Journal: Science Advances

Article Title: Ara-C suppresses H3 K27–altered spinal cord diffuse midline glioma growth and enhances immune checkpoint blockade sensitivity

doi: 10.1126/sciadv.adu3956

Figure Lengend Snippet: ( A ) qPCR was performed to analyze changes in immune checkpoint mRNA expression in PDCs with Ara-C for 0, 1, 3, and 5 days. n = 3 biological replicates per group. ( B ) Western blot showed the protein expression of different immune checkpoints in PDCs with Ara-C for 0, 1, 3, and 5 days. ( C ) Immunostaining showed the protein expression of different immune checkpoints in PDC_S01 with or without Ara-C treatment. ( D and E ) Representative images of flow cytometric results showed immune checkpoint expression on cell membranes of different PDCs. n = 3 biological replicates per group. ( F ) Representative images showed protein expression of immune-checkpoint ligands and receptors in tumors from C57 mice with or without treatment of Ara-C. n = 5 biological repeats. ( G ) Statistical comparison of immune checkpoint–positive cells between tumors with or without treatment of Ara-C. ( H ) Multiplex protein staining showed the expression of p21, PVR, and PD-L2 in spinal cord orthotopic xenograft tumors from C57 mice with or without treatment of Ara-C. ( I ) Multiplex protein staining showed the expression of CD3e, TIGIT, and PD-1 in spinal cord orthotopic xenograft tumors from C57 mice with or without treatment of Ara-C. ( J ) Multiplex protein staining showed the expression of CD3e, Granzyme B, and FOXP3 in spinal cord orthotopic xenograft tumors from C57 mice with or without treatment of Ara-C. *0.01 ≤ P < 0.05, **0.001 ≤ P < 0.01, ***0.0001 ≤ P < 0.001, and **** P < 0.0001 for all figures.

Article Snippet: The primary antibodies and dilutions used in this study were as follows: Histone H3 K27M Mutant Specific (1:1000, CST, 74829S), Tri-Methyl-Histone H3 Lys27 (1:200, CST, 9733S), Ki67 (ZSGB-BIO, ZM-0166), Cleaved Caspase-3 (1:400, CST, 9661S), p21 (1:1000, abcam, 188224), H3k9me3 (1:800, CST, 13969S), GFAP (1:300, abcam, ab68428), Nestin (1:200, abcam, ab105389), PDGFRA (1:500, abcam, ab203491), SOX2 (1:200, Proteintech, 11064), NeuN (1:500, abcam, ab177487), IBA1 (1:500, abcam, ab178846), PD-1 (1:1000, Proteintech, 66220-1), PDL2 (1:100, CST, 4918S), Galectin9 (1:300, abcam, ab69630), PVRL2 (1:500, Proteintech, 27171-1-AP), PVR (1:200, Proteintech, 83724-6), TIGIT (1:500, abcam, ab300073), CD3 (1:150, abcam, ab16669), and CD8 (1:1000, abcam, ab217344).

Techniques: Expressing, Western Blot, Immunostaining, Comparison, Multiplex Assay, Staining