Fig 2: Excision efficiency of ectopically expressed CreERT2 (MSCV CreERT2).A) Methylene Blue staining of immortalized and transformed Ssrp1fl/fl fibroblasts and immunoblot determining the levels of SSRP1 and SPT16 in primary, immortalized and transformed cells. B) Immunoblot to determine time-dependent changes in SSRP1 protein levels in immortalized Ssrp1fl/fl MSCV CreERT2 upon treatment with 2 µM 4-OHT. C) SSRP1 Immunofluorescence in immortalized and transformed Ssrp1 fl/fl +/- MSCV CreERT2 before or after treatment with 2 µM 4-OHT for 72 or 96 h. D) Quantification of SSRP1 immunofluorescence staining to calculate the Corrected Total Cell Fluorescence (CTCF). E) PCR of the genomic DNA to determine the excision of Ssrp1 after 4-OHT treatment of immortalized and transformed Ssrp1fl/fl fibroblasts with or without transduction with MSCV CreERT2.

Fig 3: UV crosslinking reveals that ICP22 interacts directly with the FACT complex. (A) Amino-acid sequence of ICP22 showing the positions at which a TAG stop codon is introduced and Bpa is incorporated within ICP22; (B) HEK293 cells were co-transfected with wild-type ICP22 cDNA (WT) or ICP22 cDNA with a TAG stop codon replacing different amino acid codons (represented by the asterisk) and suppressor tRNA/Bpa synthetase pair in the absence or presence of photo-cross-linkable Bpa. Flag immunoprecipitation was performed from total cell extracts and separated by SDS-PAGE. Western blots were probed with an anti-Flag antibody; (C) Flag immunoprecipitation of ICP22Bpa from cell extracts after cells were UV irradiated alive (+) in chilled PBS or not (-). Western blot was probed with an anti-Flag antibody; (D) HEK293 cells were co-transfected with ICP22 cDNA with a TAG stop codon replacing Y230 and suppressor tRNA/Bpa synthetase pair in the presence of photo-cross-linkable Bpa, and UV-irradiated alive (+) in ice-cold PBS. Flag immunoprecipitation was performed from total cell extracts, cytoplasmic and nuclear fractions and separated by SDS-PAGE. Western blots were probed with antibodies against Flag and FACT complex subunits SPT16 and SSRP1; (E) HEK293 cells were co-transfected with ICP22 cDNA with a TAG stop codon replacing L191 or C225 and suppressor tRNA/Bpa synthetase pair in the presence of photo-cross-linkable Bpa, and UV-irradiated alive (+) in ice-cold PBS. Flag immunoprecipitation was performed from total cell extracts and separated by SDS-PAGE. Western blots were probed with antibodies against Flag and FACT complex subunits SPT16 and SSRP1.

Fig 4: Knockdown of MYCN down-regulates the expression of FACT in neuroblastoma. (A) quantitative polymerase chain reaction (qPCR) results of MYCN, SPT16, and SSRP1. Error bars are SEM. (**p < 0.01, Student’s t test). (B) Western blots for MYCN, SPT16, and SSRP1 protein expression in BE(2)C cells treated with control and MYCN knockdown. (C) MYCN target genes are down-regulated in MYCN knockdown. (*p < 0.05, Student’s t test). (D) Heatmap showing the expression levels of the 51 MYCN target genes in BE(2)C cells treated with control and MYCN knockdown.

Fig 5: Generation of homozygous floxed Ssrp1 mice (Ssrp1 fl/fl).A) Immunoblot of protein extracts from spleen illustrating the difference in SSRP1 and SPT16 levels between Ssrp1+/+ and Ssrp1 Neo LacZ fl/+ age-matched mice (15 weeks and 28 weeks of age). B & C) Representative PCR of genomic DNA from individual pups (indicated by numbers) from a single litter of B) Ssrp1Neo LacZ fl/+ crossed with B6.129S ROSA26 FLPe (Flpe) and C) Ssrp1Neo LacZ fl/+ crossed with B6.129S ROSA26 FLPo (FlpO) mice to confirm pups with excision of the synthetic cassette (FRT Neo LacZ) shown in red. D) Immunoblot of protein extracts from spleen showing SSRP1 and SPT16 expression in Ssrp1+/+, Ssrp1fl/+, and Ssrp1fl/fl mice.