Figure 2: Multiplexed subcellular imaging of cell line models reveals primary and micronuclear rupture are associated with distinctive protein expression patterns.

A, Experimental overview. Human RPE cells were treated with LMNB1 siRNA, MPS1i, or X-ray radiation (2Gy), to induce PN or MN rupture then analyzed using plate-based CyCIF. B, Representative images of each treatment group (arrows, MN rupture; arrowheads, PN rupture). C, Quantification of BAF-positive PN and MN ruptures following treatment (mean % of cells +/− S.D) (*p<0.05) (t-test, two-sided, unpaired). D, p-CyCIF images of a ruptured, BAF-positive PN bleb in cells treated with LMNB1 siRNA, showing localized disorganization and accumulation of lamin A/C and emerin, reduced levels of lamin B1/B2 and NUP133, and absence of pH2AX, LBR, and STING. E, Percentage of BAF-negative or BAF-positive PN blebs in LMNB1-kd cells expressing each intensity of a given marker, based on semi-quantitative visual scoring (t-test, two-sided, unpaired). F, p-CyCIF images of a ruptured, BAF-positive micronucleus a cell treated with MPS1 inhibitor, exhibiting low lamin A/C, lamin B1/B2, and NUP133, and accumulation of emerin, LBR, pH2AX, cGAS, and STING. G, Percentage of BAF-negative or BAF-positive MN expressing each intensity of a given marker in MPS1i-treated cells, based on semi-quantitative visual scoring (t-test, two-sided, unpaired). H, Representative images of XR-treated cells with PN and MN ruptures associated with accumulation of cGAS and/or pH2AX. I, Representative images of XR-treated cells with occasional pH2AX-positive PN blebs. J,K, Percentage of BAF-negative or BAF-positive PN (J) or MN (K) with a given expression level of indicated markers. L, Representative p-CyCIF images of STING in RPE cells per condition (arrowheads, STING foci). M, Quantification STING foci in each experimental condition (*p<0.05, ***p=0.0503) (t-test, two-sided, unpaired).