EMBO/soda-nel huggingface dataset:
Anti-tumoral activity of the G-quadruplex ligand pyridostatin against BRCA1/2-deficient tumours (2022)
Figure 2

Figure 2. Pyridostatin activates innate immune responses in BRCA2-deficient cells.

H1299+shBRCA2DOX cells were grown for 4 days in the presence (-BRCA2) or absence (+BRCA2) of DOX and subsequently treated with 5 or 10 μM pyridostatin (PDS) for 3 days. Whole-cell extracts were immunoblotted as indicated. KAP1 and IRF3 phosphorylation sites are shown in red. SMC1 and GAPDH were used as loading controls.

H1299+shBRCA2DOX cells were grown for 4 days in the presence or absence of DOX and subsequently treated with 10 μM pyridostatin (PDS) for 1, 2 or 3 days. Whole-cell extracts were immunoblotted as indicated. KAP1 and IRF3 phosphorylation sites are shown in red. SMC1 and GAPDH were used as loading controls.

Quantitative RT-PCR of cells grown as in (A) and treated with 10 µM pyridostatin (PDS) for 3 days was performed using primers specific for the indicated genes. mRNA levels are expressed relative to GAPDH and to -DOX (+BRCA2) cells. Error bars represent the SEM of n = 3 independent experiments, each performed in technical triplicate. P values were calculated using an unpaired two-tailed t-test. *, P ≤ 0.05; ***, P ≤ 0.001.

BRCA2+/+ and BRCA2-/- RPE-1 cells were treated with 10 μM pyridostatin (PDS) for 2 days. Whole-cell extracts were immunoblotted as indicated. KAP1, IRF3 and STAT1 phosphorylation sites are shown in red. Tubulin and GAPDH were used as loading controls.

H1299+shBRCA2DOX cells treated as in (B) were fixed and prepared for immunofluorescence with antibody against cGAS. DNA was counterstained with DAPI. Scale bar represents 20 µm.

Quantification of cGAS-positive micronuclei per cells shown in (E). Graph and error bars represent the mean and SEM of of n = 3 independent experiments. A minimum of 250 cells were analysed per condition per experiment. P values were calculated using an unpaired two-tailed t-test. *, P ≤ 0.05; **, P ≤ 0.01; NS, P > 0.5.

Exact P values for (C, F) are provided in Appendix Table S8.
Anti-tumoral activity of the G-quadruplex ligand pyridostatin against BRCA1/2-deficient tumours (2022)
Figure 3

Figure 3. Pyridostatin impairs growth of PARPi-resistant BRCA1-deficient tumours and triggers DNA damage and innate immune responses in BRCA1-deficient PARPi-resistant cells.

FVB female mice were injected intramuscularly with (A) BRCA1-proficient KP3.33 (Brca1+/+) or (B) BRCA1/53BP1-deficient KB1PM5 (Brca1-/-, Tp53bp1-/-) mouse mammary tumour cells. Pyridostatin (PDS) was administered intravenously (i.v.; 7.5 mg/kg/day) and talazoparib was administered orally (p.o.; 0.33 mg/kg/day), over the indicated periods of time. Vertical dotted line indicates end of treatment. Tumour volume was measured at the timepoints shown on the graph and expressed relative to tumour volume at the beginning of treatment. Each experimental group included n = 5 mice. Error bars represent SEM. P values were calculated between treated and untreated tumours at day 16, using an unpaired two-tailed t-test. ****, P ≤ 0.0001; NS, P > 0.5.

BRCA1-proficient KP3.33 (Brca1+/+), BRCA1-deficient KB1PM5 (Brca1-/-) and BRCA1/53BP1-deficient KB1PM5 (Brca1-/-/Tp53bp1-/-) mouse mammary tumour cells were treated with 2 µM pyridostatin (PDS) for 24 hours and released into fresh medium without pyridostatin. Whole-cell extracts were prepared 0 to 72 hours after release and immunoblotted as indicated. SMC1 was used as a loading control. KAP1 phosphorylation site is indicated in red.

