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. 2020 Jul 1;117(29):17019–17030. doi: 10.1073/pnas.2002193117

Fig. 4.

Fig. 4.

DYRK1B facilitates repair of IR-induced DNA damage. (A) DYRK1B-silenced cells reconstituted with Dox-inducible expression vector (TRE-Vector-Flag) or those that harbor DYRK1B alleles were induced with doxycycline. Twenty-four hours posttreatment, cells were irradiated and allowed to recover. Cells were thereafter processed for the comet assay according to standard procedures. Relative tail moment of at least 200 cells from two independent experiments was quantified using ImageJ and results were plotted. Western blotting was performed to examine the expression of DYRK1B using the indicated antibodies. The asterisk denotes the protein band that corresponds to endogenous DYRK1B. Bars represent mean ± SEM; ****P < 0.0001. (B) Representative metaphase preparations from IR-treated HeLa derivatives are shown. HeLa cells lentivirally infected with DYRK1B gRNA (DYRK1B gRNA#1) were reconstituted with vector or DYRK1B alleles. Cells were thereafter irradiated and processed to determine the number of chromosomal breaks. At least 120 metaphases from two independent experiments were counted and results were plotted. Arrowheads denote chromosome breaks. Bars represent mean ± SEM; *P < 0.05, ****P < 0.0001. (C) Scheme depicting cell processing for the neutral comet assay following transient global inhibition of transcription using DRB. (D) Western blotting was performed to evaluate the expression of DYRK1B. (E) Representative images from single-cell electrophoresis to analyze relative tail moments. Quantification is shown and was derived from at least 200 cells from two independent experiments. Bars represent mean ± SEM; ***P < 0.001, ****P < 0.0001. (F) Scheme depicting cell processing for metaphase analyses following transient global inhibition of transcription using DRB. (G) Representative metaphase preparations from IR-treated HeLa derivatives are shown. The numbers of chromosomal breaks were quantified in HeLa cells processed as in B with or without pre-DRB treatment. Bars represent mean ± SEM; *P < 0.05, **P < 0.01.