![Oncogenic CBFs shift PU.1 sense to antisense transcription. (A) Scheme of small-hairpin RUNX1-ETO knockdown (shRUNX1-ETO) and control (shControl) designs. (B) RUNX1-ETO RT-qPCR after RUNX1-ETO knockdown at day 2 and day 9 in Kasumi-1 cells relative to GAPDH housekeeping gene (biological triplicates). (C) CD34 surface marker and viability kinetics assessed by flow cytometry after shRUNX1-ETO in Kasumi-1 cells (biological duplicates). (D) May Grünwald/Giemsa cytospins for morphology analysis of Kasumi-1 cells. (E) Heatmap of gene expression by RNA-seq after shRUNX1-ETO in Kasumi-1 at day 2 and day 9 (biological triplicates, n = 5810 genes; cutoff false discovery rate [FDR] <0.001). Gene expression of shRUNX1-ETO day 9 compared with shControl day 2 to day 9 and shRUNX1-ETO day 2. (F) Upregulated and downregulated genes involved in myeloid differentiation (red and yellow, respectively) using RNA-seq after shRUNX1-ETO in Kasumi-1 cells at day 9 vs shControl day 2 to day 9. (G) PU.1 asRNA and mRNA transcript expression after shRUNX1-ETO in Kasumi-1 cells (2 days after lentiviral transduction, n = 6) and AI-10-49 inhibitor treatment of ME-1 cells (6 hours after treatment, biological triplicates). (H) Ratio of PU.1 asRNA and mRNA transcript expression after shRUNX1-ETO in Kasumi-1 cells and AI-10-49 inhibitor treatment in ME-1 cells. (I) Aligned reads of PRO-seq with quantified peaks for PU.1 AsPr, PrPr, and AsPr/PrPr ratio after shRUNX1-ETO in Kasumi-1 cells (2 days after lentiviral transduction, n = 6). (J) Aligned PRO-seq reads after RUNX1-ETO knockdown start at the RUNX-binding site in PU.1 AsPr. Data are represented as mean ± standard error of the mean. *P < .05, ***P < .001, Student t test.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/138/15/10.1182_blood.2020008971/6/bloodbld2020008971f5.png?Expires=1735511248&Signature=K8AZnv9NGAXx9Q~WJkQ-P2PavRqMTkOzS3vAA4wfMUJqJXWHDUcl6Hne6LepAtWMbUK-UNaKkJ97xvUjQxE~kwSa2DZusR-L-azs9JXBL-TD7msCln8IaU8DS3X0x1wuoEWr9WA2-9-Saar~attQ~vnUjl2IZUe3BKkubsf~nH6hbetxXRx1OWnkIlHygItnUidKCt2EnEAVn4DjqNLNCDsyf4JKNy5hZlydOJu2~DNUHC5V4X~GvtjpvqQUcfDRejkdAEfKKYPmB~X5lYSqpxx8HRzw75t5dZ4itgC5umsIhm3S-kdLNFNwazr8pTGg2eGDZcLxSil3Ye9ISuErNQ__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Oncogenic CBFs shift PU.1 sense to antisense transcription. (A) Scheme of small-hairpin RUNX1-ETO knockdown (shRUNX1-ETO) and control (shControl) designs. (B) RUNX1-ETO RT-qPCR after RUNX1-ETO knockdown at day 2 and day 9 in Kasumi-1 cells relative to GAPDH housekeeping gene (biological triplicates). (C) CD34 surface marker and viability kinetics assessed by flow cytometry after shRUNX1-ETO in Kasumi-1 cells (biological duplicates). (D) May Grünwald/Giemsa cytospins for morphology analysis of Kasumi-1 cells. (E) Heatmap of gene expression by RNA-seq after shRUNX1-ETO in Kasumi-1 at day 2 and day 9 (biological triplicates, n = 5810 genes; cutoff false discovery rate [FDR] <0.001). Gene expression of shRUNX1-ETO day 9 compared with shControl day 2 to day 9 and shRUNX1-ETO day 2. (F) Upregulated and downregulated genes involved in myeloid differentiation (red and yellow, respectively) using RNA-seq after shRUNX1-ETO in Kasumi-1 cells at day 9 vs shControl day 2 to day 9. (G) PU.1 asRNA and mRNA transcript expression after shRUNX1-ETO in Kasumi-1 cells (2 days after lentiviral transduction, n = 6) and AI-10-49 inhibitor treatment of ME-1 cells (6 hours after treatment, biological triplicates). (H) Ratio of PU.1 asRNA and mRNA transcript expression after shRUNX1-ETO in Kasumi-1 cells and AI-10-49 inhibitor treatment in ME-1 cells. (I) Aligned reads of PRO-seq with quantified peaks for PU.1 AsPr, PrPr, and AsPr/PrPr ratio after shRUNX1-ETO in Kasumi-1 cells (2 days after lentiviral transduction, n = 6). (J) Aligned PRO-seq reads after RUNX1-ETO knockdown start at the RUNX-binding site in PU.1 AsPr. Data are represented as mean ± standard error of the mean. *P < .05, ***P < .001, Student t test.