Figure 7.

HITTERS binds to and regulates the formation of MRN complex under ER stress. A) The schematic representation of modified ChIRP‐MS. B) qPCR showed the modified ChIRP method retrieved about 30–50% of HITTERS. Western blot confirmed that HITTERS binds to both MRE11 and RAD50, but not NBS1 nor glyceraldehyde‐3‐phosphate dehydrogenase. Cells were treated with TM (10 µg mL⁻¹, 6 h) before harvesting for ChIRP‐MS. For qPCR, Student t‐test was used for each primer. ***, P < 0.001. C) RIP assay of MRE11 and RAD50. Western blot confirmed MRE11 and RAD50 were successfully precipitated. qPCR indicated HITTERS were significantly enriched by MRE11 and RAD50. Cells were treated with TM (10 µg mL⁻¹, 6 h) before harvesting for RIP. For qPCR, Student t‐test was used. ***, P < 0.001. D,E) Co‐IP results showed the interaction between MRE11 and RAD50 in CAL27 cells were reduced after HITTERS knockdown. The interaction between MRE11 and NBS1 remained no change after HITTERS knockdown. Cells were transfected with siRNA for 48 h and then treated with TM (10 µg mL⁻¹) for 6 h. H) Diagrams of full‐length HITTERS and the truncations in MS2bs‐MS2bp RNA pull‐down assay. I) The reconstructed plasmids containing 12XMS2 tag and full‐length HITTERS and truncations with the correct sizes are indicated. J,K) Immunoblot analysis for RAD50 and MRE11 in the protein samples pulled down by different HITTERS truncations. J) SCC25 and K) CAL27 cells were transfected with two plasmids for 48 h and treated with TM (10 µg mL⁻¹) for 6 h.