Fig. 6 KIFC1-S26 phosphorylation induces drug resistance. a, c–f KIFC1-S26 phosphorylation induces etoposide resistance. b, g–j Etoposide-resistance is restrained by ATM/ATR inhibitor VE-822. a, b The indicated stable cell lines were treated with etoposide (0.5 μM) or VE-822 (5 μM) for 4 days, and then were analyzed for cell viability using the MTT assay. Histogram graphs showing the percentage of surviving cells. a Two-tailed t test p values (from left to right): p= 0.0007, 0.0099, and 0.0090. b Two-tailed t test p values (from left to right): p=0.0005, 0.0110, and 0.0143. c, g Xenograft experiment with KIFC1-WT, S26A, or S26D stable cells was described in the Methods section. Tumors were collected and photographed (scale bar, 2 cm). d, e, h, i Quantification of average tumor volume (d, h) and weight (e, i). Six tumors were included in each group. d Two-tailed t test p values (from left to right): p=0.0062 and 0.00001. (e) Two-tailed t test p values: p=0.0099 and 0.00001. h Two-tailed t test p values: p=0.0053 and 0.0016. i Two-tailed t test p values: p=0.0057 and 0.0016. f, j Representative immunohistochemical images showing γ-tubulin staining (scale bar, 10 μm) with quantitative analysis of pseudo-bipolar (centrosome clustering) and multipolar mitosis of the tumor sections of xenograft tumor samples after treatment with etoposide or VE-822. Arrows point to the centrosomes. For each experimental condition, 100–161 cells were counted, and three independent experiments were performed. f Two-tailed t test p values: p=0.0016, 0.9245, and 0.5663. j Two-tailed t test p values: p=0.0006 and 0.0092. Data represent the mean ± SD of three times of independent experiments. NS=not significant, *p < 0.05, **p < 0.01, Source data are provided as a Source Data file.
Fig. 8 The ATM/ATR-KIFC1-centrosome clustering pathway promotes tumor recurrence. a Schematic drawing of tumor recurrence, as described in the Methods section. Xenograft tumors were treated with etoposide alone and in combination with PBS, VE-822, or CW069, and were allowed to grow up to a mean volume of 300mm3 before being surgically resected. After surgery, the mice were treated with the indicated drugs for another 2 weeks. After 5 months with no drug treatment, mice with tumor recurrence were identified and analyzed. b The time at which the indicated tumors reached 300mm3.
Data represent mean ± SD (n = 10). Two-tailed t test p values (from left to right): p = <0.00001 and <0.00001. c, d KIFC1-S26 phosphorylation promotes CIN in xenograft tumors. The indicated cells were isolated from the surgically resected tumors and then were cultured to assess the rate of CIN in vitro. For each experimental condition, 100–150 cells were counted, and three independent experiments were performed. c The graph shows the fraction of indicated cells with different numbers of chromosomes per cell. For each experimental condition, 100–127 cells were counted, and three independent experiments were performed. Two-tailed t test p values (from left to right): p = 0.0022, 0.0035, 0.0021, and 0.0008. d The graph shows the fraction of the indicated cells stained for centromeric DNA on chromosomes 3 and 7 with FISH analysis. For each experimental condition, 100-124 cells were counted, and three independent experiments were performed. Two-tailed t test p values (from left to right): p = 0.0092 and 0.0077. e Graph depicting the Kaplan-Meier analysis of tumor recurrence in the different groups. Survival cutoff criteria (compassionate euthanasia), when the recurrent tumors impeded ambulation, defecation, urination, or eating. Each group, n =10. Two-tailed t test p values (from left to right): p = 0.0442 and 0.0291. f Representative images showing local recurrence and distant recurrence (lung metastases). The boxed enlargements showed tumor morphology. Hematoxylin and eosin staining of tumor tissue sections. g, h KIFC1-S26 phosphorylation promotes CIN in the locally recurrent tumors. Recurrence-WT (Rec-WT) and recurrence-S26D (Rec- S26D) cells were isolated from the indicated recurrent tumors and then were re-cultured to assess the rate of CIN in vitro. The untreated cells were MDAMB- 231 cells. Data represent the mean ± SD of three times of independent experiments. For each experimental condition, 100–109 cells were counted, and three independent experiments were carried out. g The graph shows the fraction of indicated cells with different numbers of chromosomes per cell. For each experimental condition, 100–109 cells were counted, and three independent experiments were performed. Two-tailed t test p values (from left to right): p = 0.0022, 0.0031, 0.0079, and 0.0024. h The graph showed the fraction of the indicated cells stained for centromeric DNA on chromosomes 3 and 7 with FISH analysis. For each experimental condition, 100–159 cells were counted, and three independent experiments were performed. Two-tailed t test p values (from left to right): p = 0.0261, 0.0098,0.0047, and 0.0164. *p < 0.05; **p < 0.01. Source data are provided as a Source Data file.