BRCA1+/+, BRCA1-/- and BRCA1-/-/TP53BP1-/- RPE-1 cells were treated with 10 μM pyridostatin (PDS) or 2 μM olaparib for 2 days. Whole-cell extracts were immunoblotted as indicated. KAP1, IRF3 and STAT1 phosphorylation sites are shown in red. SMC1 and GAPDH were used as loading controls.

Exact P values for (A, B) are provided in Appendix Table S8.
Convergent dopamine and ALK4 signaling to PCBP1 controls FosB alternative splicing and cocaine behavioral sensitization (2022)
Figure 1

Figure 1. Dopamine and ALK4 signaling synergize to potentiate induction of ΔFosB mRNA and nuclear translocation of ΔFosB protein in NAc MSNs

(A) Schematic of the protocol used for consecutive stimulation of primary cultures of NAc MSNs. MSNs were extracted at embryonic day 16.5 (E16.5) and cultured for 7 days (7div) before 2 hours (2hr) treatment with either 1μM SKF81297 (SKF), 100ng/ml activin A (Act) or their combination (Act+SKF). Following 2 additional days (2div), the treatments were repeated and cultures fixed for imaging or collected for RNA extraction.

(B-F) Real-time quantitative PCR (RT-qPCR) analysis of ∆FosB (B), FosB (C), c-Fos (D), ARC (E) and EGR1 (F) mRNAs in MSNs cultures exposed to consecutive stimulation (same treatment in both treatment days) with activin A (Act), SKF81297 (SKF) or their combination as indicated. Results are expressed as average ± SEM of fold increase over vehicle. N=5.

(G) Representative immunofluorescence staining of ∆FosB (green) in cultured MSNs exposed to consecutive stimulation (same treatment in both treatment days) with activin A (Act), SKF81297 (SKF) or their combination as indicated. Counterstaining with MAP2 and DAPI is shown in the lower row. Scale bar, 25μM.

(H) Quantification of ∆FosB+ nuclei in cultured MSNs after the indicated treatments (same treatment in both treatment days). Results are expressed as average ± SEM of % of MAP2+ cells displaying ∆FosB in the nucleus. N=6.

(I) Representative immunofluorescence staining of ∆FosB (green) in cultured MSNs exposed to consecutive stimulation, with different 1st and 2nd treatments with activin A (Act), SKF81297 (SKF) or their combination as indicated. Counterstaining with MAP2 and DAPI is shown in the right column. Scale bar, 25μM.

(J) Quantification of ∆FosB+ nuclei in cultured MSNs after different 1st and 2nd treatments with activin A (Act), SKF81297 (SKF) or their combination as indicated. Results are expressed as average ± SEM of % of MAP2+ cells displaying ∆FosB in the nucleus. N=6.

(K) Representative immunofluorescence staining of ∆FosB (green) in cultured MSNs extracted from mutant mice lacking ALK4 in GABAergic neurons (Gad67Cre;Alk4fl/fl) and control mice (Alk4fl/fl) after exposure to consecutive stimulation (same treatment in both treatment days) with activin A (Act), SKF81297 (SKF) or their combination as indicated. Counterstaining with MAP2 and DAPI is shown in the second and fourth rows. Scale bar, 25μM.

(L) Quantification of ∆FosB+ nuclei in cultured MSNs from mutant and control mice after the indicated treatments (same treatment in both treatment days). Results are expressed as average ± SEM of % of MAP2+ cells displaying ∆FosB in the nucleus. N=6.

Data information: *, p<0.05; **, p<0.01;***, p<0.001; one-way ANOVA followed with Tukey's multiple comparisons test. N denotes biological replicates.
Convergent dopamine and ALK4 signaling to PCBP1 controls FosB alternative splicing and cocaine behavioral sensitization (2022)
Figure 3

Figure 3. PCBP1 and Smad3 interact with regulated intron 4 and adjacent sequences in FosB mRNA upon coincident activation of D1 and ALK4 receptors in NAc MSNs

(A, B) RT-qPCR analysis of FosB mRNA in PCBP1 (A) and Smad3 (B) immunoprecipitates from HeLa cells exposed to consecutive stimulation (same treatment in both treatment days) with activin A (Act), forskolin (Fsk) or their combination as indicated. Results are expressed as average ± SEM of fold increase over vehicle. N=4.

