Fig 2: OGRE/G4 deletion strongly decreases the DNA replication activity of an endogenous origin. a The OGRE/G4 sequence of an endogenous origin (Ori1) was deleted and the deletion was confirmed using a restriction site close to the targeted sequence (see “Methods” section). b G4 formation propensity profiling of the Ori1 sequence targeted for deletion. The Ori1 sequence is located on chromosome 11 and presents a strong peak in the G4-Hunter score profile (red line). Such peak is not present upon OGRE/G4 deletion (alleles 1 and 2, blue and green dotted lines, respectively), and no point above 1 or below -1 is observed. This argues against the probability of G4 formation at this mutated locus. The striped lines on the top indicate the extent of the deletion in allele 1 (red and blue) and allele 2 (green and blue). c Nascent strand enrichment of Ori1 in parental NIH 3T3 cell line (black) and in mutant clones with the deletion (grey). Replication activity was strongly decreased after deletion of the OGRE/G4 sequence, whereas the activity of the external origin (Ori 2) did not vary. The background control regions Bcgd1 and 2 are located in origin-free regions. Results are the mean ± SD of 3 independent experiments; p values were obtained using the two-tailed Student’s t test; *p = 0.05; **p = 0.01; ***p = 0.001, p > 0.05. d Deletion of the OGRE/G4 did not affect the transcription level of the Rai1 gene, associated with Ori1. As a control, the housekeeping genes Actb and Gapdh were used. Results are the mean ± SD of 3 independent experiments; p values were obtained using the two-tailed Student’s t test; p > 0.05. Primer sequences are in Supplementary Table 2. e DNA replication activity was assessed as in Fig. 1c with the EBV origin, or with the 500 bp OGRE/G4 element of Ori2, or after scrambling or deletion of the same OGRE/G4 sequence. Results are the mean ± SD of 4–5 independent experiments; p values were obtained using the two-tailed Student’s t test; *p = 0.05; **p = 0.01; p > 0.05. Note that data presented for episome containing delta oriP and OGRE/G4-containing origin were performed independently from results presented in the Fig. 1; ns not significant: p > 0.05

Fig 3: The TGF-ß pathway and other intracellular signaling pathways in LM3 cells. (A) Immunoblot analysis of phosphorylated Smad2, p38MAPK and ERK1/2, as well as the levels of total Smad2 and Smad4 in LM3 cells. Actin was used as loading control. (B) Smad4 nuclear localization by immunofluorescence in LM3 cells treated with or without 2 ng/ml TGF-ß1 (magnification, ×1000). Mv1Lu cells were used as a control of the TGF-ß response. White arrows show Smad4 nuclear localization. (C) Smad-dependent transcriptional activity in LM3 cells in response to 1 ng/ml TGF-ß1 for 16 h. Prior to treatment, cells were co-transfected with luciferase reporters SBE-Lux and 3TP-lux (see Materials and methods). NMuMG cells were employed as a control (inset). Experiments were performed in triplicates and repeated at least twice. Data represent the mean ± SD of luciferase relative units (LRU) from triplicates. (D) PAI-1 mRNA expression in LM3 cells treated with or without 2 ng/ml TGF-ß1 for 8 or 24 h, by RT-PCR. NMuMG cells were used as control of TGF-ß response. ß-actin (Actb) was employed as a control.

Fig 4: Gene expression for rat heat shock protein-70 (Hspa4), heme oxygenase (Hmox1), metallothionein (Mt1), GCLM (Gclm), and GCLC (Gclc) in isolated neonatal rat lateral and fourth choroid plexus (CP) tissues exposed in vitro to cadmium. Lateral and fourth CP tissues from four neonatal rats were harvested and pooled; this comprised a single sample (i.e., n = 1). Each set of tissues was incubated in serum-free medium (SFM) with 0 or 500 nM CdCl2 for 24 h (37 °C). The mRNA was analyzed in triplicate by qualitative RT-PCR in six separate sets of CP tissues. The mRNA expression for each test gene was normalized to mRNA for rat ß-actin (Actb) and GAPDH (Gapdh). Values are expressed as relative mRNA expression for control and cadmium-treated tissues. Data are means ± SE; n = 6 sets of tissues. * p < 0.05 vs. Control.

Fig 5: Bmal1-/- mouse embryonic stem cells (mESCs) remain undifferentiated and express higher level of Nanog protein than wild-type cells.(A) Scheme of the Bmal1 gene (black bars, exons) and the CRISPR/Cas9 strategy used. DNA sequence targeted by the guide RNA (red) and the localization of the PAM domain (blue) are indicated. (B) Alignment of DNA sequences of the Bmal1 gene upon genotyping of Bmal1-/- mESCs obtained using CRISPR/Cas9. DNA regions targeted by the guide RNA are indicated in red. (C) Microscopy images of Bmal1-/- and wild-type mESCs grown in the 2i + LIF medium. Scale bar is 100 µm. (D) RT-qPCR analysis of Bmal1 mRNA in wild-type and Bmal1-/- mESCs. ActB was used as a housekeeping control. Expression was normalized to Gapdh and RNA18s and multiplied by 10 for better representation. n.d. indicates non-detection of transcripts. Mean and SEM of three independent experiments is shown. (E) Western blot analysis of Bmal1 protein using whole-cell extracts of Bmal1-/- and wild-type mESCs. Lamin B provides a loading control. (F) mRNA expression analysis of Nanog, Oct4, and Rex1 in Bmal1-/- and wild-type mESCs by RT-qPCR. Expression was normalized to Gapdh and RNA18s and multiplied by 10 for better representation. Asterisk (*) indicates significant difference (T-student P < 0.05). (G) Western blot analysis of whole-cell extracts comparing the protein levels of Nanog and Oct4 in Bmal1-/- and wild-type mESCs. Lamin B was used as a loading control. (H) Immunofluorescence analysis of Nanog protein (green) expression in Bmal1-/- and wild-type mESCs. DAPI stains nuclear DNA (blue). Scale bar is 10 µm. (I) Histogram showing the percentage of cells with low or high intensity of Nanog staining in parental and Bmal1-/- mESCs. Mean and SEM of three independent experiments is shown. Asterisks (*) indicate significant difference (Fisher’s test, P < 0.01).