pretrox gfp mps1 wt  (Addgene inc)

Journal: Current biology : CB

Article Title: MPS1 localizes to end-on microtubule-attached kinetochores to promote microtubule release.

doi: 10.1016/j.cub.2022.10.047

Figure Lengend Snippet: Figure 1. MPS1 can localize to end-on attached kinetochores (A) Schematic diagram depicting transient MPS1 binding to microtubule-attached kinetochores. (B) Nocodazole (Noc) or MG132 arrested HeLa MPS1-GFP cells were either directly fixed or cooled to 4C for 9 min then fixed and immunostained as indicated. (C) MPS1 kinetochore cell average intensities of cells in (B). Bars show mean ± SEM. ****p % 0.0001. (D) STLC-arrested HeLa MPS1-GFP cells treated as in (B). The K-fiber marker HURP was used to visualize kinetochore attachments.19,20

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER siRNA against HEC1 5’UTR (5’-CCCUGGGUCGUGUCAGGAA-3’) Nijenhuis et al.12 N/A siRNA against BUBR1 3’UTR (5’-GCAATCAAGTCTCACAGAT-3’) Espert et al.24 N/A On-target plus Smartpool siRNA oligos against PPP2R5A (5’-GCUCAAAGAUGCCACUUCA-3’, 5’-CAAUACAAGUGCCGAAUAA-3’, 5’-UGAAUGAACUGGUUGAGUA-3’, 5’-GGAAAUGAAUGGCAAGCUU-3’) Dharmacon Cat#L-009352 On-target plus Smartpool siRNA oligos against PPP2R5B (5’-CGCAUGAUCUCAGUGAAUA-3’, 5’-UCAAGUCGCUGUCUGUCUU-3’, 5’-CAAACCAUCGUAUCACUGA-3’, 5’-GAACAAUGAGUAUAUCCUA-3’) Dharmacon Cat#L-009366 On-target plus Smartpool siRNA oligos against PPP2R5C (5’-GGAUUUGCCUUACCACUAA-3’, 5’-GGAAGAUGAACCAACGUUA-3’, 5’-CAUCAGAAUUUGUGAAGAU-3’, 5’-CAGAAGUAGUCCAUAUGUU-3’) Dharmacon Cat#L-009433 On-target plus Smartpool siRNA oligos against PPP2R5D (5’-GUACAUCGACCAGAAGUUU-3’, 5’-UCCAUGGACUGAUCUAUAA-3’, 5’-UGACUGAGCCGGUAAUUGU-3’, 5’-GUAGGCAGAUCAACCACAU-3’) Dharmacon Cat#L-009799 On-target plus Smartpool siRNA oligos against PPP2R5E (5’-UUAAUGAACUGGUGGACUA-3’, 5’-GCACAGCUGGCAUAUUGUA-3’, 5’-GACACGCUAUCUGAUCUUA-3’, 5’-GGAUAAAGUAGACGGAUUU-3’) Dharmacon Cat#L-008531 Recombinant DNA Plasmid: MPS1 N-terminal GFP CRISPR This paper N/A Plasmid: AAVS1CRISPR_inducible miRFKBP5_Mis12-GFP-FKBP3:mCherryFRB-INCENP 47-918 This paper, adapted from plasmids pERB109 and pMK232 (Addgene #28280 and #72834) Ballister et al.30; Natsume et al.36 N/A Plasmids: pcDNA5-mCherry-BUBR1 WT, LI/AA Espert et al.24 N/A Plasmids: pcDNA5-GFP-MPS1 WT, KD, N8A, N8E This paper and Hayward et al.10 N/A Plasmids: pcDNA5-HEC1-mCherryWT, 9A, 9D This paper N/A Plasmid: pcDNA5-GFP-B56 Delta This paper N/A Software and algorithms FIJI distribution of ImageJ v1.52q-v1.53s NIH image; Schindelin et al.37 https://imagej.nih.gov/ij/; RRID: SCR_003070 R v4.0.0 R Core Team http://www.r-project.org; RRID: SCR_001905 RStudio v1.3.959 RStudio Team38 http://www.rstudio.com; RRID: SCR_000432 (Continued on next page) ll OPEN ACCESS Current Biology 32, 5200–5208.e1–e8, December 5, 2022 e3 Report

Techniques: Binding Assay, Marker

Journal: Current biology : CB

Article Title: MPS1 localizes to end-on microtubule-attached kinetochores to promote microtubule release.