(C) Schematic showing the location of probes used for RNA-PLA studies on the interaction of PCBP1 and Smad3 with sequences in FosB and ∆FosB mRNAs. The top diagram shows the FosB gene with the relative position of exons (E1 to E4) and introns (I1 to I4). The location of the regulated intron 4 (rI4) subjected to alternative splicing in FosB mRNA is indicted with black dotted lines. In ∆FosB mRNA, the dotted line indicates the junction between exons 4a and 4B after rI4 splicing. The locations of probes for exon 3 (pE3), exon 4 (pE4), regulated intron 4 (prI4) and exon 4a/4b junction (pE4a/b) are indicated in green. The red bar shows the location of the stop codon that becomes in-frame in ∆FosB mRNA after rI4 splicing.

(D) Representative images of RNA-PLA (green) between PCBP1 (left) or Smad3 (right) and sequences in exon 4 (pE4) of FosB mRNA, respectively, in HeLa cells exposed to consecutive stimulation (same treatment in both treatment days) as indicated. Counterstaining with phalloidin and DAPI is shown in the second and fourth columns. Scale bars, 20μM.

(E, F) Quantification of RNA-PLA between PCBP1 (E) or Smad3 (F) and pE4 in HeLa cells after the indicated treatments. Results are expressed as average ± SEM of number of RNA-PLA dots per cell. Only nuclear dots were quantified in this analysis. N=4.

(G) Representative images of RNA-PLA (green) between PCBP1 (left) or Smad3 (right) and sequences in exon 4 (pE4) of FosB mRNA, respectively, in cultured MSNs exposed to consecutive stimulation as indicated. Counterstaining with phalloidin and DAPI is shown in the second and fourth columns. Scale bars, 20μM.

(H, I) Quantification of RNA-PLA between PCBP1 (E) or Smad3 (F) and pE4 in cultured MSNs after the indicated treatments. Results are expressed as average ± SEM of number of RNA-PLA dots per cell. Only nuclear dots were quantified in this analysis. N=4.

Data information: *, p<0.05; **, p<0.01;***, p<0.001; one-way ANOVA followed with Tukey's multiple comparisons test. N denotes biological replicates.
Convergent dopamine and ALK4 signaling to PCBP1 controls FosB alternative splicing and cocaine behavioral sensitization (2022)
Figure 4

Figure 4. PCBP1 is required for the synergistic effects of dopamine and ALK4 signaling on ΔFosB mRNA induction and nuclear translocation of ΔFosB protein in NAc MSNs

(A) RT-qPCR analysis of PCBP1 mRNA in cultures of naive MSNs (no virus) or MSNs treated with lentiviruses expressing shRNA against PCBP1 (shPCBP1) or a scrambled shRNA. Results are expressed as average ± SEM of fold increase over control uninfected cells . N=4.

(B, C) RT-qPCR analysis of ∆FosB (B) and FosB (C) mRNA in cultured MSNs treated with scrambled and shPCBP1 lentiviruses and exposed to consecutive stimulation (same treatment in both treatment days) with activin A (Act), SKF81297 (SKF) or their combination as indicated. Results are expressed as average ± SEM of fold increase over vehicle . N=4.

(D) Representative immunofluorescence staining of ∆FosB (red) in cultured MSNs treated with scrambled and shPCBP1 lentiviruses and exposed to consecutive stimulation (same treatment in both treatment days) with activin A (Act), SKF81297 (SKF) or their combination as indicated. Counterstaining with green fluorescent protein (GFP, expressed by the lentiviruses) and DAPI is shown in the second and fourth rows. Scale bar, 25μM.

(E) Quantification of ∆FosB+ nuclei in cultured MSNs treated with scrambled and shPCBP1 lentiviruses followed by the indicated treatments. Results are expressed as average ± SEM of % of GFP+ cells displaying ∆FosB in the nucleus. N=4.

