Fig. 1

Drug-induced binuclear senescent cells show nucleolar amide I higher-wavenumber peak shifts originating from β-sheets.

PrECs were treated with or without 4 µM DCB for 24 h, released for 48 h, and analyzed by the following microscopies. (a) Left, phase-contrast images confirm the effective induction of polyploid cells with ≥ 2 nuclei through DCB treatment. Right, the quantification. n = 3 (30 counts each). Hereafter, cells are defined as follows: ‘Control (Cntl),’ cells in 0 µM and with a single nucleus (< 118 µm²); ‘Senescent (Snst),’ cells in 4 µM and with double nuclei. (b) DCB-treated binucleated cells specifically stained with anti-p21 antibody (left) and with anti-p16 antibody (right). Each graph compares the percentage of stained population between mononucleated cells in 0 µM (Cntl) and binucleated cells in 4 µM (Snst). n = 3 (20 counts each). (c) Bright-field images (upper) and CARS images (lower) at wavenumber 2933 cm^− 1 (Im[χ⁽³⁾] spectral images). White lines in the figures are thought to be due to microscopic particles being trapped by the laser and tweezed within the field of view. (d) Raman spectra (Im[χ⁽³⁾] spectra) of cytoplasm (purple), nucleoplasm (green), and nucleoli (red), whose origin is indicated in (c) by arrowhead and dotted rectangles (averaged). Left, higher magnifications of amide I vibration band. The peak shift was shown in comparison to the peak value of cytoplasm at 1653 cm^− 1 (vertical line). Right, whole spectra, where magnified areas were colored. (e) Top, fluorescent images with Amylo-Glo staining, an amyloid indicator dye. Bottom, the quantification of emission intensity in the nucleoli (20 × 20 pixels). (f) Left, ratiometric and subtraction images based on Gaussian function fitting of the protein amide I vibration band. Upper, images of \(\:{g}_{2,\:\mathbb{x}}/\left({g}_{1,\:\mathbb{x}}+{g}_{2,\:\mathbb{x}}\right)\). Lower, images of \(\:{g}_{2,\:\mathbb{x}}-{g}_{1,\:\mathbb{x}}\). Right, quantification of the β-sheet by \(\:{g}_{2,\:\mathbb{x}}/\left({g}_{1,\:\mathbb{x}}+{g}_{2,\:\mathbb{x}}\right)\). A cross-sectional average of the largest nucleolus in each nucleus was used. (g) Typical result of the amide I band (1540–1740 cm⁻¹) of the nucleolar Raman spectrum fitted using three Gaussian functions for control and senescent cells. See also the Methods sectioan. ***p < 0.001. Bars, 10 μm.