Journal: bioRxiv

Article Title: Kinetochores respond to subtle changes in the stability of microtubule attachments

doi: 10.1101/2021.02.19.432040

Figure Lengend Snippet: A) Quantification of HURP fluorescence intensity. Whole spindle measurements were made on sum intensity projections of fluorescence z-stacks. U2OS cells treated for 3 hours with DMSO (circles), Taxol (squares), UMK57 (triangles), or UMK57+Taxol (diamonds). Error bars indicate SEM; n = 25 spindles per condition; *p ≤ 0.05; n.s., p ≥ 0.05 using two-tailed t test. B) Representative images of U2OS cells treated for 3 hours with DMSO or UMK57 and then fixed and stained for DNA (blue) or HURP (green). Images represent maximum intensity projections of fluorescence z-stacks. Scale bar, 10 μm. C) Quantification of Ska3 fluorescence intensity relative to ACA. U2OS cells treated for 3 hours with DMSO (circles), Taxol (squares), or UMK57 (triangles). Error bars indicate SEM; n = 100 kinetochores per condition; n.s., p ≥ 0.05 using two-tailed t test. D) Representative images of U2OS cells treated for 3 hours with DMSO, Taxol, or UMK57 and then fixed and stained for DNA (blue), Ska3 (green), and ACA (magenta). Images represent single slices of fluorescence z-stacks. Scale bar, 10 μm. E) A proposed model for the regulation of k-MT attachment stability. Aurora B influences kinetochore substrates to determine k-MT stability. There is feedback from k-MT attachment sites in response to k-MT stabilization or destabilization. This feedback may influence kinetochore protein composition directly or indirectly (through the modulation of Aurora B activity).

Article Snippet: The following primary antibodies were used for immunofluorescence: ACA (anti-centromere antibody; Geisel School of Medicine; 1:1000), Astrin (C-terminal; 1:1000) , CENP-A (#2186; Cell Signaling; 1:500), Hec1 (C-11; Santa Cruz; 8 ug/mL), HURP (gift from Erich Nigg; 1:1000) , Mad2 (PRB-452C; Covance; 1:500), pHec1 S44 (gift from Jennifer DeLuca; 1:500), Ska3 (gift from Erich Nigg; 1:1000) ( ).

Techniques: Fluorescence, Two Tailed Test, Staining, Activity Assay

Journal: Cell Death and Differentiation

Article Title: MARCH5-dependent degradation of MCL1/NOXA complexes defines susceptibility to antimitotic drug treatment

doi: 10.1038/s41418-020-0503-6

Figure Lengend Snippet: a Parental HeLaS3 and two HeLaS3 MARCH5-KO bulks generated with two different small guide RNAs were treated with paclitaxel (PTX), nocodazole (Noc), or BI2536 (BI) immediately followed by live cell imaging. Addition of propidium iodide to the medium allowed the automated assessment of dead cells using the IncuCyte software. A nonlinear regression curve of the percentage of dead cells in relation to the total number of cells present at the beginning of the imaging of four independent experiments is shown. The mean and s.d. of exemplary data points are also shown. b Parental HeLaS3 and two independent HeLaS3 MARCH5-KO clones were either left asynchronous (Asy) or synchronized by double thymidine block, released into paclitaxel and harvested at the respective time points. Samples were then prepared for immunoblot analysis. For caspase-3 the full length (fl) and cleaved (cl) form is shown. c Same as in a). Three independent experiments with U2OS cells and two U2OS MARCH5-KO bulks generated with two different small guide RNAs. d Same as in b) with U2OS cells and two U2OS MARCH5-KO bulks generated with two different small guide RNAs. e Same as in a). Three independent experiments with A549 cells and two A549 MARCH5-KO bulks generated with two different small guide RNAs. f Same as in b) with A549 cells and two A549 MARCH5-KO bulks generated with two different small guide RNAs.

Article Snippet: HeLaS3 (ATCC® CCL-2.2, gift from Erich Nigg), A549 (ATCC® CCL-185), and U2OS (ATCC® HTB-96) cells were grown in DMEM (Sigma-Aldrich, St Louis, MO, USA, D5671) supplemented with 10% FCS (Invitrogen, Waltham, MA, USA, 10270106), 1% L-Glutamine (Sigma-Aldrich, G7513), 100 U/ml Penicillin, and 100 μg/ml Streptomycin (Lonza, Basel, Switzerland, 17-602E).