doi: 10.1016/j.cub.2022.10.047

Figure Lengend Snippet: Figure 3. Aurora B-dependent localization of MPS1 to kinetochores can be uncoupled from the microtubule-attachment status (A) Diagram of the rapamycin-dependent dimerization module used to recruit INCENP-Aurora B to the kinetochore protein Mis12. (B) HeLa MPS1-GFP cells expressing the Aurora B kinetochore targeting system were arrested with nocodazole or MG132 , then treated with rapamycin for 2 min as indicated and stained for Aurora B-pT232. (C) Plot of Aurora B-pT232 centromere and kinetochore intensities. Bars represent mean ± SEM. ****p % 0.0001. (D) Movie stills of HeLa MPS1-GFP cells expressing the Aurora B kinetochore targeting system treated with MG132. Cell at 1 min is at metaphase prior to the addition of rapamycin. Tubulin and DNA were visualized with SiR-Tubulin and Hoechst.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER siRNA against HEC1 5’UTR (5’-CCCUGGGUCGUGUCAGGAA-3’) Nijenhuis et al.12 N/A siRNA against BUBR1 3’UTR (5’-GCAATCAAGTCTCACAGAT-3’) Espert et al.24 N/A On-target plus Smartpool siRNA oligos against PPP2R5A (5’-GCUCAAAGAUGCCACUUCA-3’, 5’-CAAUACAAGUGCCGAAUAA-3’, 5’-UGAAUGAACUGGUUGAGUA-3’, 5’-GGAAAUGAAUGGCAAGCUU-3’) Dharmacon Cat#L-009352 On-target plus Smartpool siRNA oligos against PPP2R5B (5’-CGCAUGAUCUCAGUGAAUA-3’, 5’-UCAAGUCGCUGUCUGUCUU-3’, 5’-CAAACCAUCGUAUCACUGA-3’, 5’-GAACAAUGAGUAUAUCCUA-3’) Dharmacon Cat#L-009366 On-target plus Smartpool siRNA oligos against PPP2R5C (5’-GGAUUUGCCUUACCACUAA-3’, 5’-GGAAGAUGAACCAACGUUA-3’, 5’-CAUCAGAAUUUGUGAAGAU-3’, 5’-CAGAAGUAGUCCAUAUGUU-3’) Dharmacon Cat#L-009433 On-target plus Smartpool siRNA oligos against PPP2R5D (5’-GUACAUCGACCAGAAGUUU-3’, 5’-UCCAUGGACUGAUCUAUAA-3’, 5’-UGACUGAGCCGGUAAUUGU-3’, 5’-GUAGGCAGAUCAACCACAU-3’) Dharmacon Cat#L-009799 On-target plus Smartpool siRNA oligos against PPP2R5E (5’-UUAAUGAACUGGUGGACUA-3’, 5’-GCACAGCUGGCAUAUUGUA-3’, 5’-GACACGCUAUCUGAUCUUA-3’, 5’-GGAUAAAGUAGACGGAUUU-3’) Dharmacon Cat#L-008531 Recombinant DNA Plasmid: MPS1 N-terminal GFP CRISPR This paper N/A Plasmid: AAVS1CRISPR_inducible miRFKBP5_Mis12-GFP-FKBP3:mCherryFRB-INCENP 47-918 This paper, adapted from plasmids pERB109 and pMK232 (Addgene #28280 and #72834) Ballister et al.30; Natsume et al.36 N/A Plasmids: pcDNA5-mCherry-BUBR1 WT, LI/AA Espert et al.24 N/A Plasmids: pcDNA5-GFP-MPS1 WT, KD, N8A, N8E This paper and Hayward et al.10 N/A Plasmids: pcDNA5-HEC1-mCherryWT, 9A, 9D This paper N/A Plasmid: pcDNA5-GFP-B56 Delta This paper N/A Software and algorithms FIJI distribution of ImageJ v1.52q-v1.53s NIH image; Schindelin et al.37 https://imagej.nih.gov/ij/; RRID: SCR_003070 R v4.0.0 R Core Team http://www.r-project.org; RRID: SCR_001905 RStudio v1.3.959 RStudio Team38 http://www.rstudio.com; RRID: SCR_000432 (Continued on next page) ll OPEN ACCESS Current Biology 32, 5200–5208.e1–e8, December 5, 2022 e3 Report

Techniques: Expressing, Staining

Journal: Nature cell biology

Article Title: Dynamic Kinetochore Size Regulation Promotes Microtubule Capture and Chromosome Biorientation in Mitosis