非整倍體分析
這些實驗是按照前面描述的那樣進行的。用Colcemid(50 ng/ml，37℃，6h)處理細胞，收集細胞，懸浮在KCl(75 mm，37℃，15min)中，固定在Carnoy『s液中30min，然後滴到玻片上，用5%Giemsa液染色。用共聚焦顯微鏡分析染色體數目。
免疫沉澱和Western blotting。
細胞裂解物製備和Western blotting如前所述52進行。簡而言之，細胞在裂解緩衝液(50 mM Tris-HCl pH 8.0，5 mM EDTA，150 mM NaCl，0.5%NP-40，1 mM PMSF)中裂解，10000 g離心5min，SDS-PAGE和Western blotting進一步分析。細胞裂解產物與抗Flag-M2瓊脂糖凝膠或蛋白A/G瓊脂糖凝膠和抗KIFC1抗體在4℃下免疫共沉澱4~6h，用裂解緩衝液洗滌，SDS樣品緩衝液煮沸，SDS-PAGE電泳分級，Western blotting分析。源數據以源數據文件的形式提供。
建立穩定的細胞系。
用慢病毒系統感染MDA-MB-231和MCF-10A細胞，分別感染KIFC1-shRNA(用0.5 mg/ml嘌呤黴素篩選)和耐shRNA的KIFC1 WT、S26A和S26D病毒(用1 mg/ml潮黴素篩選)。單克隆WT和突變細胞的蛋白表達水平與內源性KIFC1相似。使用Lenti-X™Tet-on®3G誘導表達系統(Takara Bio Inc.)52，將穩定表達FLAG-Plk4的可誘導HeLa Tet-on細胞先後感染pLVX-Tet3G病毒和pLVX-TRE3G-Plk4病毒，並用所指示的抗性標記進行篩選。為誘導FLAG-Plk4的表達，培養基中添加2μg/ml的多西環素(DOX)。為了篩選有誘導性CA(DOX+)和無誘導性CA(DOX−)的細胞系，我們選擇了高效的單克隆細胞系。
螢光原位雜交。
FISH分析按照ZytoLight規範CDKN2A/CEN 3/7/17四色探針(Zytovision，不萊梅港，德國)的用戶手冊進行。簡單地說，蓋玻片上的細胞固定在Carnoy『s固定液中，在2×SSC(2min)中洗滌，乙醇脫水，DNA探針處理，然後在75°C下變性(2min)。蓋玻片在37°C孵育過夜，4×SSC(含0.05%吐溫20，5min)洗滌，0.25×SSC 72°C孵育(2min)，4×SSC(含0.05%吐溫20，30s)洗滌，4『，6-二氨基-2-苯基吲哚(DAPI)染色。
用共聚焦顯微鏡分析了3號染色體(紅色)、7號染色體(綠色)和DNA(藍色)。
組織晶片和免疫組化染色。
購買乳腺癌(HBreD140Su04；上海Outdo Biotechnology，中國上海)和結直腸癌(COC1601；上海SuperBiotek製藥技術，上海，中國)的人體組織微陣列。所有樣本的臨床特徵均從公司網站下載。用抗KIFC1抗體進行免疫組織化學染色。如前所述，對KIFC1染色強度進行量化、評分和分級(低，0-4分；中，5-8分；高，9-12分)。為確保調查結果的公正性，採用雙盲法收集數據。源數據以源數據文件的形式提供。
患者來源的異種移植(PDX)模型。
乳腺癌PDX模型由上海立德生物技術公司(中國上海)在徵得患者知情同意的情況下製作。實驗程序經中國臨床試驗註冊倫理委員會批准(註冊號：ChiCTR1900027396)。本研究中使用的腫瘤的臨床特徵如圖所示。S2.。每隻BALB/c裸鼠(6~8周齡)6隻，每隻腫瘤組織切成3~5 mm的腫塊，植入4個皮下。每天觀察腫瘤生長情況。當腫瘤體積達到200mm3時，腹腔注射硫代巴比妥鈉(200μl)、依託泊苷(40 mg/kg)或順鉑(20 mg/kg)。24h後處死小鼠，取出腫瘤組織。石蠟包埋腫瘤組織切片行免疫組化染色，顯微鏡下觀察。
荷瘤小鼠模型。
將MDA-MB-231細胞或穩定的細胞系(3×106細胞)注射到6周齡雌性BALB/c裸鼠左側或兩側皮下。當腫瘤體積達到150mm3時，隨機分為對照組、依託泊苷、VE-822和CW069治療組。依託泊苷(20 mg/kg/周)或CW069(200 mg/kg，每周連續4天)腹腔注射。VE-822(20 mg/kg，每周連續4天)灌胃給藥。每隔5天用卡尺測量腫瘤大小。在圖7中，從裸鼠身上提取腫瘤，並在第60天進行分析。如圖3所示。S9，各指標組以含多西環素600 mg/kg的飼料自由餵養(Harlan Teklad日糧)，第45天從裸鼠體內取出腫瘤進行分析。在圖8中，當腫瘤體積達到300mm3時，手術切除腫瘤，並用指定的藥物再治療2周。
在沒有藥物治療的5個月後，對腫瘤復發的小鼠進行鑑定和分析。動物在華東師範大學動物中心的監督下，按照高標準的倫理和科學標準對待動物。