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. 2022 May 18;119(21):e2114324119. doi: 10.1073/pnas.2114324119

Fig. 1.

Fig. 1.

Forward genetics selection with VBIM identifies increased H3K9 methylation as a possible mechanism of Enz resistance. (A) Schematic representation of the validation-based insertional mutagenesis (VBIM)-seq workflow. (B) Bubble plot representing the top VBIM-enriched genes in response to Enz (10 µM) treatment of VBIM-infected LNCaP cells for 72 d. Circle size proportionately correlates with the FPKM value of the associated gene in the vehicle-treated samples. (C) Proliferation assay of EV or full-length EHMT1-overexpressing LNCaP cells treated with vehicle (DMSO) or Enz (10 µM) for the indicated times. Results shown are representative of three biological repeats. (D) Schematic representation of EHMT1 isoform 1 (iso1: full length) and isoform 2 (iso2: lacking the catalytic SET domain); the corresponding genes were cloned into a double cassette lentiviral vector coexpressing GFP under a separate ubiquitin promoter (Ub). (E) Immunoblots showing the expression of the indicated proteins in lentivirally transduced LNCaP cell populations that harbor ∼20% cells expressing GFP, EHMT1 iso1, or HA-EHMT1 iso2. Arrow indicates the position of HA-EHMT1-iso2. (F) Representative contour plots of a flow cytometry–based competition assay showing the distribution of EHMT1 iso1-GFP or iso2-GFP expressing cells versus the GFP-expressing control cells. Cells were treated with DMSO or Enz (5 µM) for 80 d before the flow analysis. The percentage of GFP-positive (GFP +ve) cells is shown for each group. (G) RT-qPCR and (H) immunoblot analyses comparing the relative expression of H3K9 methyltransferases and readers in Enz-naïve and -resistant LNCaP cells that were treated with either Enz (10 µM) or DMSO (Veh) for the indicated times. Transcript levels in G were normalized first to RPLP0 and then to vehicle-treated cells for all comparisons. Significance was calculated using two-way ANOVA; error bars represent SD. Results are representative of three biological repeats, performed in triplicate. ns, not significant. (I) Immunoblots comparing the indicated histones in histone extracts purified from LNCaP cells treated with Enz (5 µM) or DMSO (Veh), as indicated, for 180 d. SIN, self-inactivating; SFFV, spleen focus-forming virus; SET, Su(var)3-9, enhancer-of-zeste and trithorax; ANK, ankyrin repeat; C3HC4, C3HC4 type zinc finger.