1na pp1  (MedChemExpress)

Journal: Frontiers in Chemistry

Article Title: Bulky PP1 analogs exert cellular effects independently from analog-sensitive kinase inhibition

doi: 10.3389/fchem.2026.1812827

Figure Lengend Snippet: PP1 analogs inhibit cell migration. Examples of HaCaT and MDA-MB-231 cell gaps observed upon wound making and 48 h later, in presence of DMSO or 5 µM of 1NM- and 1NA-PP1, respectively.

Article Snippet: We prepared 10 mM stock solutions of 1NA-PP1 (MedChemExpress, HY-13941), 1NM-PP1 (MedChemExpress, HY-13942), 3MB-PP1 (Aobious, AOB3854), 3IB-PP1(Sigma, 529598) and 3MSB-PP1 in 100% DMSO and we stored aliquots at −20 °C.

Techniques: Migration

Journal: The EMBO Journal

Article Title: The Spo13/Meikin pathway confines the onset of gamete differentiation to meiosis II in yeast

doi: 10.15252/embj.2021109446

Figure Lengend Snippet: Imaging of SPBs (Cnm67‐RFP) and Spc72‐GFP in P HSL1 ‐CDC20 ama1Δ spo13Δ control cells and cells containing cdc5‐as or ime2‐as treated with CMK or 1Na‐PP1, respectively, at metaphase I (7 h in SPM). Top, time‐lapse series. Bottom, Spc72's presence at SPBs was quantified in cells synchronized in silico to SPB separation at entry into metaphase I ( t = 0). Whole‐cell extracts from P HSL1 ‐CDC20 ama1Δ CDC5‐ha3 Ha3‐SPO13 cells were subjected to α‐Ha immunoblotting. Secondary antibodies were detected by near‐infrared fluorescence imaging. Top, progression into metaphase I. Bottom, serial two‐fold dilutions of the sample collected at t = 10 h in SPM. The Cdc5‐ha3/Ha3‐Spo13 ratio is ~ 20. Cdc5 interacts with Ime2. Cdc5‐ha3, Ime2, and Spo13 were detected by immunoblotting in whole‐cell extracts and α‐Ha immunoprecipitates from the indicated strains. Cdc5's polo‐box domain (PBD) binds to Ime2. Ha3‐tagged versions of the wild‐type PBD or the phosphopeptide‐binding mutant PBD‐FAA were expressed at t = 5.5 h in SPM in cells depleted of Cdc20 and Cdc5. PBD‐ha3 and Ime2 were detected by immunoblotting in whole‐cell extracts and α‐Ha immunoprecipitates. Cdc5‐dependent modification of Ime2. P HSL1 ‐CDC20 ama1Δ control cells and cells containing spo13Δ or cdc5‐as were treated with CMK (to inhibit Cdc5‐as) at t = 8 h in SPM (arrowheads). The panel shows immunoblot analysis of whole‐cell extracts. C, sample from proliferating cells. Increased gel mobility of Ndt80 at t ≥ 10 h confirms inhibition of Cdc5‐as. The arrow marks the modified form of Ime2. Data information: Data in (A‐E) are representative of two independent experiments. Source data are available online for this figure.

Article Snippet: To inhibit and reactivate Cdk1, cdc28‐as2 cells treated with 1Na‐PP1 (10 μM) were collected by filtration (Whatman 10400772, 3 μm pore size), washed with conditioned SPM (cSPM, 10 culture volumes, obtained from cultures in SPM without inhibitor), and inoculated into cSPM.

