rabbit anti mouse p21  (Proteintech)

Journal: bioRxiv

Article Title: Transient lagging chromosomes cause primary microcephaly

doi: 10.1101/2024.05.02.592199

Figure Lengend Snippet: ( A ) Time- lapse single-plane images RPE1 cells stained with SiR-Tubulin (red) and SpY-DNA (white) showing examples of transient or persistent lagging chromosomes (green arrows). Scale bars = 5 µm. ( B ) Quantification of transient lagging chromosomes in siWDR62- treated RPE1; n = 46-57 cells, * p= 0.0368 in Chi-square test. ( C ) Immunofluorescence images of interphase RPE1 cells treated with siCTRL , siWDR62 or Doxorubicin, and stained with DAPI, 53BP1 and p21 antibodies. Blue dotted lines represent 53BP1-negative nuclei, red dotted lines represent 53BP1-positive nuclei. Scale bars = 10 µm. ( D ) Quantification of 53BP1 positive cells. Connected dots represent paired experiment. N = 4 independent experiments, n = 648 – 1440 cells; ****, p < 0.0001 in Fisher’s exact test ( E ) Quantification of p21 positive cells. Connected dots represent paired experiment. Blue dotted lines represent p21-negative nuclei, red dotted lines represent p21-positive nuclei. N = 6, n = 570-1010 cells; ****, p < 0.0001 in Fisher’s exact test; ( F ) Example of FACS CFSE analysis data ( G ) Quantification of cell divisions after 5 days beyond serum starvation based on CFSE intensity, N = 4, n = 18194 - 82963 cells; **, p = 0.0029, in one-way ANOVA. Error bars represent SEM.

Article Snippet: The following primary antibodies were used: recombinant human anti-α-tubulin (1:500; ( )), rabbit anti-CAMSAP1(1:1500; Novus Biologicals NBP1-26645), rabbit anti-WDR62 (1:1000; Bethyl A301- 560A), rabbit anti-MCAK (1:1000; ( )), rabbit anti-53BP1 (1:1000; Cell Signalling Technology 4937), mouse anti-p21 (1:1000; Cell Signalling Technology 2947), mouse anti-yH2Ax (1:2000; EMD Millipore, 05-636), mouse anti-AuroraB (1:2000; BD Biosciences 611083), rabbit anti-NPL4(1:500; Novus Biologicals NBP1-82166), rabbit anti-UBASH3B (1:500; Proteintech 19563-1-AP).

Techniques: Staining, Immunofluorescence

Journal: bioRxiv

Article Title: Transient lagging chromosomes cause primary microcephaly

doi: 10.1101/2024.05.02.592199

Figure Lengend Snippet: ( A ) Immunofluorescence images of metaphase RPE1 cells treated with siCTRL and siWDR62 and stained with DAPI and WDR62 antibodies. Scale bars = 5 µm. ( B ) Quantification of WDR62 levels in RPE1 cells treated with siCTRL or siWDR62 cells normalized to siCTRL values. N = 4 independent experiments, n = 49-81 cells, ****, p < 0.0001, unpaired t-test. Error bars represent SEM. ( C ) Live stills of GFP-centrin1/GFP-CENP-A RPE1 anaphase cells treated with siCTRL or siWDR62 . White arrow indicates transient lagging chromosome in siWDR62 -treated cell. ( D ) Quantification of GFP- Centrin1/GFP-CENP-A RPE1 cells with transient lagging chromosomes in anaphase after siCTRL or siWDR62 treatment. n = 29-48 cells; * p = 0.0317 in Fischer’s exact test. Note that the data represented here are a duplicate of the quantification in . ( E ) Schematic representation of automated 53BP1 (upper panel) and p21/yH2AX analysis (lower panel). Nuclei were segmented using DAPI staining, and, for 53BP1, foci were counted as positive if the spots displayed an intensity than was higher than the nuclear mean plus 2 standard deviations; for p21/yH2AX the intensity of nuclei in corresponding channel were measured and counted as positive if their intensity was higher than the mean plus one standard deviation in siCTRL -treated cells. ( F ) Immunofluorescence images of interphase RPE1 cells, treated with siCTRL , siWDR62 or 100 nM Doxorubicin, stained with DAPI and ψH2AX antibody. Blue dotted circles represent ψH2AX-negative nuclei, red ones ψH2AX-positive nuclei. Scale bars = 10 µm. ( G ) Quantification of yH2AX positive cells after siCTRL, siWDR62 or Doxorubicin treatment. N = 6, n = 834 – 2234 cells; siCTRL vs. Doxorubicin, ****, p < 0.0001, Fisher’s exact test. ( H ) Schematic representation the Carboxyfluorescein succinimidyl ester (CFSE) assay: CFSE is a cell-permeable dye that covalently couples to intramolecular molecules via its succinimidyl group; since after a CFSE pulse its intensity decreases by half after each cell division, it can be used as a pulse-chase assay to quantify cell proliferation. In all our assays we compared the indicated siRNA- treatments to serum starvation. To calculate the number of cell divisions above serum starvation we determined by FACS the peak intensity of a serum-starved cells (A) and divided it by the peak intensity of the condition of interest (X). The log2 of (A/X) indicates the number of additional divisions cells have undergone in comparison to serum-starvation.

