Fig 1: Condensin gene silencing induces increases in CIN phenotypes in hTERT cells. (A) Cumulative NA distribution frequencies following condensin gene silencing in hTERT cells relative to siControl (black). KS tests reveal reduced expression induces statistically significant changes in NA distributions, with the exception of NCAPG (p = 0.07) (N/A, not applicable; N/S, not significant (p > 0.01); ****, p < 0.0001). (B) Column graph presenting the frequency of MN formation following silencing. The fold increase in mean MN formation relative to siControl is presented above each column, while the horizontal line identifies the minimum threshold to be considered a significant increase. (C) Venn diagram presenting the results of the NA and MN formation assays performed in both HCT116 and hTERT cells. Note that SMC2, NCAPD3, and NCAPG2 silencing induced significant phenotypes in all four assay/cellular contexts. (D) Representative Western blots showing SMC2 (left), NCAPD3 (middle) and NCAPG2 (right) levels following silencing in hTERT cells with both individual and pooled siRNAs; Cyclophilin B is the loading control. Semi-quantitative image analysis was performed, and residual protein levels are presented relative to siControl (1.00). See Figure S2 for detailed information. (E) Bar graph showing the frequency of enumerable mitotic spreads versus those with severe or mild chromosome decompaction phenotypes. Note that the majority of SMC2 silenced cells present with decompaction phenotypes, including ~83% and ~11% with severe and mild phenotypes, respectively. (F) Dot plot presenting the individual chromosome numbers for each enumerable mitotic chromosome spread, with the horizontal line identifying the modal chromosome number (46) of hTERT cells. Note that only 17% of spreads generated following SMC2 silencing could be enumerated due to the prevalent severe decompaction phenotype. KS tests reveal significant differences in the distribution of chromosome numbers relative to siControl (N/A, not applicable; ****, p < 0.0001). (G) Bar graph depicting increases in the overall frequency of aberrant chromosome numbers relative to siControl, which includes small-scale losses, small-scale gains and large-scale gains. Note that only 17/100 mitotic chromosome spreads could be enumerated following SMC2 silencing, 14 of which, exhibited numerical deviations from the modal number.
Fig 2: SMC2 silencing induces CIN phenotypes in HCT116 cells. (A) Western blot showing SMC2 levels following silencing with either individual (siSMC2-1, -2, -3, or -4) or pooled (siSMC2-P) siRNA duplexes; Cyclophilin B is the loading control. Semi-quantitative image analysis was performed where SMC2 levels were first normalized to the respective loading control and are presented relative to siControl (1.00). See Figure S1 for detailed information. (B) Representative images depicting visual increases in NA heterogeneity and MN formation (arrowheads) following SMC2 silencing relative to siControl. (C) Cumulative NA distribution graph for SMC2 silenced conditions relative to siControl. KS tests reveal statistically significant increases in cumulative NA distribution frequencies following SMC2 silencing (siSMC2-2, -3, and -P) relative to siControl (N/A, not applicable; ****, p < 0.0001). (D) Column graph presenting the mean MN formation + standard deviation (SD) following SMC2 silencing; fold increase relative to siControl is presented above each column. The horizontal line indicates the minimum threshold (mean + 2 SD of siControl) required to be considered a significant increase in MN formation relative to siControl. (E) Representative images of various phenotypes observed in mitotic chromosome spreads including, normal (top left, inset presents a single magnified normal chromosome), small-scale chromosome losses (top middle), small-scale gains (top right) and large-scale gains (bottom left), along with severe (bottom middle, inset presents a magnified portion of a decondensed chromosome) and mild (bottom right) chromosome decompaction phenotypes. Chromosome numbers are presented in the bottom right corner of those spreads that could be accurately enumerated. (F) Bar graph showing the frequency of enumerable mitotic chromosome spreads (+ SD) versus those with severe or mild chromosome decompaction phenotypes. Note that a subset of the mild, but none of the severe decompaction phenotypes, could be accurately enumerated. (G) Dot plot presenting the individual chromosome numbers for each enumerable mitotic chromosome spread. Horizontal line identifies the modal chromosome number (45) for HCT116. KS-tests reveal statistically significant changes in the cumulative frequency distributions of chromosome numbers following SMC2 silencing relative to siControl (****, p < 0.0001). (H) Bar graph showing overall increases in the frequency of aberrant chromosome numbers following SMC2 silencing relative to siControl, which includes small-scale losses, small-scale gains, and large-scale gains.