Techniques: Imaging, In Silico, Western Blot, Fluorescence, Binding Assay, Mutagenesis, Modification, Inhibition

Journal: The EMBO Journal

Article Title: The Spo13/Meikin pathway confines the onset of gamete differentiation to meiosis II in yeast

doi: 10.15252/embj.2021109446

Figure Lengend Snippet: A Imaging of SPBs (Cnm67‐RFP) and Spc72‐GFP in ndt80Δ and ndt80Δ spo13Δ cells induced to express P EST ‐IME2‐ΔC plus P EST ‐CDC5 at 4 h in SPM ( t = 0). Top, time‐lapse series. Bottom, quantification of cells with Spc72 at SPBs. In ndt80Δ cells, the SPO13 deletion causes only a small advance in Spc72 removal (19 min; 95% CI, 8–30; P = 0.001; Welch's t ‐test). B Immunoblot detection of Spo13, Spo13‐10A, and Spo13‐10D in whole‐cell extracts from P HSL1 ‐CDC20 ama1Δ cells progressing into metaphase I. Cdc28‐as2 was inhibited with 1Na‐PP1 at 8 h in SPM (arrow). C, sample from proliferating cells. C, D Imaging of SPBs (Cnm67‐RFP) and Spc72‐GFP in P HSL1 ‐CDC20 ama1Δ cells containing different SPO13 alleles. (C) Time‐lapse series of spo13‐10A and spo13‐10D cells. (D) Spc72's presence at SPBs was quantified in spo13‐10A , spo13‐10D , spo13Δ , and control cells synchronized in silico to SPB separation at entry into metaphase I ( t = 0). spo13‐10D cells remove Spc72 with similar timing after entry into metaphase I as spo13Δ cells ( P = 0.64, Welch's t ‐test). Data information: Data are representative of two (A) or three (C, D) independent experiments. Source data are available online for this figure.

Article Snippet: To inhibit and reactivate Cdk1, cdc28‐as2 cells treated with 1Na‐PP1 (10 μM) were collected by filtration (Whatman 10400772, 3 μm pore size), washed with conditioned SPM (cSPM, 10 culture volumes, obtained from cultures in SPM without inhibitor), and inoculated into cSPM.

Techniques: Imaging, Western Blot, In Silico

Journal: The EMBO Journal

Article Title: The Spo13/Meikin pathway confines the onset of gamete differentiation to meiosis II in yeast

doi: 10.15252/embj.2021109446

Figure Lengend Snippet: P HSL1 ‐CDC20 ama1Δ cdc28‐as2 cells were treated with 1Na‐PP1 at t = 3 h in SPM to inhibit Cdc28‐as2. At t = 5 h (left) or t = 9 h (right), Cdc28‐as2 was activated by washing cells with conditioned SPM (cSPM) lacking 1Na‐PP1 (washout). Top, time‐lapse series from the imaging of Mpc70‐GFP and RFP‐tubulin. The weak, nuclear signal from Ndt80‐GFP serves to confirm entry into meiosis. Middle, quantification of spindle formation and Mpc70 loading to SPBs. Bottom, quantification of control experiments. Cells were washed with cSPM plus 1Na‐PP1 at t = 5 h (left) or t = 9 h (right) in SPM. TEM analysis of SPBs. P HSL1 ‐CDC20 ama1Δ cdc28‐as2 cells were treated with 1Na‐PP1 at 3 h in SPM. At 9 h in SPM, cells were washed with cSPM containing (top) or lacking 1Na‐PP1 (washout, bottom). TEM samples were collected at 13 h in SPM. Washout of 1Na‐PP1 causes MP formation ( P < 0.0001, Fisher's exact test). Scale bar, 0.2 μm. P HSL1 ‐CDC20 ama1Δ cdc28‐as2 control cells and cells containing spo13Δ or cdc5‐as were treated with 1Na‐PP1 at t = 3 h in SPM to inhibit Cdc28‐as2. At t = 9 h in SPM, Cdc28‐as2 was activated by washing cells with cSPM lacking 1Na‐PP1 (washout). cdc5‐as cells were washed with cSPM plus CMK to activate Cdc28‐as2 and inhibit Cdc5‐as. Top, time‐lapse series from the imaging of Mpc70‐GFP and RFP‐tubulin. The weak, nuclear signal originates from Ndt80‐GFP. Bottom, quantification of spindle formation and Mpc70 loading to SPBs. P HSL1 ‐CDC20 ama1Δ cells containing SPC72 , spc72‐7 , or spc72‐7 plus cdc5‐as were shifted from 24 to 36°C at t = 4.2 h in SPM (to inactivate Spc72‐7, arrowheads) and treated with CMK at t = 6 h in SPM (to inhibit Cdc5‐as). Top, time‐lapse series from the imaging of SPBs (Cnm67‐RFP) and Mpc70‐GFP. Bottom, quantification of SPB separation and Mpc70 loading to SPBs. Inhibition of Cdc5‐as in spc72‐7 cells advances the time of Mpc70 loading by 119 min (95% CI, 76–162; P < 0.0001; Welch's t ‐test). Data are representative of two independent experiments.