Article Snippet: The following primary antibodies were used: recombinant human anti-α-tubulin (1:500; ( )), rabbit anti-CAMSAP1(1:1500; Novus Biologicals NBP1-26645), rabbit anti-WDR62 (1:1000; Bethyl A301- 560A), rabbit anti-MCAK (1:1000; ( )), rabbit anti-53BP1 (1:1000; Cell Signalling Technology 4937), mouse anti-p21 (1:1000; Cell Signalling Technology 2947), mouse anti-yH2Ax (1:2000; EMD Millipore, 05-636), mouse anti-AuroraB (1:2000; BD Biosciences 611083), rabbit anti-NPL4(1:500; Novus Biologicals NBP1-82166), rabbit anti-UBASH3B (1:500; Proteintech 19563-1-AP).

Techniques: Immunofluorescence, Staining, Standard Deviation, CFSE Assay, Pulse Chase, Comparison

Journal: bioRxiv

Article Title: Transient lagging chromosomes cause primary microcephaly

doi: 10.1101/2024.05.02.592199

Figure Lengend Snippet: ( A ) Immunofluorescence images of interphase RPE1 cells treated with siCTRL , siCAMSAP1 , siWDR62 , siCAMSAP1+siWDR62 and Doxorubicin, stained with DAPI, 53BP1 and p21 antibodies. Blue dotted circles represent 53BP1- negative nuclei, red ones 53BP1-positive nuclei. Scale bars = 10 µm. ( B ) Quantification of 53BP1 positive cells after indicated siRNA treatments. Connected dots represent paired experiment. N = 4, n = 648 – 1440 cells; siCTRL vs. siWDR62 , ***, p = 0.0002; siCTRL vs. siCAMSAP1 , *, p = 0.0127; siWDR62 vs. siCAMSAP1 + siWDR62, *** p = 0.0006; Fisher’s exact test. ( C ) Quantification of p21 positive cells in indicated siRNA treatments. Connected dots represent paired experiment. Blue dotted circles represent p21-negative nuclei, red one p21-positive nuclei. N = 5, n = 1020 – 1330 cells; ****, p < 0.0001; Fisher’s exact test. ( D ) Quantification of cell divisions above serum starvation in CFSE assay in indicated siRNAs, N = 4, n = 18194-82963 cells; *, siCTRL vs. siWDR62, p = 0.0224: siWDR62 vs. siCAMSAP1 + WDR62 , p = 0.0319 RM (repeated measurements) mixed effect analysis; error bars represent SEM. ( E ) Quantification of persistence of transient lagging chromosomes in siCAMSAP1 and siWDR62-treated RPE1 GFP-CenpA/GFP-Centrin1 cells recorded by fluorescent time lapse microscopy, N = 4, n =43 - 59 cells; ** p = 0.0011, Wilcoxon test. ( F ) Time of 53BP1 appearance in siCAMSAP1 and siWDR62 -treated RPE1 GRP-53BP1 live cells having experienced a lagging chromosome, N = 8, n =15-19 cells; **, p = 0.0062, Mann-Whitney test.

Article Snippet: The following primary antibodies were used: recombinant human anti-α-tubulin (1:500; ( )), rabbit anti-CAMSAP1(1:1500; Novus Biologicals NBP1-26645), rabbit anti-WDR62 (1:1000; Bethyl A301- 560A), rabbit anti-MCAK (1:1000; ( )), rabbit anti-53BP1 (1:1000; Cell Signalling Technology 4937), mouse anti-p21 (1:1000; Cell Signalling Technology 2947), mouse anti-yH2Ax (1:2000; EMD Millipore, 05-636), mouse anti-AuroraB (1:2000; BD Biosciences 611083), rabbit anti-NPL4(1:500; Novus Biologicals NBP1-82166), rabbit anti-UBASH3B (1:500; Proteintech 19563-1-AP).

