EMBO/soda-nel huggingface dataset:
Inhibition of DNA methylation promotes breast tumor sensitivity to netrin-1 interference. (2016)
Figure 3

Figure 3. Netrin-1 blocking antibody net1-mAb triggers apoptosis in hypomethylated MDA-MB-231 and HMLER breast cancer cell lines.

A, B Cells were treated with DAC (10 μM, 72 h), and/or net1-mAb (10 μg/ml, 48 h) and/or recombinant netrin-1 (5 μg/ml, 48 h). TUNEL assays from 3 independent experiments. *** P<0.0001 one-way ANOVA.

C Cellular mortality was assessed using the toxilight assay. Data represent mean ± s.e.m. from 3 independent experiments. **** P<0.0001, one-way ANOVA.

D Loss of DNA methylation of the NTN1 and DAPK1 promoters in decitabine treated tumors, compared with PBS treated tumors. >1700 sequences were analyzed per group in 2 independent experiments. **** P< 0.0001, two-way ANOVA and post-hoc Tukey-test.

E, F After one week of treatment, tumors from xenografted mice were fixed in formalin, embedded in paraffin, and sliced into 4 μm sections. (E) Levels of DAPK1, UNC5B, and netrin-1 were measured in 4 independent tumors per treatment group by immunohistochemistry staining, and expressed as a percentage of total tumor surface normalized against the control PBS-group. Data represented as means ± s.e.m. **** P<0.0001, two-way ANOVA and post-hoc Tukey-test. (F) Representative tumor sections corresponding to MDA-MB-231 xenografts, scale bars = 50 μm.

G, H When tumors reached 100 mm3, mice were injected subcutaneously with decitabine (0.4 mg/kg) or PBS and/or intraperitoneally with net1-mAb (10 mg/kg). (G) Tumor volumes were measured twice a week. The statistical significance of the differences obtained between the control PBS-group and treated (DAC + net1-mAb) group was determined by two-way ANOVA and post-hoc Tukey-test. *** P<0.0001. (H) Tumor weights were measured 3 days after the end of the experiment. The statistical significance of the differences obtained between the groups was determined by one-way ANOVA, *** P<0.0001. Error bars = s.e.m.
Inhibition of DNA methylation promotes breast tumor sensitivity to netrin-1 interference. (2016)
Figure 4

Figure 4. DAC treatment triggers upregulation of genes in the netrin-1/dependence receptor signaling pathway, and, when combined with net1-mAb, induces apoptosis and tumor growth inhibition in a mouse model bearing a patient-derived breast tumor.

A Loss of DNA methylation of the NTN1 and DAPK1 promoters in decitabine treated PDX tumors, compared with the control PBS-group. The percentage of mean DNA methylation of the 11 DAPK1-CpGs was 94% (550 amplicons analyzed), while that of the 7 NTN1-CpGs was 64% (213 amplicons analyzed). Two-way ANOVA and post-hoc Tukey-test.

B Expression of DAPK1, UNC5B, and NTN1 was measured by Q-RT-PCR in PDX tumors after 7 days of in vivo DAC treatment (0.4 mg/kg). The level of PBGD expression was used as an internal control. Data are expressed as mean ± s.e.m. for at least 3 grafts per group. **** P<0.0001,, two-way ANOVA and post-hoc Tukey-test.

C Levels of DAPK1, UNC5B, and netrin-1 were measured in at least 3 independent paraffin embedded xenografts per treatment group by immunohistochemistry. Data are expressed as the percentage of the total tumor surface normalized against the median tumor surface of the PBS-group. **** P<0.0001, one-way ANOVA. Error bars = s.e.m.

D Cleaved caspase-3 and DNA fragmentation (TUNEL) median number of cells per mm2 were measured in treated xenografts. Data represent mean ± s.e.m. from at least 3 tumors per group. **** P<0.0001, one-way ANOVA.

E Representative sections of the xenograft PDX analyzed in (D). Scale bars = 50 μm. Arrows indicate positive staining.