Techniques: Generated, Live Cell Imaging, Software, Imaging, Clone Assay, Blocking Assay, Western Blot

Journal: Cell Death and Differentiation

Article Title: MARCH5-dependent degradation of MCL1/NOXA complexes defines susceptibility to antimitotic drug treatment

doi: 10.1038/s41418-020-0503-6

Figure Lengend Snippet: a Parental HeLaS3 and HeLa BAX/BAK knockout cells were transfected with control siRNA (GL2) or MARCH5 siRNA. Cells were left asynchronous (Asy) or synchronized by a double thymidine block, released into paclitaxel and harvested at the indicated times. Samples were then prepared for immunoblot analysis. b Parental HeLaS3 cells and two independent MARCH5-KO clones were left asynchronous (Asy) or synchronized by double thymidine block, released into paclitaxel together with DMSO or Q-VD and harvested at the indicated time points.

Article Snippet: HeLaS3 (ATCC® CCL-2.2, gift from Erich Nigg), A549 (ATCC® CCL-185), and U2OS (ATCC® HTB-96) cells were grown in DMEM (Sigma-Aldrich, St Louis, MO, USA, D5671) supplemented with 10% FCS (Invitrogen, Waltham, MA, USA, 10270106), 1% L-Glutamine (Sigma-Aldrich, G7513), 100 U/ml Penicillin, and 100 μg/ml Streptomycin (Lonza, Basel, Switzerland, 17-602E).

Techniques: Knock-Out, Transfection, Control, Blocking Assay, Western Blot, Clone Assay

Journal: Cell Death and Differentiation

Article Title: MARCH5-dependent degradation of MCL1/NOXA complexes defines susceptibility to antimitotic drug treatment

doi: 10.1038/s41418-020-0503-6

Figure Lengend Snippet: a Parental HeLaS3 cells and two independent MARCH5-KO clones were either left asynchronous (Asy) or synchronized by a double thymidine block and released into paclitaxel. Once the mitotic index reached about 30%, mitotic cells were shaken off and re-plated in the same medium in a fresh dish. 10 h later another shake off was performed and all detached cells were harvested as “Arrested”. Adherent cells were trypsinized and harvested as “Slipped”. Numbers below the blots show the quantification of the respective bands. Quantification was normalized to the GAPDH signal and to the untreated sample (Asy) of the parental HeLa cells. b U2OS cells were transfected with a control siRNA (GL2) or a siRNA targeting MARCH5. Cells were otherwise treated like in a). Quantification was normalized to the GAPDH signal and to the untreated sample (Asy) of the U2OS cells transfected with the control siRNA (GL2). c Analysis of the percentage of U2OS cells that died in interphase after slipping out of mitotic arrest. U2OS cells were transfected with the indicated siRNAs, synchronized with a single thymidine block, released into paclitaxel and followed by live cell imaging for 72 h. 100 cells entering mitotic arrest were assessed, but only cells that slipped were taken into account for this analysis. All data displayed are mean ± s.d. of five (GL2, siMARCH5), four (siNOXA + siMARCH5), or three (siNOXA, siBIM, siBIM + siMARCH5) independent experiments, indicated as dots. One-way ANOVA, followed by the Holm–Sidak’s multiple comparisons test was performed with p < 0.05 considered as statistically significant.

Article Snippet: HeLaS3 (ATCC® CCL-2.2, gift from Erich Nigg), A549 (ATCC® CCL-185), and U2OS (ATCC® HTB-96) cells were grown in DMEM (Sigma-Aldrich, St Louis, MO, USA, D5671) supplemented with 10% FCS (Invitrogen, Waltham, MA, USA, 10270106), 1% L-Glutamine (Sigma-Aldrich, G7513), 100 U/ml Penicillin, and 100 μg/ml Streptomycin (Lonza, Basel, Switzerland, 17-602E).

Techniques: Clone Assay, Blocking Assay, Transfection, Control, Live Cell Imaging

Journal: Cell Death and Differentiation

Article Title: MARCH5-dependent degradation of MCL1/NOXA complexes defines susceptibility to antimitotic drug treatment

doi: 10.1038/s41418-020-0503-6

Figure Lengend Snippet: a Parental HeLaS3 (WT) and two clonal HeLaS3 MARCH5-KO lines created with two different small guide RNAs targeting MARCH5 (MARCH5-KO#1 and -KO#2) were treated with ABT737, etoposide (Eto), or staurosporine (STS) for 24 h. For the UV-R treatment cells were subjected to 2 mJ of UV-R and then incubated for 24 h. Cells were then harvested and propidium iodide uptake was used to measure cell death by flow cytometry. All data displayed are mean ± s.d. from five independent experiments, indicated as dots. Two-way ANOVA was used with Holm–Sidak’s multiple comparisons test to compare the WT as the control group against the two MARCH-KO lines for each treatment. All comparisons not indicated are statistically not significant ( p > 0.05). b Parental HeLaS3 and HeLaS3 MARCH5-KO cells were transfected with either control siRNA targeting luciferase (GL2), NOXA and GL2 siRNA (siNOXA), MCL1 and GL2 siRNA (siMCL1) or NOXA and MCL1 siRNA (DKD) for 48 h. Cells were then harvested and prepared for immunoblot analysis. The full length (fl) and the cleaved (cl) form of caspase-3 are shown. c Parental HeLaS3 and two independent HeLaS3 MARCH5-KO clones were treated with Cycloheximide (CHX), harvested after the indicated time points and prepared for immunoblot analysis. Numbers below the blots show the quantification of the respective bands. Quantification was normalized to the GAPDH signal and to the untreated sample (0) of the respective genotype.

