pmk411 (ostir1(f74g) maid-egfp-nluc) cassette  (Addgene inc)

Journal: Biology of the Cell

Article Title: Chromosome Segregation in Closed Mitosis Under an Excess of Nuclear Envelope

doi: 10.1111/boc.70011

Figure Lengend Snippet: Interphase chromatin protrusions after Nem1 depletion. The strain (FM2748) is isogenic to the one shown in Figure , but carries the following labels: Hta2‐mCherry (chromatin), Net1‐eCFP (rDNA), and TetR‐YFP/ tetO:1061 (cXIIr telomere). It also carries the nem1:aid* allele and the OsTIR1 system for depleting Nem1‐aid* after auxin (IAA) addition. (A) Representative micrographs of log‐phase FM2748 cells without and with rDNA chromatin protrusions. Cells in G1, S/G2 and G2/M cells are shown. In all cases, protrusions (white arrows) correspond to the rDNA locus. (B) Quantification of chromatin protrusions relative to cell cycle stages shown in (A), growth media (YPD vs. SCcshl), and − vs. + IAA for 24 h (mean ± SEM, n = 3). FM2748 ( nem1:aid*) and its wild type (WT) parental FM2707 (as FM2748 but with a wild type NEM1 ) were assessed. Note that depletion of Nem1 causes a single rDNA protrusion in most cases. They are observed in G1 and S/G2/M. (C) Characterization of rDNA shape in the +IAA (24 h) protrusions (mean ± SEM, n = 3). (D) Relative location of the cXIIr telomere in the +IAA (24 h) protrusions (mean ± SEM, n = 3). (E) As in (B) but before (0 h) and just 3 h after IAA addition in the FM2748 strain (mean ± SEM, n = 3). (F) Characterization of the rDNA shape in the +IAA (3 h) protrusions (mean ± SEM, n = 3). (G) Relative position of the cXIIr telomere in the +IAA (3 h) protrusions (mean ± SEM, n = 3). (H) Protrusion length after 3 and 24 h in IAA (mean ± SD). One way ANOVA followed by the Tukey test was applied for statistical assessment (*** p < 0.001; ** p < 0.01; * p < 0.05; ns, p > 0.05).

Article Snippet: To investigate the role of Nem1 in chromosome segregation, we utilized a nem1‐aid* OsTIR1 strain (FM2748), which allows conditional degradation of Nem1 upon the addition of the auxin indole‐3‐acetic acid (IAA) (Matos‐Perdomo et al. ).

Techniques:

Journal: Biology of the Cell

Article Title: Chromosome Segregation in Closed Mitosis Under an Excess of Nuclear Envelope

doi: 10.1111/boc.70011

Figure Lengend Snippet: Chromosome XII and rDNA segregation patterns in cells depleted of Nem1 while growing in nutrient rich and complete minimum media. (A) Cell cycle distribution of log‐phase cultures in YPD and SCcshl with (−IAA) or without (+IAA) Nem1 (mean ± SEM, n = 3). (B) Schematic illustration of cXII segregation. In early anaphase (unresolved rDNA): (a) the nucleus commences its elongation through the division axis while the nucleolus/rDNA remains on one edge; (b) cXIIr resolves from centromere to telomere, which unzips the rDNA to form a bridge between mostly segregated nuclear masses. In late anaphase (segregated rDNA): (c), distal cXIIr regions are the last to be resolved and lag behind the rest of the genome; (d and e) rDNA compaction pulls distal regions to complete segregation. This latter process can occur asynchronously so that one sister cXIIr can appear in an advanced segregation state (d). (C) Representative micrographs of the drawings of (B). (D) Quantification of the different rDNA and cXIIr segregation figures (mean ± SEM, n = 3). Note that Nem1 depletion increases the percentage of late anaphase cells with lagging cXIIr. The growth media had little influence on this pattern (unpair t test for “type e” in −IAA vs. +IAA render p < 0.001 for both YPD and SC). (E) Transitions between the five anaphase types in the presence and absence of Nem1 ( N , number of cells followed by time‐lapse super‐resolution microscopy; each colored line corresponds to a cell). Time zero corresponds to when the cell entered anaphase (type a).

Article Snippet: To investigate the role of Nem1 in chromosome segregation, we utilized a nem1‐aid* OsTIR1 strain (FM2748), which allows conditional degradation of Nem1 upon the addition of the auxin indole‐3‐acetic acid (IAA) (Matos‐Perdomo et al. ).