Techniques: Immunofluorescence, Staining, CFSE Assay, Time-lapse Microscopy, MANN-WHITNEY

Journal: bioRxiv

Article Title: Transient lagging chromosomes cause primary microcephaly

doi: 10.1101/2024.05.02.592199

Figure Lengend Snippet: (A) Brains extracted from D. melanogaster larvae of w 1118 , PatroninRNAi , WDR62RNAi , and PatroninRNAi + WDR62RNAi , stained with DAPI. The dotted red circle represents the area of the right lobe from w 1118 brain superposed on PatroninRNAi , WDR62RNAi , PatroninRNAi+ WDR62RNAi brains. (B) Brain lobes area quantification of D. melanogaster larvae brains extracted from w 1118 (n= 73 lobes), PatroninRNAi (n= 56), WDR62RNAi (n= 76), PatroninRNAi+ WDR6RNAi (n= 23) larvae., w 1118 vs WDR62RNAi p < 0.0001, w 1118 vs PatroninRNAi + WDR62RNAi p < 0.0001, WDR62RNAi vs PatroninRNAi + WDR62RNAi p < 0.0001, one-way Anova test. (C) Quantification of neuroblast stained with Miranda antibodies in brains extracted from w 1118 (n= 9 brains), PatroninRNAi (n= 8), WDR62RNAi (n= 15), PatroninRNAi+ WDR6RNAi (n= 10) larvae, w 1118 vs WDR62RNAi p = 0.0041, WDR62RNAi vs PatroninRNAi + WDR62RNAi p = 0.0324, one-way Anova test. (D) Extracted D. melanogaster larvae brains from w 1118 , PatroninRNAi , WDR62RNAi , PatroninRNAi+ WDR6RNAi and stained for Miranda and DAPI. The dotted white line highlights the shape of lobes. (E) Immunofluorescence from a larva brain stained for Miranda and α-tubulin. Dotted lines represent the pole-to-pole axe and the cellular axe perpendicular to the Miranda signal (spindle orientation angle). The magenta line represents the angle between the 2 axes. (F) Quantification of the spindle orientation angles (as determined in ) in neuroblasts from D. melanogaster larvae brain lobes from w 1118 (n= 48 cells), PatroninRNAi (n= 20), WDR62RNAi (n= 44), PatroninRNAi+ WDR6RNAi (n= 35) larvae, all non-significant, Kruskal-Wallis test. (G) Scheme of larvae turning behaviour assay: the number of larva contraction before each change in direction was determined (see Supplementary Fig5C for examples of tracking). (H) Quantification of the contractions before change of direction in w 1118 (n=16 larvae), PatroninRNAi (n= 12), WDR62RNAi (n= 16), Patronin RNAi+ WDR6RNAi (n= 15) larvae. w 1118 vs PatroninRNAi , w 1118 vs WDR62 RNAi p = 0.0001, WDR62 RNAi vs PatroninRNAi + WDR62RNAi p < 0.0001, Kruskal-Wallis test. (I) Scheme of larvae fructose choice assay: the proportion of larvae on the half containing fructose was counted after 5 minutes of free movements (See Supplementary Fig5D for examples of tracking). (J) Quantification of larvae number on the Fructose- containing half after 5 minutes of movements of w 1118 (n=68 larvae), PatroninRNAi (n=58), WDR62RNAi (n=54), PatroninRNAi+ WDR6RNAi (n=53) larvae. w 1118 vs PatroninRNAi , w 1118 vs WDR62RNAi P = 0.0319, WDR62RNAi vs PatroninRNAi + WDR62RNAi P = 0.0390, Fisher’s exact test. (K) Proposed model for the origin of primary microcephaly origin: We postulate that cells can can detect lagging chromosomes via the Aurora B activity gradient. If these lagging chromosomes are only briefly in contact with Aurora B, as is the case in many wild-type or more frequently in CAMSAP1- depleted cells (fast poleward microtubule flux speed), this may lead to a transient activation of 53BP1, but not an activation of p21. If these lagging chromosomes remain longer, as is the case in WDR62- depleted cells (slow poleward microtubule flux speed) this will lead to a rapid 53BP1 activation and p21 induction resulting in a cell cycle delay which can exhaust the neuroprogenitor cell pool. Re- equilibrating poleward microtubule flux rates by co-depleting CAMSAP1 and WDR62 prevents transient lagging chromosomes and rescues the primary microcephaly phenotype.

Article Snippet: The following primary antibodies were used: recombinant human anti-α-tubulin (1:500; ( )), rabbit anti-CAMSAP1(1:1500; Novus Biologicals NBP1-26645), rabbit anti-WDR62 (1:1000; Bethyl A301- 560A), rabbit anti-MCAK (1:1000; ( )), rabbit anti-53BP1 (1:1000; Cell Signalling Technology 4937), mouse anti-p21 (1:1000; Cell Signalling Technology 2947), mouse anti-yH2Ax (1:2000; EMD Millipore, 05-636), mouse anti-AuroraB (1:2000; BD Biosciences 611083), rabbit anti-NPL4(1:500; Novus Biologicals NBP1-82166), rabbit anti-UBASH3B (1:500; Proteintech 19563-1-AP).