Article Snippet: HeLaS3 (ATCC® CCL-2.2, gift from Erich Nigg), A549 (ATCC® CCL-185), and U2OS (ATCC® HTB-96) cells were grown in DMEM (Sigma-Aldrich, St Louis, MO, USA, D5671) supplemented with 10% FCS (Invitrogen, Waltham, MA, USA, 10270106), 1% L-Glutamine (Sigma-Aldrich, G7513), 100 U/ml Penicillin, and 100 μg/ml Streptomycin (Lonza, Basel, Switzerland, 17-602E).

Techniques: Incubation, Flow Cytometry, Control, Transfection, Luciferase, Western Blot, Clone Assay

Journal: Cell Death and Differentiation

Article Title: MARCH5-dependent degradation of MCL1/NOXA complexes defines susceptibility to antimitotic drug treatment

doi: 10.1038/s41418-020-0503-6

Figure Lengend Snippet: a Parental HeLaS3 and two independent HeLaS3 MARCH5-KO clones were either left asynchronous (Asy) or synchronized by double thymidine block and released into paclitaxel. Once cells were mitotic (M), they were treated with Cycloheximide (CHX) or solvent control (DMSO) for the indicated times in minutes. Numbers below the blots show the quantification of the respective bands. Quantification was normalized to the GAPDH signal and to the early mitotic arrest sample without CHX (M) of the respective genotype. b Same as in a) with U2OS cells and two U2OS MARCH5-KO bulks generated with two different guide RNAs. For NOXA a short and a long exposure are shown. c Same as in a) with A549 cells and two A549 MARCH5-KO bulks generaed with two different guide RNAs.

Article Snippet: HeLaS3 (ATCC® CCL-2.2, gift from Erich Nigg), A549 (ATCC® CCL-185), and U2OS (ATCC® HTB-96) cells were grown in DMEM (Sigma-Aldrich, St Louis, MO, USA, D5671) supplemented with 10% FCS (Invitrogen, Waltham, MA, USA, 10270106), 1% L-Glutamine (Sigma-Aldrich, G7513), 100 U/ml Penicillin, and 100 μg/ml Streptomycin (Lonza, Basel, Switzerland, 17-602E).

Techniques: Clone Assay, Blocking Assay, Solvent, Control, Generated

Journal: Oncotarget

Article Title: POPX2 is a novel LATS phosphatase that regulates the Hippo pathway

doi: 10.18632/oncotarget.26689

Figure Lengend Snippet: Lysates from HEK293 cells expressing GST-vector or GST-POPX2 together with ( A ) Flag-NDR1, ( B ) Myc-MOB1, ( C ) Flag-MST1, ( D ) Flag-LATS1, ( E ) Flag-YAP, and ( F ) Flag-TAZ were subjected to immunoprecipitation with glutathione sepharose beads and western analysis. Proteins associated with GST-POPX2 were detected by Western blotting (WB) with either anti-Flag or anti-Myc antibodies.

Article Snippet: The following plasmids were from Addgene: pPS2977-NDR1 (from Pamela Silver, Addgene plasmid # 8927), pClneoMyc human MOB1 (from Yutaka Hata, Addgene plasmid # 37024), pcDNA3 Lats1 (Nigg HS189) (from Erich Nigg, Addgene plasmid # 41156, 3XFlag pCMV5-TOPO TAZ WT (from Jeff Wrana, Addgene plasmid # 24809).

Techniques: Expressing, Plasmid Preparation, Immunoprecipitation, Western Blot

Journal: Oncotarget

Article Title: POPX2 is a novel LATS phosphatase that regulates the Hippo pathway

doi: 10.18632/oncotarget.26689

Figure Lengend Snippet: ( A ) Endogenous MST1 from HEK293 cells was immunoprecipitated. The IP complexes were separated by SDS PAGE, followed by Western analysis using POPX2 antibody. The Western blot reveals endogenous MST1-POPX2 interaction. ( B ) Similar experiment as in (A) was performed using LATS1 antibody for immunoprecipitation. The Western blot reveals endogenous LATS1-POPX2 interaction. ( C ) Reciprocal endogenous POPX2 immunoprecipitation followed by Western analysis using MST1 and LATS1 antibodies reveal endogenous POPX2-MST1 and POPX2-LATS1 interactions. Endogenous immunoprecipitation experiments are representative of 2 independent experiments; representative images were shown.

