aavs1 locus  (Addgene inc)

Journal: iScience

Article Title: GPX4-dependent ferroptosis sensitivity is a fitness trade-off for cell enlargement

doi: 10.1016/j.isci.2025.112363

Figure Lengend Snippet: Increased lipid peroxidation in large cells (A) Digital holography images of RSL3 treatment time course for control, doxorubicin-treated, and palbociclib-treated RPE1 cells. (B) Quantification of imaging parameters at 0 and 4 h after RSL3 addition. The blue numbers in the optical volume indicate the increase in cell volume (swelling) between 0- and 4-h time points. (C) Lipid peroxidation in RPE1 cells using C11-bodipy lipid peroxidation sensor and flow cytometry. Probe oxidation results in a shift of the fluorescence emission peak from red (PE-A) to green (FITC-A) channel. Cells treated with palbociclib were treated with or without 1 μM RSL3 and 1 μM Fer-1 as indicated. Data shown are mean ± SD, n = 3. (D and E) (D) Same as (C) but cells were treated with 50 nM doxorubicin. Statistical analysis in (C and D) was ANOVA followed by Tukey’s test. (E) Digital holographic quantification of cell areas and phase shifts for single GPX4 KO cells treated with (blue line) or without (red line) doxorubicin for 3 days after which they were imaged for 6 h in the presence of ferroptosis inhibitor Fer-1. (F) Same treatment as in (E), but Fer-1 was washed out immediately before imaging. Mean values are in solid line, with error bars showing standard deviation; between 52 and 660 cells were tracked. (G) Tetracycline-inducible expression of mGreenLantern-p21. The cell size distribution with and without Tet-induction for 3 days was measured with coulter counter. (H) WB analysis of mGreenLantern-p21 with and without Tet-induction for 3 days, followed by 100 nM RSL3 for 24 h. (I) Senescence-associated β-galactosidase staining of mGreenLantern-p21 cells. Data shown are mean ± SD, n = 3. ANOVA with Tukey’s test. See also Figure S3 .

Article Snippet: The RPE1 mGreenLantern-p21 cells were established by cloning mGreenLantern in frame with p21 coding sequence into AAVS1-Tet plasmid (Addgene plasmid #158663, a kind gift from Masato Kanemaki).

Techniques: Control, Imaging, Flow Cytometry, Fluorescence, Standard Deviation, Expressing, Staining

Journal: Genome biology

Article Title: Deciphering the role of RNA in regulating CTCF's DNA binding affinity in leukemia cells.

doi: 10.1186/s13059-025-03582-x

Figure Lengend Snippet: Fig. 1 Establish a cellular model to interfere with CTCF-RNA interactions to study the impact on CTCF’s DNA binding affinity. A Schematic diagram illustrating three techniques to disrupt CTCF-RNA interactions. On the left is an illustration of how the ectopic HA-tagged CTCF swap system works in combination with acute protein degradation of endogenous CTCF. The homozygous miniAID-mClover3 knockin SEM cell lines CTCFAID2/WT and CTCFAID2/dRBR were previously generated [11]. When added to cell culture, the 5-Ph-IAA auxin analog acts as a ligand to bind to the miniAID tag (fused to endogenous CTCF protein) and OsTIR1(F74G) protein to promote acute protein degradation through ubiquitination by the SCF complex. After 6 h of 5-Ph-IAA treatment, the CTCF HA-tagged WT or CTCF-HA-dRBR ectopic proteins were induced by doxycycline for a total of 18 h of doxycycline and 24 h of 5-Ph-IAA treatment. In the middle is an illustration of transcription inhibition by the natural product, triptolide. Triptolide was added to live cell culture to block the PolII activity and global nascent transcription. On the right is a diagram showing how RNase A was used during the ChIP-seq procedure, either added before (pre-fixation treatment) or after (post-fixation treatment) the chromatin fixation, to degrade global RNAs. B Immunoblot analysis of endogenous (CTCFAID2.0) and induced exogenous (HA-CTCF) expression of CTCF using an antibody for CTCF. CTCFAID2.0 expression can be seen in all untreated samples (−, −). After 6 h of 10 μM 5-Ph-IAA treatment, CTCFAID2 protein expression is degraded. Exogenous expression of HA-tagged CTCF wildtype and dRBR mutant is comparable to endogenous CTCF following 18 h of 1 μg/mL doxycycline with concurrent 10 μM 5-Ph-IAA treatment (+, +). GAPDH was included as a loading control. C Quality control of RNA inhibition upon triptolide and RNase A treatment. Total RNAs were collected after drug treatment, followed by reverse transcription. The cDNA was fragmented, amplified, and quantified by a bioanalyzer. Three replicates were included for each treatment

Article Snippet: In brief, CTCFAID2 cells have an in-frame miniAID-mClover3 tag at the C-terminus of endogenous CTCF and constitutively express OsTIR1 F74G (Addgene 232800).

Techniques: Binding Assay, Knock-In, Generated, Cell Culture, Ubiquitin Proteomics, Inhibition, Blocking Assay, Activity Assay, ChIP-sequencing, Western Blot, Expressing, Mutagenesis, Control, Reverse Transcription, Amplification