Data information: *, p<0.05; **, p<0.01;***, p<0.001; one-way ANOVA followed with Tukey's multiple comparisons test in (A), two-way ANOVA followed with Tukey's multiple comparisons test in (B, C, and E). N denotes biological replicates.
Convergent dopamine and ALK4 signaling to PCBP1 controls FosB alternative splicing and cocaine behavioral sensitization (2022)
Figure 6

Figure 6. ALK4 is required in NAc MSNs of adult mice for sustained induction of ΔFosB mRNA and protein during cocaine sensitization

(A-F) RT-qPCR analysis of FosB (A, D), c-Fos (B, E) and ∆FosB (C, F) mRNAs in NAc of Gad67Cre;Alk4fl/fl (A-C) mutant mice 1 hour (A-C) or 24 hour (D-F) after the second saline (Sal) or cocaine (Coc) injection of TIPS. Results are expressed as average ± SEM of fold increase over the saline group (Sal/Sal) of Gad67Cre mice. N=5.

(G) Representative immunofluorescence staining of ∆FosB in Nac of control (Gad67Cre) and mutant (Gad67Cre;Alk4fl/fl) mice, 24 hours after the second injection of the TIPS protocol. Scale bars 20 μm.

(H) Quantification of ∆FosB positive cells 24 hours after the second injection of TIPS protocol in the Nac of Gad67Cre and Gad67Cre;Alk4fl/fl mice. Results are expressed as average ± SEM of the number of ∆FosB+ cells per mm2. At least 10 images from both left and right hemispheres were analyzed per mouse and condition. N=5.

(I) Western blot analysis of FosB and ∆FosB in in NAc of Gad67Cre and Gad67Cre;Alk4fl/fl mice 24 hour after the second injection of TIPS. Blots were probed with an antibody that recognizes both FosB (upper band, 48kDa) and ∆FosB (lower band, 38 kDa). Lower blot shows reprobing with anti-β-actin.

(J, K) Quantification of FosB (J) and ∆FosB (K) levels in NAc of Gad67Cre and Gad67Cre;Alk4fl/fl mice 24 hour after the second injection of TIPS. Results are expressed as average ± SEM of fold increase of FosB and ∆FosB over the saline group (Sal/Sal) of Gad67Cre mice normalized to β-actin levels. N=4s.

Data information: *, p<0.05; **, p<0.01;***, p<0.001; one-way ANOVA followed with Tukey's multiple comparisons test. N denotes biological replicates.
p53 transcriptionally regulates SQLE to repress cholesterol synthesis and tumor growth (2021)
Figure 1

Fig. 1 p53 suppresses cholesterol biosynthesis

A-G p53+/+ and p53-/- C57BL/6N male mice were treated as in A. Data are means ± SD (n=6 to 8). B Representative liver photos of mice livers. C Representative changes in liver weight. D H&E (hematoxylin-eosin) staining (left) and oil red O staining (right) of livers. Scale bar, 50μm. E Serum cholesterol concentrations of each group. Liver cholesterol concentrations (F) and triglyceride levels (G) of each group were examined.

H and I Cholesterol levels in p53+/+ and p53-/- HepG2 cells (H) or HCT116 cells (I) were examined. Protein expression was shown by western blotting (bottom panel).

J Cholesterol concentrations of p53+/+ and p53-/- HCT116 cells were determined by FilipinⅢ staining.

K Heat map analysis of 17 sterol biosynthesis genes and 3 other SREBP2 target genes from RNA-seq data using mice livers of p53+/+ and p53-/- mice fed with normal or HFD diet. Expression levels were normalized to the mean level of each gene among all samples and compared to p53+/+ normal diet mice. Color scale indicates the expression fold change of target gene.

L Enrichment of cholesterol biosynthesis genes in the liver of normal diet (left) and HFD diet (right). FDR: false discovery rate; NES: normalized enrichment score.