F The combination of net1-mAb and DAC reduces human breast tumor growth in immuno-compromised mice. After anesthesia, mice were engrafted in the interscapular area with a ≈ 60 mm3 patient-derived tumor. When the tumors reached 120-150 mm3, mice were injected subcutaneously with decitabine (0.4 mg/kg) or PBS and/or intraperitoneally with net1-mAb (10 mg/kg) or with a human IgG1 control isotype antibody (Ctrl IgG1, 10 mg/kg) from day 1 to day 21. Tumor volumes were measured twice a week; n = 7 mice per group. The statistical significance of the differences obtained between the DAC + Ctrl IgG1-group and the DAC + net1-mAb-group was determined by two-way ANOVA2 and a post-hoc Tukey-test, **** P<0.0001. Error bars = s.e.m.
Inhibition of DNA methylation promotes breast tumor sensitivity to netrin-1 interference. (2016)
Figure 5

Figure 5. Sensitivity of MDA-MB-231 cell lines stably transfected with shRNA targeting DAPK1, UNC5B, and NTN1 to treatments combining DAC and net1-mAb.

A, D Stably transfected MDA-MB-231 cells bearing a control (A), DAPK1 (B), UNC5B (C), or NTN1 shRNA (D) were injected into the mammary fat pad of immuno-compromised mice. When tumors reached 100-120 mm3, mice were injected subcutaneously with DAC (0.4 mg/kg) or PBS and/or intraperitoneally with net1-mAb (10 mg/kg). Tumor volumes were measured twice a week, n = 8 mice per group. The statistical significance of the differences obtained between DAC-group and DAC + net1-mAb-group for shControl, DAC-group and DAC + net1-mAb-group for shDAPK1 and shUNC5B, and PBS-group and DAC-group for shNTN1, respectively, were determined by two-way ANOVA and a post-hoc Tukey-test. **** P<0.0001, , ns = not significant. Error bars = s.e.m.
PIF1 helicase promotes break-induced replication in mammalian cells (2020)
Figure 2

Figure 2. PIF1 is required for BIR to repair DSBs at broken forks to promote replication restart.

(A) U2OS WT or PIF1-KO cells were treated with the indicated concentrations of HU (left) or APH (right) for 72 hr, and the cell viability assay was performed.

(B) U2OS WT or PIF1-KO cells were labeled with CldU for 30 min followed by incubation with 2 mM HU for 2 hr and then IdU for another 30 min. Labeled cells were processed for DNA fiber analysis. Representative images of stalled or restarted forks and forks with new origin firing were shown (left). The percentage of restarted forks was quantified by analyzing of 110-130 fibers for each experiment (right). Experiments were repeated four times for each sample.

(C) Schematic drawing of the EGFP-BIR-5085 reporter and the repair steps leading to the repair product expressing EGFP after Cas9n/sgRNA-5085 expression.

(D) U2OS (EGFP-BIR-5085) cell lines carrying Dox-inducible Cas9/sgRNA-5085 (Dox-Cas9) or Cas9n/sgRNA-5085 (Dox-Cas9n) were incubated with or without Dox (5 µg/ml) and assayed by FACS analysis 2 days later (left). U2OS (EGFP-BIR-5085, Dox-Cas9 or Dox-Cas9n) cells expressing shRNAs RAD51, POLD3 and PIF1 or shRNA vector (Ctrl) were incubated with 5 µg/ml Dox, and FACS analysis was performed after 2 days (right).

(E) Track length of single EGFP-positive clones derived from U2OS (EGFP-BIR-5085) cells after Cas9/sgRNA-5085 (n=47) or Cas9n/sgRNA-5085 (n=39) expression was analyzed by sequencing of the PCR products from genomic DNA covering the repair junctions.

Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as **P < 0.01, ***P < 0.001.
PIF1 helicase promotes break-induced replication in mammalian cells (2020)
Figure 3

Figure 3. PIF1 is required for BIR at Flex1 upon replication and oncogenic stress.

