Immunocytochemistry and PLA Analysis of DNA Damage Response

BT549 EV and β4 cells (2.5 X 10⁴) were seeded on glass coverslips coated with 5µg/ml Cultrex mouse laminin-1 (Trevigen, Gaithersburg, MD) overnight and then treated with 10µM cisplatin for 24h. For immunocytochemistry, cells were then fixed, permeabilized, and immunostained as described previously (36 (link)) using the following antibodies: p-p53 S15 and p-53BP1 S1778 (Cell Signaling Technology), Cy3- and Cy2-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA). DAPI was used to stain nuclei. For PLA assays, cells were fixed and permeabilized according to the Duolink^® PLA Fluorescence Protocol (MilliporeSigma). Primary antibodies (1:100, Cell Signaling Technology) used were mouse or rabbit anti-p53, mouse anti-DNA-PKcs, and rabbit anti-53BP1. PLA assays were carried out with Duolink^®In Situ Detection Reagents Orange (#DUO92007), Duolink^®In Situ PLA^® Probe Anti-Rabbit PLUS/MINUS (#DUO92002/DUO92005) and Duolink^®In Situ PLA^® Probe Anti-Mouse PLUS/MINUS (#DUO92001/DUO92004) from MilliporeSigma. Cells were imaged using a Nikon Eclipse Ti2 Confocal microscope and Nikon NIS Elements software version 3.2.

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Chen M., Marrs B., Qi L., Knifley T., Weiss H.L., D’Orazio J.A, & O’Connor K.L. (2022). Integrin α6β4 signals through DNA damage response pathway to sensitize breast cancer cells to cisplatin. Frontiers in Oncology, 12, 1043538.

Publication 2022

Duolink®In Situ Detection Reagents Orange Nikon Eclipse Ti2 Confocal microscope NIS Elements software version 3.2

53bp1 Anti dna Anti igg Antibodies Assays Cells Cisplatin Confocal microscope Dapi Donkey Fluorescence Immunocytochemistry Laminin 1 Mouse Nuclei Pkcs Rabbit Stain

Corresponding Organization : Markey Cancer Center

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Variable analysis

independent variables

Cultrex mouse laminin-1 coating concentration (5 µg/ml)
Cisplatin treatment (10 µM)

dependent variables

Immunostaining of p-p53 S15 and p-53BP1 S1778
Proximity Ligation Assay (PLA) between p53, DNA-PKcs, and 53BP1

control variables

Cell type (BT549 EV and β4 cells)
Cell seeding density (2.5 x 10^4 cells)
Overnight incubation time for cell attachment to laminin-1 coated coverslips
Fixation and permeabilization conditions for immunocytochemistry and PLA
Imaging system (Nikon Eclipse Ti2 Confocal microscope) and software (Nikon NIS Elements version 3.2)

controls

Positive control: Not explicitly mentioned.
Negative control: Not explicitly mentioned.

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