Fig 3: KIF4A, but not the T799/S801 phosphorylation site mutant, promotes prometaphase chromosome congression. (A) Localization of KIF4A and KID in mitosis. CRISPR-tagged EGFP-KIF4A cells were fixed and stained for endogenous KID. Images were acquired with an Airyscan system. (B) HeLa cells were transfected for 48 h before thymidine synchronisation with control, siKIF4A, siKID, and siKIF4A + KID. After thymidine release and fixation, cells were stained for endogenous KIF4A. Arrows mark chromosomes and chromosome arms showing delayed congression to the metaphase plate. (C) mScarlet-KIF4A WT, FF1220AA, or T799A/S801A were expressed in a CRISPR-edited SMC2-EGFP cell line codepleted for KIF4A and KID. Cells were imaged undergoing mitosis, and representative maximum projections of the SMC2-EGFP signal are shown. Arrows mark chromosomes and chromosome arms showing delayed congression to the metaphase plate. (D) The plot shows the mean time and individual data points from nuclear envelope breakdown (NEBD) to anaphase onset in SMC2-EGFP cells transfected with either siControl or siKIF4A + KID and rescued with KIF4A WT and mutants (siControl n = 22, siKIF4A+KID n = 59, rescue KIF4A WT n = 16, FF1220AA n = 14, and T799A/S801A n = 20). Error bars indicate the SD. A nonparametric Kruskal-Wallis test (P < 0.0001) with a 95% confidence interval was performed, and conditions were compared after analysis by Dunn's test (**, P < 0.001; *, P < 0.1). (E) HeLa cells codepleted of KIF4A and KID and transfected with GFP-KIF4A constructs were fixed and stained for DNA and tubulin. The bar graph displays the extent of chromosome alignment for KIF4A and KID codepleted cells and for cells expressing GFP-KIF4A WT and mutants (siKIF4A+KID n = 103, rescue KIF4A WT n = 98, FF1154AA n = 73, FF1220AA n = 56, T799A/S801A n = 60, and T799A/S801A n = 66). Error bars indicate the SD (for three independent experiments).
Fig 4: KIF4A localizes to chromosomes axes and the anaphase central spindle. (A and B) Superresolution images of EGFP-KIF4A and PRC1 (A) or SMC2-GFP and mScarlet-KIF4A (B) were acquired using a Zeiss LSM880 microscope with Airyscan. DNA was detected using Hoechst 33258. Metaphase and anaphase localizations are shown. Scale bars represent 5 µm. (C) Immunoprecipitation (IP) of WT FLAG-KIF4A (WT), deletion mutants, and alanine point mutants from anaphase HeLa cells. (D) The scatterplot represents the maximum length reached by the spindle in anaphase B and was obtained measuring data acquired by live-cell imaging of HeLa cells treated with KIF4A siRNA and rescued with KIF4A WT and mutant constructs. The graph shows the mean with individual data points marked, and error bars indicate the SD (WT n = 15, FF1154AA n = 18, FF1220AA n = 15, and ΔCRD n = 12). A nonparametric Kruskal-Wallis test to KIF4A WT was performed (P < 0.0001), and conditions were compared with the post analysis by Dunn's test (***, P < 0.001). (E) The distribution of detected endogenous PRC1 staining throughout the length of the spindle in coverslips treated with siKIF4A and rescued with KIF4A WT or specific point mutants. Curves were centered to match the maximum intensity point (WT n = 12, FF1154AA n = 16, FF1220AA n = 11, and ΔCRD n = 7). Error bars indicate the SD.
Fig 5: Marked proliferation is characterized by enhancement in cell cycle transcripts. (a) Gene ontology enrichment identified cell cycle, immune system process, and nucleosome assembly as the most significantly upregulated processes 72 hours after injury. (b) The top 50 differentially expressed genes (identified by lowest p-value) organized by fold change. (c) Heat map showing differences in normalized counts of the top 50 differentially expressed genes between no injury (n = 5) and 72 hours (n = 6). (d) Protein-protein interaction network of top 50 differentially expressed genes via STRING analysis identified protein clusters specific to proliferation and inflammation. (e) Ki67 expression 72 hours following injury (green) by immunohistochemistry. VE-cadherin and DAPI denote endothelial junctions (red) and nuclei (blue), respectively. Scale bar: 60 μm (f) Immunohistochemistry reveals Smc2 expression 72 hours following injury (green). VE-cadherin and DAPI reveal endothelial junctions (red) and nuclei (blue), respectively. Scale bar: 60 μm.
Supplier Page from Abcam for Anti-SMC2 antibody