(A) Schematic drawing of the EGFP-Flex1-BIR-5086 reporter. Flex1: 0.3 kb Flex1(AT)34 derived from CFS FRA16D.

(B) U2OS (EGFP-Flex1-BIR-5086) cells were treated with or without 2 mM HU for 24 hr, and the percentage of EGFP positive cells was quantified by FACS analysis 3 days after HU removal.

(C) U2OS (EGFP-Flex1-BIR-5086) cells expressing shRNAs for RAD51, POLD3 and PIF1 or shRNA vector (Ctrl) were treated with 2 mM HU for 24 hr. The percentage of EGFP positive cells by HU induction was quantified by FACS analysis 3 days after HU removal.

(D) U2OS (EGFP-Flex1-BIR-5086) cells were infected by retrovirus expressing H-RAS-V12-FLAG (RAS) or Cyclin E-HA (Cyc E), and the percentage of EGFP positive cells was quantified by FACS analysis 4 days after retrovirus infection (top). The expression of RAS or Cyclin E is shown by Western blot analysis (bottom).

(E) U2OS (EGFP-Flex1-BIR-5086) cells expressing shRNAs for RAD51, POLD3 and PIF1 or shRNA vector (Ctrl) were infected by retroviruses expressing RAS, and the percentage of EGFP positive cells was quantified by FACS analysis 4 days after retrovirus infection (top). Expression level of RAD51 and POLD3 is shown by Western blot analysis (bottom).

(F) U2OS WT or PIF1-KO cells were infected by retrovirus expressing RAS or empty vector. Cell profiling was plotted by counting cell numbers every 24 hr, and normalized to the cell number on the first day.

Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as **P < 0.01.
PIF1 helicase promotes break-induced replication in mammalian cells (2020)
Figure 4

Figure 4. BIR is used at replication-associated DSBs even for STGC.

(A) Proposed models for repair of DSBs generated at broken replication forks (see text for details). seDSB: single-ended DSB; deDSB: double-ended DSB. Pink arrows: endonucleases to remove Flex1 or other secondary structures at DSB ends or DNA tails at the fork junctions.

(B) Schematic drawing of the EGFP-STGC-1731 reporter and proposed pathways to repair DSBs generated by endonuclease cleavage (I-SceI or Cas9, left) or converted from nicks (Cas9n, right).

(C) U2OS (EGFP-STGC-1731) cell lines carrying Dox-inducible Cas9/sgRNA-1731 (Dox-Cas9) or Cas9n/sgRNA (Dox-Cas9n) were incubated with or without Dox (5 µg/ml), and the percentage of EGFP positive cells was quantified by FACS analysis 2 days later (top). U2OS (EGFP-STGC-1731, Dox-Cas9 or Dox-Cas9n) cells expressing shRNAs for RAD51, POLD3 and PIF1 or shRNA control (Ctrl) were incubated with Dox (5 µg/ml), and the percentage of EGFP positive cells was quantified by FACS analysis after 2 days (bottom).

(D) Schematic drawing of the EGFP-Flex1-STGC-1541 reporter and the proposed mechanism to repair DSBs at Flex1 generated upon fork breakage by SDSA which involves two DSB ends.

(E) U2OS (EGFP-Flex1-STGC-1541) cells were treated with or without 2 mM HU for 24 hr, and the percentage of EGFP positive cells by HU induction was quantified by FACS analysis 4 days after removal of HU (left). U2OS (EGFP-Flex1-STGC-1541) cells expressing shRNAs for RAD51, POLD3 and PIF1 or shRNA vector (Ctrl) were treated with 2 mM HU for 24 hr, and the percentage of EGFP positive cells was quantified by FACS analysis 4 days later (right).

(F) U2OS (EGFP-Flex1-STGC-1541) cells were infected by retroviruses expressing RAS or empty vector, and the percentage of EGFP positive cells was assayed by FACS 5 days following infection (left). U2OS (EGFP-Flex1-STGC-1541) cells expressing shRNAs for RAD51, POLD3 and PIF1 shRNAs or shRNA vector were infected by retroviruses expressing RAS, and the percentage of EGFP positive cells was assayed by FACS 5 days after infection (middle). Expression level of RAD51 and POLD3 is shown by Western blot analysis (right).