Article Snippet: To inhibit and reactivate Cdk1, cdc28‐as2 cells treated with 1Na‐PP1 (10 μM) were collected by filtration (Whatman 10400772, 3 μm pore size), washed with conditioned SPM (cSPM, 10 culture volumes, obtained from cultures in SPM without inhibitor), and inoculated into cSPM.

Techniques: Imaging, Inhibition

Journal: The EMBO Journal

Article Title: The Spo13/Meikin pathway confines the onset of gamete differentiation to meiosis II in yeast

doi: 10.15252/embj.2021109446

Figure Lengend Snippet: Inhibition of Cdk1 at anaphase I. CDC20‐mAR and CDC20‐mAR cdc28‐as1 cells were released from the metaphase I‐arrest with CuSO 4 at 7 h in SPM ( t = 0) and treated with 1NM‐PP1 at t = 40 min to inhibit Cdc28‐as1 (arrows). Top, time‐lapse series from the imaging of Mpc70‐GFP, SPBs (Cnm67‐RFP), and nuclei (TetR‐RFP). Bottom, quantification of meiotic events. cdc20 ts ‐mAR P DMC1 ‐AMA1 P HSL1 ‐CDH1 cells were released from the metaphase I‐arrest with CuSO 4 ( t = 0) at 25°C. Subsequent incubation at 25°C allows progression through meiosis II (left), while a shift to 37°C at t = 50 min causes arrest at metaphase II (right). Mpc70‐GFP and the PSM marker RFP‐Spo20 51–91 were imaged in cells fixed at the indicated times. RFP‐Spo20 51–91 is expressed from an episomal plasmid (Diamond et al , ). Images represent ≥ 90% of cells harboring the plasmid. Scale bar, 2 μm. cdc20 ts ‐mAR P DMC1 ‐AMA1 P HSL1 ‐CDH1 cells containing CDC28 or cdc28‐as2 were released from metaphase I at t = 0 and shifted to 37°C at t = 50 min to cause arrest at metaphase II. 1Na‐PP1 was added at t = 100 min to inhibit Cdc28‐as2. Microtubules (GFP‐Tub1), Mpc70‐GFP, and RFP‐Spo20 51–91 were imaged in cells fixed at the indicated times. Images represent ≥ 90% of cells carrying the RFP‐Spo20 51–91 plasmid. Scale bar, 2 μm. The regulatory network controlling MP assembly at different stages of meiosis. Green arrows: activation. Dashed, green arrows: latent activation overwhelmed by inhibition. Red, bar‐headed lines: inhibition. Items in light gray are inactive or absent. Block arc: SPB outer layer. X: Cdc20 substrate that blocks PSM formation at metaphase II. See text for details. Data information: Data in (B) and (C) are representative of two independent experiments.

Article Snippet: To inhibit and reactivate Cdk1, cdc28‐as2 cells treated with 1Na‐PP1 (10 μM) were collected by filtration (Whatman 10400772, 3 μm pore size), washed with conditioned SPM (cSPM, 10 culture volumes, obtained from cultures in SPM without inhibitor), and inoculated into cSPM.

Techniques: Inhibition, Imaging, Incubation, Marker, Plasmid Preparation, Activation Assay, Blocking Assay