American Society of Hematology

Figure 3

$Figure 3. Aggregation of constitutively active lymphoma-associated TIR mutants in lymphoma cell lines. (A-B) HEK293 cells were transfected with plasmids for mCitrine-TIR (yellow pseudocolor) or mutants and full-length MyD88-mCerulean (blue pseudocolor, B). Twenty-four hours later, cells were fixed and visualized under the confocal microscope at ×1000 magnification. Shown are representative confocal images of at least 3 independent experiments. (C-G) Augmented amount of MyD88 in aggregates in different DLBCL cell lines. Cell lysates were fractionated by centrifugation and lysate and pellet fraction analyzed by SDS-PAGE followed by anti-MyD88 (C-E) or IRAK1 (G) and anti-tubulin immunoblot (see blots in panels E and G). Relative MyD88 or IRAK1 and tubulin levels were quantified by charge-coupled device detection and plotted as the tubulin-normalized ratios of lysate (WCL) vs pellet MyD88 or IRAK1. (C-D) Data were combined from 9 experiments and are shown as min-to-max box-and-whisker plots. In panel C, WT MyD88 DLBCL cell lines (white, OCI-LY19, BJAB, U2392) were compared with MyD88-mutated cell lines (burgundy, OCI-LY10, TMD8, HBL1, OCI-LY3 with L265P; HLY1 with S119C, SUDHL2 with S122R). In panel D, cells were plotted according to L265P mutational status. White, WT MyD88 (OCI-LY19, BJAB, U2392); red, L265P heterozygous (OCI-LY10, TMD8, HBL1); burgundy, L265P homozygous (OCI-LY3). (E) Comparison of unmutated OCI-LY19 (white) vs L265P homozygous OCI-LY3 (red) lysate vs pellet ratio from 9 combined experiments and showing a representative immunoblot. (F) Differences in MyD88 lysate expression in L265P-mutated OCI-LY3 cells are not due to lower mRNA expression as assessed by quantitative PCR in OCI-LY19 (white) and OCI-LY3 (burgundy) cells. One representative of 3 independent experiments was done in triplicate (mean + upper limit). (G) MyD88-containing aggregates also contain IRAK1. Comparison of unmutated OCI-LY19 (white) vs L265P homozygous OCI-LY3 (red) lysate vs pellet ratio from 3 combined experiments and showing a representative immunoblot. IB, immunoblot; WCL, whole-cell lysate.$

Aggregation of constitutively active lymphoma-associated TIR mutants in lymphoma cell lines. (A-B) HEK293 cells were transfected with plasmids for mCitrine-TIR (yellow pseudocolor) or mutants and full-length MyD88-mCerulean (blue pseudocolor, B). Twenty-four hours later, cells were fixed and visualized under the confocal microscope at ×1000 magnification. Shown are representative confocal images of at least 3 independent experiments. (C-G) Augmented amount of MyD88 in aggregates in different DLBCL cell lines. Cell lysates were fractionated by centrifugation and lysate and pellet fraction analyzed by SDS-PAGE followed by anti-MyD88 (C-E) or IRAK1 (G) and anti-tubulin immunoblot (see blots in panels E and G). Relative MyD88 or IRAK1 and tubulin levels were quantified by charge-coupled device detection and plotted as the tubulin-normalized ratios of lysate (WCL) vs pellet MyD88 or IRAK1. (C-D) Data were combined from 9 experiments and are shown as min-to-max box-and-whisker plots. In panel C, WT MyD88 DLBCL cell lines (white, OCI-LY19, BJAB, U2392) were compared with MyD88-mutated cell lines (burgundy, OCI-LY10, TMD8, HBL1, OCI-LY3 with L265P; HLY1 with S119C, SUDHL2 with S122R). In panel D, cells were plotted according to L265P mutational status. White, WT MyD88 (OCI-LY19, BJAB, U2392); red, L265P heterozygous (OCI-LY10, TMD8, HBL1); burgundy, L265P homozygous (OCI-LY3). (E) Comparison of unmutated OCI-LY19 (white) vs L265P homozygous OCI-LY3 (red) lysate vs pellet ratio from 9 combined experiments and showing a representative immunoblot. (F) Differences in MyD88 lysate expression in L265P-mutated OCI-LY3 cells are not due to lower mRNA expression as assessed by quantitative PCR in OCI-LY19 (white) and OCI-LY3 (burgundy) cells. One representative of 3 independent experiments was done in triplicate (mean + upper limit). (G) MyD88-containing aggregates also contain IRAK1. Comparison of unmutated OCI-LY19 (white) vs L265P homozygous OCI-LY3 (red) lysate vs pellet ratio from 3 combined experiments and showing a representative immunoblot. IB, immunoblot; WCL, whole-cell lysate.

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