Fig 2: Cross-reactivities of anti-human CMTM6 and CMTM4 antibodies with canine CMTM6 and CMTM4. Binding of anti-human CMTM6 antibody (HPA026980, Sigma-Aldrich) and anti-human CMTM4 antibody (LS-C368889, LifeSpan Biosciences) to canine CMTM6- and CMTM4-expressing cells was examined by flow cytometry. Rabbit IgG was used as isotype-matched control antibody. Using a bicistronic expression vector, AcGFP1 expression was utilized to predict CMTM6 or CMTM4 expression. Antibody binding to the AcGFP1+ cell fraction was considered a specific reaction.

Fig 3: Comparison of the CMTM4 gene among mammalian species. (A) Multiple alignment of the deduced amino acid sequences of CMTM4. The predicted MARVEL domain in canine CMTM4 (49–170) is shown. (B) Phylogenetic analysis of canine CMTM4 in relation to those of other mammalian species. The bootstrap consensus tree was inferred from 1,000 replicates. The bootstrap percentages are shown next to the branches. The GenBank accession numbers of each reference sequence are indicated.

Fig 4: Gene knockdown of CMTM6 or CMTM4 in a PD-L1–expressing canine cell line. DH82 cells were transfected with siRNAs against canine CMTM6 or CMTM4 and cultured for 48 h before the analysis. (A) The effects of CMTM6 knockdown on CMTM6 mRNA expression were examined by qRT-PCR. Data are shown as the means ± SD. (B) The effects of CMTM6 knockdown on PD-L1 expression was analyzed by flow cytometry. Data are shown as the means of MFI ± SD. (C) The effects of CMTM4 knockdown on CMTM4 mRNA expression were examined by qRT-PCR. Data are shown as the means ± SD. (D) The effects of CMTM4 knockdown on PD-L1 expression on the cell surface was analyzed by flow cytometry. Data are shown as the means of MFI ± SD. The statistical analysis was performed using Tukey's test. *p < 0.05.

Fig 5: CMTM6, CMTM4, and PD-L1 protein expressions in canine cancers. Canine malignant melanoma (n = 4) and osteosarcoma (n = 4) samples were analyzed for CMTM6, CMTM4, and PD-L1 expression by immunohistochemistry. For negative control, the sections were incubated with isotype-matched control antibody. Representative results for malignant melanoma (Sample #1) and osteosarcoma (Sample #5) are shown (scale bar: 50 μm).