Data information: In C, E, F, G, H, I, J Bars represent mean ± s.d., *P<0.05; **P<0.01; ***P<0.001; For B, D, n=6-8 biologically independent samples; For F, G, H, I, J n=3 biologically independent samples; Statistical significance was determined by two-tailed unpaired t-test.
p53 transcriptionally regulates SQLE to repress cholesterol synthesis and tumor growth (2021)
Figure 2

Fig. 2 a SREBP2-independent mechanism for p53-mediated SQLE inhibition

A Heat map analysis of 17 sterol biosynthesis genes and 3 other SREBP2 target genes from RNA-seq data using p53+/+ and p53-/- HCT116 cells. Expression levels were normalized to the mean level of each gene among all samples and compared to p53+/+ cells. Color scale indicates the expression fold change of target gene.

B mRNA expression of SQLE in the mouse liver of p53+/+ and p53-/- mice with normal or HFD diet.

C mRNA and protein levels of SQLE in different tissues of p53+/+ or p53-/- mice were determined by qRT-PCR and western blot respectively.

D and E SQLE protein expression and mRNA levels were examined in p53+/+ and p53-/-HCT116 cells (D) and HepG2 cells (E). Actin was used as loading control.

F and G p53+/+ and p53-/- HepG2 cells were cultured for 48 hours in medium containing fetal bovine serum (Serum) or lipoprotein-depleted FBS (LPDS). Protein expression was shown by western blotting (F). mRNA levels of SQLE were examined by qRT-PCR (G). P: premature SREBP2; M: mature SREBP2.

H, I and J Protein expression and mRNA levels of p53+/+ and p53-/- HepG2 cells treated with control siRNA or three separated sets of SREBP2 siRNAs for 72 hours as indicated.

K p53+/+ and p53-/- HepG2 cells were treated with control siRNA or SREBP2 siRNA, followed by ectopic expressed RNA-resistant SREBP2 for 48 hours. mRNA and protein expression were examined by qRT-PCR and western blotting.

Data information: In B, C, D, E, G, H, I, J, K, Bars represent mean ± s.d., *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001; For B, n=4 biologically independent samples; For C, D, E, G, H, I, J, K, n=3 biologically independent samples; Statistical significance was determined by two-tailed unpaired t-test.
p53 transcriptionally regulates SQLE to repress cholesterol synthesis and tumor growth (2021)
Figure 3

Fig. 3 SQLE is a transcriptional target of p53

A Schematic representation of human SQLE genomic structure. Shown are the exon/intron organization and two potential p53 response elements (RE1 and RE2) and the corresponding mutant response elements.

B p53+/+ and p53-/- HCT116 cells were analyzed by chromatin immunoprecipitation (ChIP) assay using normal IgG and anti-p53 antibody as indicated.

C p53+/+ HCT116 cells transfected with control siRNA or SREBP2 siRNA for 72 hours were analyzed by ChIP assay with the indicated antibodies.

D Control and SREBP2 knockout MCF-7 cells using sgRNA CRISPR/Cas9 were analyzed by ChIP assay using anti-p53 or normal mouse IgG antibodies.

E The electrophoretic mobility shift assay (EMSA) of SQLE-RE2 or RE2 mut in the presence or absence of p53 antibodies as indicated. Blue box (shift band) means the binding between nuclear extracts and RE2, red box (super-shift band) means p53 as the protein presented in the EMSA band.

F Luciferase reporter constructs containing RE1, RE2, RE1mut or RE2mut were transfected into p53-/- HCT116 cells together with control (PRK5-flag vector) or p53 expression vector (PRK5-flag p53) for 48 hours. Renilla vector pRL-CMV was used as a transfection internal control. Relative levels of luciferase are shown. Data represent three independent experiments. Protein expression is shown

G Luciferase reporter constructs containing RE1, RE2, RE1mut or RE2mut were transfected into HEK293T cells together with control (PRK5-flag vector) or p53 expression vector (PRK5-flag p53) for 48 hours. Renilla vector pRL-CMV was used as a transfection internal control. Relative levels of luciferase are shown. Protein expression is shown.

