Fig 2: BAZ1B homozygous knockout cells exhibit a unique nuclear phenotype. Representative images of RPE1 (a), BAZ1B-N6 (b) and BAZ1B-O4 (c) nuclei counterstained with DAPI. White arrow-heads in (b) and (c) point to block-containing nuclei. White arrows point to the DAPI-dense Barr body. Images were captured using settings defined for RPE1 that result in pixel saturation for block-containing BAZ1B -/- nuclei. (d) RPE1 and BAZ1B -/- nuclei stained with DAPI showing the appearance of large DAPI-dense blocks in a fraction of mutant cells, as indicated to the left.

Fig 3: WSTF-RPB1 is strongly associated with the anti-AML activity. Triptolide downregulates the WSTF and RPB1 signaling pathway without affecting TAB1/P38 signaling in a dose dependent manner in MV-4-11 (A) and THP-1 (B). Time-dependent experiments show that triptolide inhibits the WSTF-RPB1 signaling pathway with no effect on TAB1/P38 signaling in MV-4-11 (C) and THP-1 (D). (E) The different expression levels of WSTF, XPB, and RPB1 is shown.

Fig 4: A speculative two-step model in which NM1 bridges the pol I machinery and chromatin via an interaction with SNF2h that competes with actin.(I) NM1 interacts with polymeric actin and with the rDNA via its C-terminus, generating local force that pulls the polymerase along active gene. (II) Upon NM1 dissociation from actin, NM1 interacts with SNF2h in a WSTF-dependent manner, a mechanism that provides a way to stabilize the WICH complex on the rDNA, to recruit PCAF, and to maintain the levels of H3K9 acetylation required for transcription activation.

Fig 5: A functional NM1 is required for the activation of pol I transcription.(A) RNAi-mediated NM1 gene knockdown in HeLa cells analyzed by double immunostaining and confocal microscopy with antibodies to NM1 and fibrillarin, after transfection of NM1-specific siRNA or control oligos (scrRNAi). Scale bars, 5 µm. (B) Steady state expression levels for NM1, WSTF, SNF2h and actin monitored on immunoblots of total lysates prepared from HeLa cells subjected to NM1 gene knockdown (NM1 RNAi) or from cells subjected to scrRNAi. (C) Semiquantitative densitometric quantification of NM1 steady state protein expression relative to actin. (D) qRT-PCR analysis of 45S pre-rRNA performed on total RNA prepared from HeLa cells subjected to NM1 gene knockdown (NM1 RNAi) or from cells subjected to control siRNA oligonucleotides (scrRNAi). The 45S pre-rRNA levels are relative to GAPDH mRNA. (E–F) FUrd incorporation assays on living HeLa cells subjected to (E) NM1 gene knockdown by RNAi, or (F) treated with BDM. Transcription was monitored by short FUrd pulses to follow incorporation into nascent nucleolar transcripts. Quantification of the FUrd foci after immunostaining and confocal microscopy was performed by measurements on randomly selected nucleolar regions in the images. The signal was quantified using ImageJ software. The average of the mean grey values in control cells was determined, and defined as hundred percent of signal. The average of the mean grey values measured after treatment was expressed proportionally. n = number of cells in each experiment. Error bars represent standard deviations. (G) rRNA synthesis in HEK293T cells stably expressing V5-wtNM1, V5-RK605AA, V5-?C or V5-?IQ NM1 mutants. For the analysis, relative 45S pre-rRNA levels were monitored from total RNA preparations by RT–qPCR using GAPDH mRNA as internal control. Error bars represent the standard deviation of three independent experiments. Significances [p RK605AA NM1 = 0.019 (*), p ?C NM1 = 0.0006 (***), p ?IQ NM1 = 0.05 (*)] were obtained by Student's T-test, two-sample, equal variance.