IL-1α production in ALK⁻ ALCL cell lines and primary cases. (A) Relative IL-1α and IL-1β mRNA expressions, measured by real-time polymerase chain reaction (PCR) and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) values, in indicated lymphoma cell lines. Error bars denote standard error of the mean (SEM) of triplicates. (B) IL-1α production, measured by in-cell ELISA (cell lysate) and normalized to cell numbers, in indicated lymphoma cell lines. Error bars denote SEM of triplicates. (C) MAC1 and MAC2A lines were transduced with IL-1α or ctrl sgRNAs, selected, and expression induced. IL-1α production was measured by in-cell ELISA as in panel B. P was calculated comparing the ctrl sgRNA (sgCTRL) and sgIL-1α groups; ∗∗P < .01. Error bars denote SEM of triplicates. (D) Immunofluorescence confocal microscopy analysis of the distribution of endogenous IL1R1 and IL-1α in the indicated lines. Antibodies used are indicated on the top. (E) Indicated ALCL lines were transduced with Ctrl or IL-1α sgRNAs and selected. Relative proliferations upon sgRNA induction were measured by the Promega CellTiter Cell Proliferation Assay (MTS) and normalized to the Ctrl sgRNA groups. P was calculated comparing sgCTRL and sgIL-1α groups; ∗P < .05; ∗∗P < .01. Error bars denote SEM of triplicates. (F) Immunohistochemistry (IHC) of IL-1α, p-IRAK4, and CD30 expression of 2 pC ALK⁻ ALCL cases. Sections were examined microscopically using 100× original magnification. The depicted images are representative of the 19 ALK⁻ ALCL cases examined. (G) IL-1α and p-IRAK4 IHC scores (detailed at supplemental Table 2) in 3 subgroups of primary ALK⁻ ALCL cases. ∗P < .05; ∗∗P < .01.