Denaturing solution

Immunostaining was carried out using the Histofine Simple Stain MAX-PO (Nichirei Biosciences Inc, Tokyo, Japan), according to the manufacturer’s instructions. In brief, sections (4 µm slices) were treated with 0.3% H₂O₂ in methanol and then incubated with anti-phospho-p44/42 MAPK (ERK1/2) overnight at 4°C. Sections were rinsed and incubated with peroxidase-labeled polymer at room temperature for 30 minutes. As a chromogen, diaminobenzidine (DAKO, Carpinteria, CA, USA) was used. For double staining immunohistochemistry, the first staining sections were washed with denaturing solution (Biocare Medical, Concord, CA, USA), rinsed and incubated with anti-CD68 or anti-pan-cytokeratin overnight at 4°C. Sections were rinsed and stained using Histofine SAB-AP (R) kit (Nichirei). First red (Nichirei) was used as a chromogen. Counter-staining was performed with hematoxylin.

IHC was performed as previously described [26 (link)]. For IHC co-stain, FFPE samples were deparaffinized, antigens retrieved at 99°C for 40 minutes, incubated with the first stain antibody (UBC9) O/N at 4°C, and visualized using the 3,3’-diaminobenzidine (DAB) chromogen. Then, samples were treated with Denaturing Solution (Biocare) to avoid cross-reaction, stained with the second antibody (SUMO1), and visualized with LSAB-AP and Vulcan Fast Red. Finally, slides were counterstained with haematoxylin and coverslipped. The following primary antibodies were used: anti-UBC9 (H-81, Santa Cruz Biotechnology), anti-SUMO1 (Santa Cruz Biotechnology), anti-SUMO2/3 (Abcam), anti-p62 (2C11, Abnova), anti-Ki-67(MIB-1, Dako). Images were generated with a BX51 Upright Microscopes from Olympus America Inc at 20x magnification. For morphological pathologist assessment, the Low Grade or High Grade dysplasia were obtained using morphological grading system and the percentage of positive staining in the tumor cells was quantified by blinded reading of the slide. Computational analysis was carried out as previously described [26 (link)].

Blocks were sectioned at a thickness of 3 μm. Antibodies targeting CD68 (clone PG-M1, Dako), phosphorylated signal transducer and activator of transcription 1 (phospho-STAT1; monoclonal, clone, and 58D6, Cell Signaling Technology, Danvers, MA, USA), and c-Maf (clone EPR16484, Abcam, Cambridge, UK) were used for analyses. Double staining was performed using a Dako Envision+ system with dextran polymers conjugated with horseradish peroxidase (Dako), as previously described.^{12 (link)} First, sections were stained with anti-CD68 antibodies for 30 min at room temperature, generating a brown color. Denaturing solution (BioCare Medical-CA, USA) was added for 5 min at room temperature for elution during double staining. Antigen retrieval was performed by heat treatment for 45 min with HIER T-EDTA Buffer (Dako). After incubation, sections were reacted with phospho-STAT1- or c-Maf-specific reagents using dextran polymers conjugated with horseradish peroxidase (Dako) overnight at 4 °C, using a Vina Green Chromogen Kit (BioCare Medical-CA), which produced green staining. Finally, slides were washed in Wash Buffer (Dako) for 3 min. Sections were counterstained with hematoxylin. The antibodies used in this study are listed in Supplementary Table 1.