Extended Data Figure 2. Analysis of TRIM37 protein levels and centrinone efficacy in different cancer cell lines and comparison of mitosis in RPE1 and CHP134 neuroblastoma cells following centrinone treatment.

(a) Immunoblots used to quantify TRIM37 protein level across different cell lines. TRIM37 immunoblots are shown above the corresponding Ponceau-stained blots. MCF7 cells have the highest TRIM37 transcript levels and copy number in the CCLE (Extended Data Fig. 1i). Serial dilutions of MCF7 extract were loaded next to extracts from other cell lines on each blot and TRIM37 band intensities across a serial dilution of MCF7 cell extract were used to generate a standard curve (graphs below each blot); measured intensities for other cell line extracts were converted into relative expression values using the standard curve. TRIM37 protein level in HepG2 cells was set to 1 and values measured for other cell lines were plotted relative to the HepG2 level in Fig 1d. (b) Schematics of protocols used to measure TRIM37 protein levels (left) and conduct passaging-based proliferation analysis of cancer cell lines (right). (c) Comparison of TRIM37 mRNA and protein levels across cancer cell lines. mRNA levels are from the CCLE and were transformed from a logarithmic (base 2) to a linear scale. Protein levels are the mean value of two measurements conducted as in (a) and are plotted relative to the amount of TRIM37 in HepG2, a non-amplified cancer cell line. Inset graph excludes MCF7, which shows exceptionally high TRIM37 mRNA and protein levels. (d) Quantification of centrosome number in the indicated cell lines and conditions (n = 100 for each condition). Centrosomes were defined as co-localized foci of CEP192 and γ-tubulin in fixed interphase cells. In the absence of any treatments, there is mild centrosome amplification in the breast cancer cell lines and in one neuroblastoma cell line. Following 8-day centrinone treatment, a significant proportion of the cells from cell lines with relatively low sensitivity to centrinone lacked centrosomes. (e) Schematic of method used to generate a pool of cells expressing H2b-mRFP for each of the indicated cell lines. TRIM37 protein level relative to RPE1, measured by semi-quantitative immunoblotting. (f) Images are stills from timelapse sequences of H2b-RFP expressing mitotic RPE1 and CHP134 cells. Both cell lines exhibit rapid mitosis (~30 minutes) with no segregation failure in DMSO. Following centrinone treatment, CHP134 cells exhibited significantly more delayed mitosis and higher rates of segregation failure compared to RPE1 cells. Scale bar is 10 μm. (g) Quantification of mitotic duration and segregation failure comparing RPE1 and CHP134 cells. (h) Schematic of protocols used to analyze mitotic duration, segregation failure and viability of the CHP134-derived cell line panel with different levels of TRIM37 protein.