed by 2 h treatment with Dex (100 nM) or vehicle in the continued presence of inhibitor, as indicated.

Techniques: Transfection, Knockdown, Confocal Microscopy, Quantitative RT-PCR