American Society of Hematology

Figure 5.

$Cooperation of the JAK- STAT3 mutation and the IL-1R pathway in ALCL. (A) Indicated ALCL lines were transduced with STAT3 or JAK2 sgRNAs along with GFP. The fraction of viable GFP+/sgRNA+ cells relative to the live cell fraction is plotted at the indicated times after sgRNA induction, normalized to day 0 values. Error bars denote SD of triplicates. P was calculated comparing day 0 to each time point of indicated sgRNA induction in the MAC1 and MAC2A lines. ∗∗P < .01. (B-C) Indicated ALCL lines were transduced with MYD88, IL-1α, or ctrl sgRNAs, selected, and expression induced. Lysates were analyzed by immunoblotting for the indicated proteins. (D) Using CRISPR site-specific knockin to generate a MAC1 line carrying the STAT3 D661Y mutation. The 661st amino acid in WT and mutated STAT3 sequences and their coding sequences are marked red. To prevent the continuous DNA cutting by the Cas9 protein, the guiding RNA targeting protospacer adjacent motif (PAM) was also switched from “TGG” to “TCG” to produce a silent mutation. (E) MAC1 parental or STAT3 D661Y knockin lines were transduced with MYD88 or ctrl sgRNAs along with GFP. The fraction of viable GFP+/sgRNA+ cells relative to the live cell fraction is plotted at the indicated times after sgRNA induction, normalized to day 0 values. Error bars denote SD of triplicates. P was calculated by comparing the parental and STAT3 D661Y knockin lines with the indicated MYD88 sgRNA transduction. ∗∗P < .01. (F) MAC1 parental or STAT3 D661Y knockin lines were transduced with MYD88 or ctrl sgRNAs, selected, and expression induced. Lysates were analyzed by immunoblotting for the indicated proteins.$

Cooperation of the JAK- STAT3 mutation and the IL-1R pathway in ALCL. (A) Indicated ALCL lines were transduced with STAT3 or JAK2 sgRNAs along with GFP. The fraction of viable GFP+/sgRNA+ cells relative to the live cell fraction is plotted at the indicated times after sgRNA induction, normalized to day 0 values. Error bars denote SD of triplicates. P was calculated comparing day 0 to each time point of indicated sgRNA induction in the MAC1 and MAC2A lines. ∗∗P < .01. (B-C) Indicated ALCL lines were transduced with MYD88, IL-1α, or ctrl sgRNAs, selected, and expression induced. Lysates were analyzed by immunoblotting for the indicated proteins. (D) Using CRISPR site-specific knockin to generate a MAC1 line carrying the STAT3 D661Y mutation. The 661st amino acid in WT and mutated STAT3 sequences and their coding sequences are marked red. To prevent the continuous DNA cutting by the Cas9 protein, the guiding RNA targeting protospacer adjacent motif (PAM) was also switched from “TGG” to “TCG” to produce a silent mutation. (E) MAC1 parental or STAT3 D661Y knockin lines were transduced with MYD88 or ctrl sgRNAs along with GFP. The fraction of viable GFP+/sgRNA+ cells relative to the live cell fraction is plotted at the indicated times after sgRNA induction, normalized to day 0 values. Error bars denote SD of triplicates. P was calculated by comparing the parental and STAT3 D661Y knockin lines with the indicated MYD88 sgRNA transduction. ∗∗P < .01. (F) MAC1 parental or STAT3 D661Y knockin lines were transduced with MYD88 or ctrl sgRNAs, selected, and expression induced. Lysates were analyzed by immunoblotting for the indicated proteins.

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