Fig 2: Strategy for generation of tetracycline (Tet)-inducible Baf53a conditional knockout (cKO) ES cells. (A) The targeting site of Baf53a. The targeting site (5′-GTT GAT TTC CCC ACG GCT AT-3′) of the Baf53a gene was shown. The target sequences are located on Exon 3 of the gene. (B) Alignment of the target sites of wild-type Baf53a and CRISPR/Cas9 resistant Baf53a (Baf53aR). The targeting site of the Baf53a cDNA was changed to 5′-GTT GAc TTt CCt ACc GCc AT-3,’ which encodes an identical amino acid sequence. (C) Schematic view of Tet-inducible Baf53a cKO ES cells. Expression of exogenous Baf53aR was induced in the absence of Tet and repressed in the presence of Tet. Both alleles of the Baf53a gene were mutated by the CRISPR/Cas9 system. (D) Sequences of deleted alleles. Genomic DNA of the deleted regions was sequenced. One allele contained a 7 bp deletion and the other contained a 23 bp deletion.

Fig 3: SMARCB1 is essential in maintaining the SWI/SNF complex integritya–b. Immunoprecipitation (IP) of the SWI/SNF complex subunits SMARCC1, SMARCA4, or ARID1A from the nuclear extracts of G401 (a) and BT16 (b) cell lines with or without Doxycycline (Dox)-induced SMARCB1 re-expression. Immunoblotted for subunits SMARCB1, SMARCC1, SMARCA4, ARID1A, ARID1B, SMARCC2, SMARCD1, SMARCE1, ACTL6A, and DPF2. Actin is a loading control.c. Mass spectrometry showing increased recovered peptides of SWI/SNF complex subunits by IP of SMARCA4 and SMARCC1 in G401 cells after SMARCB1 re-expression (Dox vs. No Dox).d. Glycerol sedimentation (10–30%) assay of SWI/SNF complex (~2MDa) from SMARCB1-deficient G401 cells without (top half) or with Dox (bottom half) immunoblotted for the indicated SWI/SNF complex subunits. BMI1 is a PRC1 complex subunit serving as a control.e. Immunoprecipitation (IP) of the SWI/SNF complex by SMARCC1 or SMARCA4 from the nuclear extracts of WT or Smarcb1 deficient mouse embryonic fibroblasts immunoblotted for the indicated SWI/SNF complex subunits.

Fig 4: Expression of Baf53b rescues the survival of Baf53a-deficient ES cells. (A) Morphological signatures of Baf53b-expressing Baf53a cKO ES cells. Baf53a cKO ES cells were transfected with expression vectors for empty (ev), Baf53a, or Baf53b. Transfected ES cells were divided into two dishes at 48 h after transfection and cultured with or without 1 μg/mL Tet. Cell morphologies were observed after 6 days. Scale bar is 250 μm. (B,C) Colony formation assay. (B) Control and Baf53a- or Baf53b-expressing Baf53a cKO ES cells as described in panel A were cultured for 6 days with or without 1 μg/mL Tet; and these samples were stained with crystal violet. (C) The colonies were dissolved in 1.0% SDS solution and absorbance was measured at 600 nm to quantify the number of surviving colonies. The representative results shown are expressed as the means ± standard deviation of three independent experiments. (D) Expression of exogenous Myc-tagged Baf53a and Baf53b protein. Expression of either Myc-Baf53a or Myc-Baf53b was confirmed by Western blot analysis at 2 days after Tet treatment. αTubulin was used as a loading control. The result is representative of three independent experiments.

Fig 5: ACTL6A suppresses p21Cip gene transcription to maintain the mesothelioma cancer phenotype.A, B ACTL6A knockdown increases Meso-1 cell p21Cip1 mRNA expression and protein level. C ACTL6A knockout Meso-1 cells express increased p21Cip1 mRNA. D Map of the full-length p21Cip1 promoter showing the p53-1 and p53-2 sites in the distal promoter and six Sp1 site in the proximal promoter. The top sequence indicates the authentic p53 site sequences. Our previous report indicates the mutations that inactivate these sites [28]. E ACTL6A knockout Meso-1 cells display enhanced p21Cip1 promoter activity for the full-length (p21-2326) and proximal (p21-124) promoter, and mutation of the six Sp1 sites eliminates the response. F Chromatin IP, using Meso-1 extracts, shows ACTL6A binding to the p21Cip1 promoter p53 binding sites and the proximal promoter Sp1 sites. G–I ACTL6A knockdown in NCI-Meso-17 cells increases p21Cip1 mRNA level and p21Cip1 promoter activity and this is associated with ATL6A binding to the p21Cip1 promoter p53 and Sp1 sites. As observed for Meso-1 cells, mutation of the proximal Sp1 sites eliminates transcriptional activity. Double asterisks indicate a significant increase (n = 3, p ≤ 0.001).