Techniques: Super-Resolution Microscopy

Journal: Biology of the Cell

Article Title: Chromosome Segregation in Closed Mitosis Under an Excess of Nuclear Envelope

doi: 10.1111/boc.70011

Figure Lengend Snippet: The NE‐vacuole axis in anaphase with and without Nem1. An asynchronous log‐phase culture of FM2748 was stained with MDY‐64 dye under conditions that label both the NE and the vacuolar membrane. MDY‐64 is read in the CFP channel, and its brightness overshadowed Net1, which could not be assessed here. (A) Seven representative anaphase cells. Note the close interaction between the NE and vacuoles. Lagging cXIIr often bends around or is attached to vacuole(s). (B) Single cell analysis of the NE perimeter at different cell cycle stages in the presence and absence of Nem1 (mean ± SD). One way ANOVA was applied for statistical assessment. The effect of Nem1 for each cell cycle stage was the only post hoc comparison included (* p < 0.05; ns, p > 0.05). (C) As in (B), but only counting late anaphase cells (i.e., elongated or binucleated Hta2 with two tetO:1061 ). The two‐tailed Student's t test was applied. (D) Vacuole number per cell (all cell cycle stages) with and without Nem1 (mean ± SEM, n = 3; unpair t test for “>3 vacuoles” renders a p = 0.0134).

Article Snippet: To investigate the role of Nem1 in chromosome segregation, we utilized a nem1‐aid* OsTIR1 strain (FM2748), which allows conditional degradation of Nem1 upon the addition of the auxin indole‐3‐acetic acid (IAA) (Matos‐Perdomo et al. ).

Techniques: Staining, Membrane, Single-cell Analysis, Comparison, Two Tailed Test

Journal: Biology of the Cell

Article Title: Chromosome Segregation in Closed Mitosis Under an Excess of Nuclear Envelope

doi: 10.1111/boc.70011

Figure Lengend Snippet: Nvj1‐Vac8 nucleus‐vacuole junctions with and without Nem1. (A) Representative log‐phase cells from a NVJ1‐YFP VAC8‐mCherry NEM1 strain (FM2793) growing in SCcshl. The top images were taken by wide‐field fluorescence microscopy (WF), while the bottom images were taken by Airyscan 2 super‐resolution microscopy (SR). Four mitotic cells are numbered in the WF example. Note the variability of NE labelling by Nvj1. Cells #3 and #4 have Nvj1/Vac8 spots indicating NVJs. In the SR example, the cell on the left is in early anaphase and shows an Nvj1‐Vac8 patch. (B) As in (A) but with the addition of MDY‐64 dye. Both examples are from SR images and include cells in late anaphase. The upper cell contains an Nvj1‐Vac8 spot, whereas the lower cell does not show NVJ, but the NE is still tightly associated with the vacuole (white arrowhead). Scale bar corresponds to 5 µm. (C) Percentage of cells displaying Nvj1‐Vac8 spots in a NVJ1‐YFP VAC8‐mCherry nem1:aid* strain (FM3311) grown overnight in SCcshl with (−IAA) and without (+IAA) Nem1 (mean ± SEM, n = 3). Cells were grouped according to the cell cycle stage they were in (anaph is anaphase). (D) Length of Nvj1 patches (mean ± SD; all cells included regardless of cell cycle stage). The two‐tailed Student's t ‐test was used to compare between the presence and absence of Nem1.

Article Snippet: To investigate the role of Nem1 in chromosome segregation, we utilized a nem1‐aid* OsTIR1 strain (FM2748), which allows conditional degradation of Nem1 upon the addition of the auxin indole‐3‐acetic acid (IAA) (Matos‐Perdomo et al. ).

Techniques: Fluorescence, Microscopy, Super-Resolution Microscopy, Two Tailed Test

Journal: Biology of the Cell

Article Title: Chromosome Segregation in Closed Mitosis Under an Excess of Nuclear Envelope

doi: 10.1111/boc.70011

Figure Lengend Snippet: Summary and models of how an excess of NE leads to lagging chromosome arms. In the presence of Nem1 (upper diagram), the NE is maintained at a size appropriate for NE elongation in anaphase. We propose that the NE surface is short enough to exert some tension on the NE bridge of the long hourglass late anaphase nucleus, which might favor karyokinesis and stabilization of type e late anaphases. In the absence of Nem1 (lower diagram), the excess of NE would be absorbed by the bridge, resulting in less tension and affecting karyokinesis and NE retraction, and thus the recoiling of cXIIr (more type c late anaphases). This in turn would lead to the perpetuation of the rDNA/cXIIr protrusions across generations.

Article Snippet: To investigate the role of Nem1 in chromosome segregation, we utilized a nem1‐aid* OsTIR1 strain (FM2748), which allows conditional degradation of Nem1 upon the addition of the auxin indole‐3‐acetic acid (IAA) (Matos‐Perdomo et al. ).

Techniques:

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