Journal: bioRxiv

Article Title: A targetable dependency on nonsense-mediated decay for proteostasis and immune control in small cell lung cancer

doi: 10.64898/2026.03.31.715503

Figure Lengend Snippet: A) DNA Sanger sequencing of the amplified B2M locus in representative H841 CRISPR/Cas9 WT and B2M-KO clones aligned to the reference sequence (NCBI, NG_012920.2) with sgRNA and the identified insertion mutation in H841 B2M-KO cells highlighted. B-D) Pharmacological NMD inhibition with either KVS0001 or SMG1i-11j compounds in healthy PBMC donors upon T cell artificial activation. ( B ) Proportion of viable CD45+ cells (left) and T cells (right) within PBMC populations after 4 days treatment with a T cell artificial activation cocktail (Act = CD3+CD28+IL-2) vs unstimulated conditions (Naive). ( C ) Proliferation of CD4+ (left) and CD8+ (right) T cells derived from T cell counts expressed as fold change (FC) for activated (Act) conditions relative to the unstimulated (Naive) control. ( D ) Proportion of T cells (CD3+), B cells (CD19+), NK cells (CD56+) and Myeloid cells (CD11b+) within PBMC populations (CD45+) from healthy donors directly after thawing (d-1), at the beginning of stimulation (CD3+CD28+IL-2) (d0) 4 days post-stimulation (d4). Graphs represent mean + SEM (n = 7). ***P<0,001; **P<0.01; *P<0.05; ns=non-significant (One-way ANOVA). E) Representative flow cytometry dot plots for the staining of CD45 vs CD56 (for NK cells) and CD8 vs CD4 (both for T cells) following 4 days co-culture of healthy donor PBMCs with WT H841 tumor cells. F) In vivo tumor growth in immunocompetent C57BL/6J mice transplanted with murine RP1380 TetO-shCTRL fed with normal or doxycycline-containing diet (n ≥ 4). G) Flow cytometry immunophenotyping of RP1380 TetO-shCTRL tumors harvested at the end of experiments shown in panel F (n ≥ 4). H-I) MHC-I surface expression quantified by flow cytometry in the indicated human and murine cell lines following genetic NMD inhibition via siRNA-mediated SMG1-KD and UPF1-KD ( H ) or doxycycline-inducible SMG1-KD ( I ). J) In vivo assessment of MHC-I (H2-Kb) surface expression in control vs NMD-inhibited murine RP1380 TetO-shSMG1 tumors (following doxycycline diet, DOXY) grown subcutaneously in C57BL/6 mice. K) Flow cytometry data for murine RP1380 TetO-shSMG1 allograft models grown in C57BL/6 mice significantly correlating tumor-specific H2-Kb surface expression with levels of immune cell infiltration (CD45+ infiltration). L) In vivo levels of tumor-specific MHC-I (H2-Kb) surface expression in control vs NMD-inhibited RP1380 TetO-shSMG1 tumors (DOXY) grown subcutaneously in RAG1-KO C57BL/6 mice. K) Flow cytometry data for murine RP1380 TetO-shSMG1 allograft models grown in RAG1-KO C57BL/6 mice correlating tumor-specific H2-Kb surface expression with levels of immune cell (CD45+) infiltration. L) Representative flow cytometry contour plot showing T cells (CD45+/CD3+) and B cells (CD45+/CD19+) in the blood from WT C57BL/6 and RAG1-KO C57BL/6 mice.

Article Snippet: Murine primary antibodies used were CD45 (Miltenyi #130110665), CD3 (Miltenyi #130119793), Nkp46 (BD Biosciences #3122669), CD19 (Miltenyi #130112037), CD11b (Miltenyi #1301138063), CD14 (Miltenyi #130115559), H2-Kb (Miltenyi #130115586), IFN-γ (ThermoFisher #48731182), KI-67 (Miltenyi #130120418).

Techniques: Sequencing, Amplification, CRISPR, Clone Assay, Mutagenesis, Inhibition, Activation Assay, Derivative Assay, Control, Flow Cytometry, Staining, Co-Culture Assay, In Vivo, Expressing