Extended Data Fig. 5: IgM interacts with intracellular TLR9 in ABC DLBCL lines.

a, Whole-cell lysates of indicated DLBCL cell lines were immunoprecipitated with anti-IgM or isotype control antibodies before being immunoblotted with IgM or indicated TLR antibodies, representative blots; n = 3. b, ABC DLBCL cell lines HBL1 and TMD8 were incubated on ice with IgM or isotype control antibodies and lysed. Lysates were immunoprecipitated (plasma membrane) with IgM or isotype control. Unbound lysates (cytosolic) were then immunoprecipitated with IgM or isotype control antibodies before all immunoprecipitated lysates were immunoblotted with the indicated antibodies; representative blots, n = 2. c, Left, confocal images of PLA reaction between IgM and TLR9 in HBL1 and TMD8 cells transduced with control SC4, CD79A or TLR9 shRNAs. Cells were puromycin selected and shRNAs induced with dox for two days before being fixed and imaged. Right, quantification data from three separate experiments. Data are pooled biologically independent experiments of n > 100 cells scored per condition. Box plots represents median and 25–75% of data, whiskers display 10–90 percentile. **P < 0.001, ***P < 0.001, one-way ANOVA with Dunnett’s post test. d, An IgM–TLR9 PLA (red) was performed in a panel of ABC and GCB DLBCL cell lines and the presence of chronic active BCR signalling (‘+’ denotes present), MYD88 mutational status and IgH isotype (μ = IgM, γ = IgG) are displayed. Nuclei were stained with DAPI (blue) and membranes were visualized by WGA (green). e, The number of puncta per cell of IgM–TLR9 PLA is quantitated. Box and whisker plots display mean and interquartile data, whiskers display 10–90 percentile. Data are from three fields of cells quantified from one representative experiment of three biologically independent replicates. f, The data from Extended Data Fig. 5e segregated by ABC (blue, n = 9) and GCB (orange, n = 9) lines. Box plots represent median and 25–75% of data, whiskers display range. **P < 0.01, Mann–Whitney unpaired one-tailed t-test. g, IgG–TLR9 PLA (red) was performed in indicated DLBCL cell lines co-stained with DAPI (blue) and IgG-AlexaFluor488 (green). MYD88 mutational status, IgH isotype and presence of chronic active BCR signalling (‘+’ denotes present; ‘−’ denotes absent) are displayed. Representative data from two independent biological replicates. h, To define the cytoplasmic location of the BCR–TLR9 interaction, we counterstained ABC cells for LAMP1, a marker of late endolysosomes, in which TLR9 resides, and performed PLA between IgM–TLR9, IgM–LAMP1 and IgM–SYK. The PLA signal is in red, LAMP1 is counterstained in blue. Representative data from three independent biological experiments. i, To quantify the association between PLA signals and LAMP1 staining, we calculated the Pearson’s correlation coefficients across all pixels in each imaged cell (n = 25 cells per PLA pair). The highest correlation was between an IgM–LAMP1 PLA and LAMP1 staining (R = 0.471), whereas the correlation between an IgM–SYK PLA signal and LAMP1 was much lower (R = 0.153). The correlation between the IgM–TLR9 PLA signal and LAMP1 staining was intermediate (R = 0.310), indicating that a significant component of the IgM–TLR9 interaction is in LAMP1⁺ vesicles. Quantified data are from one of three independent biological experiments. j, Quantification of the IgM–TLR9 PLA signal after ectopic expression of either empty vector, TLR9, wild-type MYD88 or MYD88(L265P). Data pooled from 3 (HBL1) or 2 (TMD8) biologically independent replicates of n ≥ 100 cells scored per condition. Box plots represents median and 25–75% of data, whiskers display 10–90 percentile. *P < 0.05, **P < 0.01, ***P < 0.001; one-way ANOVA with Dunnett’s post test. See ‘statistics and reproducibility’.

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