doi: 10.1038/s41556-018-0130-3

Figure Lengend Snippet: (a) Immunofluorescence images of ZW10 and HEC1 in HeLa cells treated with nocodazole or STLC. (A, Attached; U, Unattached). The experiment was repeated at least three times with similar results. (b) Overview of the secondary structure of human Spindly with predicted coiled-coils (grey bars) and disordered regions, and with sequence logos of four conserved motifs. See also . (c) Quantification of the kinetochore levels of p150 Glued in nocodazole-treated HeLa cells transfected with siRNAs to Spindly and expressing the indicated GFP-Spindly variants. The graph in shows the mean kinetochore intensity (±SD) normalized to the values of Spindly FL . Each dot represents one cell: FL ( n = 206 cells), ΔCC2 ( n = 200 cells), ΔSB ( n = 192 cells), ΔCCS ( n = 194 cells) pooled from seven independent experiments. ΔCC1 ( n = 122 cells) pooled from four independent experiments. Asterisks indicate significance (one-way ANOVA followed by Tukey's test; F (4, 909) = 238.5). ****P < 0.001. Representative images of cells are shown in . (e,f) Representative images (e) and quantification (f) of metaphase HeLa cells transfected with siRNA to Spindly and expressing the indicated GFP-Spindly variants. The graph shows the mean fold change in kinetochore intensity (±SD) normalized to the values of Spindly FL . Each dot represents one cell: FL ( n = 139 cells), ΔCC2 ( n = 136 cells), ΔSB ( n = 141 cells), ΔCCS ( n = 165 cells) pooled from five independent experiments. ΔCC1 ( n = 97 cells) pooled from four independent experiments. Asterisks indicate significance (one-way ANOVA followed by Tukey's test; F (4, 673) = 362.5). ****P < 0.001. Representative images of all the mutants are shown in . (g) Representative immunofluorescence images of HeLa cells transfected with siRNA to Spindly and expressing the indicated GFP-Spindly variants, and immuno-stained for the indicated antigens. The experiment was repeated at least three times with similar results. See siSpindly control in . (h) Representative images of the morphology of Spindly and CENP-C in metaphase kinetochores expressing the indicated GFP-Spindly variants. Stripping activity is based on ability of Spindly variants to recruit dynein/dynactin. The experiment was repeated at least three times with similar results.

Article Snippet: To generate stably expressing mCherry-Tubulin and DARPIn GFP -mCherry-MPS1 Δ200 Hela FlpIn cell lines, pmCherry-α-tubulin-IRES-puro2 (Addgene #21043) or pCDNA4-DARPIn GFP -mCherry-Mps1 Δ200 variants were transfected and selected in Puromycin (Sigma, P7255) for three weeks.

Techniques: Immunofluorescence, Sequencing, Transfection, Expressing, Staining, Control, Stripping Membranes, Activity Assay

Journal: Nature cell biology

Article Title: Dynamic Kinetochore Size Regulation Promotes Microtubule Capture and Chromosome Biorientation in Mitosis

doi: 10.1038/s41556-018-0130-3

Figure Lengend Snippet: (a) Timeline of the treatments with kinase inhibitors and nocodazole (Noco) of the experiments shown in b-f. (b) Representative images of ZW10 immunostainings of cells treated as indicated in (a). The intensity levels of the zoom ins (on right) were equalized to facilitate the direct comparison of the size of the kinetochores. The experiment was repeated at least three times with similar results. (c,d) Representative images (c) and quantification (d) of kinetochore localization of the indicated GFP-Spindly variants in HeLa cells treated with nocodazole and Cpd-5 as indicated in (a). The graph shows the mean kinetochore intensity (±SD) normalized to the values of Spindly FL . Each dot represents one cell: FL in DMSO ( n = 87 cells), FL in Cpd-5 ( n = 92 cells), ΔN in DMSO ( n = 87 cells), and ΔN in Cpd-5 ( n = 93 cells), pooled from three independent experiments. Asterisks indicate significance (one-way ANOVA followed by Tukey's test; F (3, 355) = 255.6). ****P < 0.001. (e,f) Representative images (e) and volume quantification (f) of immunostained kinetochores of HeLa cells expressing the indicated versions of GFP-Spindly and treated as indicated in (a). In (e), imaging acquisition was set to obtain similar mean intensity levels for the different conditions. The graph shows the mean kinetochore volume (±SD). Each dot represents a pair of sister kinetochores: FL in DMSO ( n = 37 kinetochores), FL in Cpd-5 ( n = 48 kinetochores), ΔN in DMSO ( n = 43 kinetochores), and ΔN in Cpd-5 ( n = 60 kinetochores), pooled from three independent experiments. Asterisks indicate significance (one-way ANOVA followed by Tukey's test; F (3, 194) = 68.18). ****P < 0.001. (g) Representative images of HeLa cells overexpressing GFP-Spindly FL and an active (WT) or kinase dead (KD) version of mCherry-MPS1 Δ200 targeted to GFP-Spindly by DARPin αGFP . The experiment was repeated two times with similar results.

Article Snippet: To generate stably expressing mCherry-Tubulin and DARPIn GFP -mCherry-MPS1 Δ200 Hela FlpIn cell lines, pmCherry-α-tubulin-IRES-puro2 (Addgene #21043) or pCDNA4-DARPIn GFP -mCherry-Mps1 Δ200 variants were transfected and selected in Puromycin (Sigma, P7255) for three weeks.

Techniques: Comparison, Expressing, Imaging