Fig 1: Obtention of a PBX1 KD clone. Panel (a): Western Blot demonstrating the diminution of both PBX1a (black arrowhead) and PBX1b (white arrowhead) expression PBX1 KD clone (clone KD2). WT HEK cells transfected with control siRNA (WT + si CTRL) or PBX1 siRNA (WT + si PBX1) served as negative and positive controls, respectively. WT: non-transfected HEK cells. Panel (b): Sequence of PBX1 exon 3 in WT HEK cells (HEK WT) and PBX1 KD clone (clone KD2) showing an 8-bp insertion (dashed box) at the sgRNA’s target sequence (green box) in KD2. Uncropped Western Blot is presented in Supplemental Data (Figure S10).
Fig 2: Schematic view of PBX1 with previously published variants. Blue arrow represents the CRISPR-engineered frameshift variant in clone KD2. Isoform: PBX1a - NM_002585.4 - NP_002576.1. All variants impact all PBX1 isoforms, except p.(Thr88Ile)(absent from NP_001340059). Yellow dotted boxes: Nuclear Localization Signal (NLS).
Fig 3: Heatmaps comparing the differential expression between WT HEK and PBX1 KD cells transfected with similar PBX1 plasmids. HEK + WT/mutant: WT HEK cells transfected with WT or mutant PBX1a plasmid. Clone KD2 + WT/mutant: KD2 cells transfected with WT or mutant PBX1a plasmid.
Fig 4: PBX1 partial LoF impairs cell proliferation, not fully rescued by PBX1a transfection. (a) PBX1 LoF impairs cell proliferation (Methylen blue test). OD: optical density. H: hours. Bars represent standard deviations (SD). (b) WB assessing PBX1a overexpression (black arrowhead) in transfected HEK WT cells (HEK WT) and KD2 clones. White arrowhead: PBX1b. (c) Transfection with either PBX1a WT or 320G>A plasmids enhances proliferation in WT HEK cells in comparison with control (PMiR-ZIP, a GFP-encoding plasmid), but not in PBX1 KD cells, after 3 days of culture. Bars represent SD. (d) PBX1 KD clones are unable to grow as a monolayer and reach confluency, a phenotype slightly rescued by both WT and mutant PBX1a overexpression. For each quantitative experiment, 3 biological replicates were realized. Each biological replicate comprised 3 technical replicates per sample. * = t test between 0.01 and 0.05, ** = t test between 0.001, and 0.01, *** = t test between 0.0001 and 0.001.
Fig 5: PBX1 transcripts in HEK cells and clone KD2. (a) Sashimi plot of PBX1 in HEK cells + WT PBX1a (1), HEK cells + PBX1a-320G>A (2), clone KD2 + WT PBX1a (3), and clone KD2 + PBX1a-320G>A. Exon junction coverages < 5 are hidden for lisibility. Black arrows represent exons of PBX1a isoform (NM_002585). (b) Schematic view of PBX1 transcripts. Red triangle represents the location of the c.320G>A mutation. Vertical dotted purple line represents the location of the CRISPR-generated frameshift insertion.
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