Mantra snap software 1

Endoscopic biopsy specimens and surgically resected ESCC tumors were stained with UBQLN4 monoclonal antibody (mAb; #sc‐136145; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and/or MRE11A polyclonal antibody (pAb; #4895S; Cell Signaling Technology, Danvers, MA, USA) as previously described [33]. Images were taken by the BX43 upright microscope (Olympus, Tokyo, Japan) with a magnification of 20× using the mantra snap Software 1.03 (Perkin Elmer, Waltham, MA, USA). H‐scores were calculated using the inform 2.4 software (Perkin Elmer) and following manual instructions. Briefly, the tumor area was segmented from the stromal area and nuclei/cytoplasm compartments were distinguished by detecting the intensity of hematoxylin and/or 3,3'‐diaminobenzidine (DAB) staining. The optical signal threshold to classify the score into 4‐bins was set to 0.05, 0.12, and 0.2. Five slides per case were analyzed and the average H‐score was then used as the final value. The cutoff values for UBQLN4 and MRE11A were determined by considering H‐score values for the mean value observed in normal adjacent epithelia esophagus tissues plus 10 SD.

All of the FFPE tissues analysed were provided by SJHC. The cohort consists of 80 FFPE tissues (primary melanomas (n = 22), metastatic stage III (n = 12) and metastatic stage IV (n = 46)) from melanoma patients. FFPE tissues from patients with nevus (n = 7) were collected as the normal control. The clinical information for the melanoma patients is described in Table S1. IHC was performed as previously described,²⁵, ³³ using the mouse anti‐human ILF2 Ab (1:250 dilution, Santa Cruz, Cat# sc‐365068) and U2AF2 Ab (1:200 dilution, Santa Cruz, Cat# sc‐53942). Images were taken by the BX43 upright microscope (Olympus, Tokyo) at 20× magnification and with the Mantra Snap Software 1.03 (Perkin Elmer, Waltham). The images were analysed using inForm 2.4 software (Perkin Elmer). H‐scores were calculated following the inForm software instructions available at https://www.perkinelmer.com/ Content/LST_Software_ Downloads/inFormUserManual_2_3_0_rev1.pdf.