Article Snippet: The following plasmids were from Addgene: pPS2977-NDR1 (from Pamela Silver, Addgene plasmid # 8927), pClneoMyc human MOB1 (from Yutaka Hata, Addgene plasmid # 37024), pcDNA3 Lats1 (Nigg HS189) (from Erich Nigg, Addgene plasmid # 41156, 3XFlag pCMV5-TOPO TAZ WT (from Jeff Wrana, Addgene plasmid # 24809).

Techniques: Immunoprecipitation, SDS Page, Western Blot

Journal: Oncotarget

Article Title: POPX2 is a novel LATS phosphatase that regulates the Hippo pathway

doi: 10.18632/oncotarget.26689

Figure Lengend Snippet: ( A ) In vitro phosphatase assay. Bacterially expressed POPX2 (4 µg and 8 µg) was incubated with phospho-Flag-MST1 for 45 mins at 30°C. The reaction mixtures were subjected to SDS-PAGE and Western analysis using phospho-MST1-Thr183 antibodies. Calf-intestinal phosphatase (CIP) was used as a negative control. ( B ) HEK293 cells were transfected with GFP-POPX2 or GFP-Flag vector constructs. The cells were treated with okadaic acid (1 µM) for 1 hr to inhibit PP2A and induce MST1 autophosphorylation. Cell lysates were separated on SDS-PAGE and subjected to Western analysis to detect phospho-MST1-Thr183. ( C ) In vitro phosphatase assay. Bacterially expressed POPX2 was incubated with phospho-Flag-LATS1 instead of Flag-MST1 as in (A). The reaction mixtures were subjected to SDS-PAGE and Western analysis using phospho-LATS1-Thr1079 antibodies. Calf-intestinal phosphatase (CIP) was used as a negative control. CIP can remove phosphate group from phospho-tyrosine, serine and threonine, but with a preference for phospho-tyrosine. In our experiment, CIP was used as a negative control because when CIP was used in similar experimental condition as POPX2 for phosphatase assays, it was found not as efficient as POXP2 in dephosphorylating LATS1. ( D ) Densitometry quantification of (C) from three independent experiments. POPX2 dephosphorylates LATS1 in a dose dependent manner. ( E ) HEK293 cells were transfected with Flag-LATS1 + Myc-MOB1 together with GST-vector, GST-PP1, GST-PP2A or GST-POPX2. Cell lysates were harvested and separated by SDS-PAGE and subjected to Western analysis. Equal amounts of total protein lysates were loaded into each well of the gel. Total LATS1 (left panel) and pLATS1 (right panel) were probed on separate membranes. ( F ) Densitometry quantification of phospho-LATS1. Three independent experiments were performed. Error bars represent standard deviation. Student t -test was performed to determine statistical significances.

Article Snippet: The following plasmids were from Addgene: pPS2977-NDR1 (from Pamela Silver, Addgene plasmid # 8927), pClneoMyc human MOB1 (from Yutaka Hata, Addgene plasmid # 37024), pcDNA3 Lats1 (Nigg HS189) (from Erich Nigg, Addgene plasmid # 41156, 3XFlag pCMV5-TOPO TAZ WT (from Jeff Wrana, Addgene plasmid # 24809).

Techniques: In Vitro, Phosphatase Assay, Incubation, SDS Page, Western Blot, Negative Control, Transfection, Plasmid Preparation, Construct, Standard Deviation

Journal: Oncotarget

Article Title: POPX2 is a novel LATS phosphatase that regulates the Hippo pathway

doi: 10.18632/oncotarget.26689

Figure Lengend Snippet: ( A ) High levels of POPX2 lead to dephosphorylation of LATS1 on Thr1079 and inactivation of LATS1 kinase activity. Absence of LATS1 activity prevents YAP/TAZ degradation and promotes YAP/TAZ nuclear translocation. Active YAP/TAZ will then promote transcription of target genes. ( B ) When POPX2 is absent, LATS1 is phosphorylated and activated. Active LATS1 is capable of phosphorylating YAP/TAZ, which are then targeted for degradation by proteosomes.

Article Snippet: The following plasmids were from Addgene: pPS2977-NDR1 (from Pamela Silver, Addgene plasmid # 8927), pClneoMyc human MOB1 (from Yutaka Hata, Addgene plasmid # 37024), pcDNA3 Lats1 (Nigg HS189) (from Erich Nigg, Addgene plasmid # 41156, 3XFlag pCMV5-TOPO TAZ WT (from Jeff Wrana, Addgene plasmid # 24809).

Techniques: De-Phosphorylation Assay, Activity Assay, Translocation Assay