Techniques: Staining, Immunofluorescence, Activity Assay, Activation Assay

Journal: Cell reports

Article Title: Cells use multiple mechanisms for cell-cycle arrest upon withdrawal of individual amino acids.

doi: 10.1016/j.celrep.2023.113539

Figure Lengend Snippet: Figure 4. Amino acid signaling funnels through cyclin D1, p21, and p27 to regulate cell-cycle entry (A) Schematic of key mediators involved in cell-cycle commitment. (B–E) Population average and 95% confidence interval of CDK2 activity and endogenous p21 (B and C) or endogenous cyclin D1 (D and E) in MCF10A cells grown in regular horse serum. Cells were first imaged in full-growth media for 16 h before the indicated amino acid was acutely withdrawn. In (B) and (D), cells were selected for plotting if they completed anaphase 1–2 h before amino acid withdrawal (a G1-phase withdrawal). In (C) and (E), cells were selected for plotting if they completed anaphase 10–12 h before amino acid withdrawal (a G2-phase withdrawal), marked by the gray bar. All plots contain at least 75 cells per condition. (F–H) Quantification of percentage of MCF10A cells with hyper-phosphorylated Rb (Ser807/811) in wild-type (WT), cyclin D1 overexpression (F), p21 knockout (G), or p27 small interfering RNA (siRNA) knockdown (H) conditions. Cells were grown in regular horse serum; the indicated amino acid was withdrawn for 48 h. Error bars indicate 95% confidence intervals. Statistical analyses were performed using permutation test: ****p < 0.0001. All plots contain at least 18,000 cells per condition.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit anti-ATF4 at 1:250 Cell Signaling Technology 11815 Rabbit anti-phospho-S6 (Ser240/244) at 1:250 Cell Signaling Technology 2215 Rabbit anti-Cyclin D1 (SP4) at 1:500 Lab Vision RM-9104-S0 Rabbit anti-p21 at 1:250 Cell Signaling Technology 2947 Mouse anti-p27 at 1:250 BD Biosciences 610241 Rabbit anti-phospho-Rb (Ser807/811) (D20B12) at 1:500 Cell Signaling Technology 8516 Alexa Fluor 546 anti-mouse secondary at 1:500 Thermo Fisher A-11030 Alexa Fluor 647 anti-mouse secondary at 1:500 Thermo Fisher A-21236 Alexa Flour 546 anti-rabbit secondary at 1:500 Thermo Fisher A-11035 Alexa Fluor 647 anti-rabbit secondary at 1:500 Thermo Fisher A-21245 Chemicals, Peptides, and Recombinant Proteins Hoechst Biotium 40046 Mek inhibitor Selleckchem PD-0325091 Cycloheximide Enzo Life Science ALX-380-269-G001 Chloroquine Sigma-Aldrich AAJ6445914 5-(N-Ethyl-N-isopropyl)-Amiloride Selleckchem S9849 Trimethoprim Thermo Fisher AAJ6305303 DharmaFECT 1 Dharmacon T-2001-02 DMEM/F-12 Thermo Fisher 11039047 DMEM/F-12 w/o Amino Acids, L-Glutamine, Glucose, Sodium Pyruvate USBiological D9807-11 Horse Serum Life Tech 16050–122 Dialyzed Equine Serum Valley Biomedical AS3053 EGF Peprotech AF-100-15 Hydrocortisone Sigma-Aldrich H0888 Insulin Sigma-Aldrich I1882 Cholera Toxin Sigma-Aldrich C8052 Collagen Advanced BioMatrix 5015 Critical commercial assays ViewRNATM ISH Cell Assay Kit Thermo Fisher QVC0001 CCND1 FISH probes Thermo Fisher VA6-16943 Experimental models: Cell lines MCF10A ATCC CRL-10317 RPE-hTERT ATCC CRL-4000 MCF10A H2B-mTurq DHB-mVenus DHFR-mCherry This study N/A MCF10A H2B-mTurq DHB-mVenus DHFR-mCherry-Cyclin D1 (Yang et al., 2017)32 N/A mCitrine-CCND1 MCF10A (Gookin et al., 2017)25 N/A mCitrine-CDKN1A MCF10A (Moser et al., 2018)21 N/A p21-null MCF10A (Bachman et al., 2004)36 N/A Oligonucleotides siCDKN1A IDT hs.Ri.CDKN1A.13.1 hs.Ri.CDKN1A.13.3 (Continued on next page) Cell Reports 42, 113539, December 26, 2023 11

Techniques: Activity Assay, Over Expression, Knock-Out, Small Interfering RNA, Knockdown