H Luciferase reporter constructs containing RE2 were transfected into HEK293T cells together with control, wild-type p53 or p53 R175H expression vector for 48 hours. Renilla vector pRL-CMV was used as a transfection internal control. Relative levels of luciferase are shown.

I Luciferase reporter assay with indicated response element constructs in human HEK293T cells co-transfected with or without p53 expression vector.

J Luciferase reporter constructs containing RE2 were transfected into HEK293T cells treated with or without 20μm PFTα for 24 hours. Renilla vector pRL-CMV was used as a transfection internal control. Relative levels of luciferase are shown.

Data information: In B, C, D, F, G, H, I, J Bars represent mean ± s.d., *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001; n=3 biologically independent samples; Statistical significance was determined by two-tailed unpaired t-test.
p53 transcriptionally regulates SQLE to repress cholesterol synthesis and tumor growth (2021)
Figure 4

Fig. 4 p53 inhibits cholesterol metabolism through SQLE

A-C Squalene (A), lanosterol (B) and cholesterol (C) abundance in p53+/+ and p53-/- HepG2 cells transfected with control or SQLE siRNA for 48 hours were determined via ultra-high pressure liquid chromatography coupled to mass spectrometry (UHPLC-MS). Protein levels were analyzed by western blotting with specific antibodies (A bottom panel).

D p53+/+ and p53 -/- HCT116 cells were transfected with control or SQLE siRNA for 48 hours as indicated. Relative cholesterol concentrations were examined as described in methods. Protein expression were determined by western blot.

E and F Cholesterol levels of HCT116 cells (E) and HepG2 cells (F) treated with or without 30μm terbinafine for 72 hours.

G and H Triglyceride levels in p53+/+ and p53-/- HCT116 cells (G) and HepG2 cells (H) treated with SQLE siRNA or control siRNA for 48 hours.

Data information: A-H, Bars represent mean ± s.d., *P<0.05; **P<0.01; ***P<0.001; n=3 biologically independent samples; Statistical significance was determined by two-tailed unpaired t-test.
p53 transcriptionally regulates SQLE to repress cholesterol synthesis and tumor growth (2021)
Figure 5

Fig. 5 SQLE supports tumor growth through cholesterol

A Protein expression of SQLE in human liver tumors (C) and non-cancerous adjacent tissue (N) were analyzed by western blotting.

B SQLE gene expression in HCC and adjacent normal tissue was analyzed in TCGA Liver Hepatocellular Carcinoma (normal [n=50] versus tumors [n=371]).

C SQLE gene expression in COAD and adjacent normal tissue was analyzed in TCGA Colon Cancer (normal [n=41] versus tumors [n=286]).

D-F p53+/+ HepG2 cells were stably overexpressed SQLE or vector control. Cell proliferation (D), number of colonies (E) and cholesterol concentration (F) are shown respectively. Protein expression was analyzed using western blotting (F bottom panel).

G and H p53+/+ HepG2 cells transfected with control siRNA or SQLE siRNA were cultured in LDPS medium containing 5μg cholesterol for six days as indicated. Cell number (G) and cholesterol concentration (H) were determined. Protein expression was analyzed by western blotting after siRNA transfection 48 hours (G bottom panel).

I p53+/+ HCT116 cells transfected with control siRNA or SQLE siRNA for 48 hours and then cells were cultured in LDPS medium containing 5μg cholesterol for 6 days as indicated. Cell proliferation is shown. Protein expression was analyzed by western blotting after siRNA transfection 48 hours (bottom panel).

J - M p53+/+ HCT116 cells (J and K) and p53+/+ HepG2 cells (L and M) were transfected with SQLE siRNA or control siRNA in the presence or absence of exogenous SQLE cDNA for 48 hours. Cholesterol concentration (upper panel) and protein expression (bottom panel) were determined (J and L). Cell proliferation is shown (K and M).

Data information: B, C, D-M, Bars represent mean ± s.d., *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001; n=3 biologically independent samples; Statistical significance was determined by two-tailed unpaired t-test.