(G) Proposed models for activating BIR at replication-dependent and replication-independent DSBs (see text for details).

Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as **P < 0.01 and n.s. (not significant) P>0.05.
PIF1 helicase promotes break-induced replication in mammalian cells (2020)
Figure 6

Figure 6. PIF1- and POLD3-mediated BIR is important for repairing DSBs at Flex1 caused by

FANCM deficiency.

(A) U2OS (EGFP-Flex1-BIR-5086) cells were infected by lentiviruses expressing FANCM shRNA or shRNA vector (Ctrl) (left) or cells from the same reporter cell line expressing shRNAs for RAD51, POLD3 and PIF1 or shRNA vector (Ctrl) were infected by FANCM shRNA lentiviral viruses (right). The percentage of EGFP positive cells after spontaneous recombination was quantified by FACS analysis 5 days after lentiviral infection of FANCM shRNA. Knock down of FANCM by shRNA is shown by qPCR and Western blot in Appendix Fig S12A.

(B) U2OS (EGFP-Flex1-STGC-1541) cells expressing shRNAs for RAD51, POLD3 and PIF1 or shRNA vector were infected by lentivirus expressing FANCM shRNA. The percentage of EGFP positive cells was quantified by FACS analysis 5 days after FANCM shRNA lentiviral infection. Knock down of FANCM by shRNA is shown by qPCR and Western blot in Appendix Fig S12B.

(C) Proposed model for concerted roles of FANCM-dependent fork reversal and PIF1/POLD3-dependent BIR in protection of Flex1 stability. Pink arrow: endonuclease cleavage to remove Flex1.

(D) Growth curves of WT or FANCM-KO HCT116 cells were plotted after expressing POLD3 (left) or PIF1 (right) shRNA or shRNA vector (Ctrl). Cell number was normalized to that on day one.

Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as **P < 0.01.
Role of PCIF1-mediated 5′-cap N6-methyladeonsine mRNA methylation in colorectal cancer and anti-PD-1 immunotherapy (2022)
Figure 1

Figure. 1 PCIF1 is upregulated in CRC.

A. Representative images of IHC staining of PCIF1 in three CRC tissues and adjacent noncancerous tissues. Scale bar, 50 μm (left). Analysis of the PCIF1 protein expression in normal and CRC tissues. Each dot represents one sample (right). Data are the mean ± SEM. **P < 0.01, by Student's t-test.

B. Kaplan-Meier plots of overall survival of 27 CRC patients with low tumor PCIF1 expression (0-4.5 staining score, n = 18) or high tumor PCIF1 expression (4.6-9.0 staining score, n = 9). Data are the mean ± SEM. ***P < 0.001 by Student's t-test.

C. Western blot analysis of PCIF1 in normal colon cells (CCD841CON) and various CRC cell lines. GAPDH served as a loading control.

D. qRT-PCR analysis of PCIF1 mRNA in the indicated cell lines. Levels were normalized to GAPDH expression. Data are the mean ± SD of n=3 biological replicates. *P < 0.05, **P < 0.01, ***P < 0.001 by Student's t-test.

E. Representative immunofluorescence images of HT29 and HCT116 cells stained for endogenous PCIF1 protein (green). Nuclei were stained with DAPI (blue). Scale bar, 30 μm.

F. Western blot analysis of PCIF1 in whole extract (W), cytosolic (C), and nuclear (N) fractions of HCT116 and HT29 cells. Tubulin and lamin A/C served as cytosolic and nuclear markers, respectively.
Role of PCIF1-mediated 5′-cap N6-methyladeonsine mRNA methylation in colorectal cancer and anti-PD-1 immunotherapy (2022)
Figure 2

Figure. 2 PCIF1 depletion reduces CRC tumorigenesis.

A. Western blot analysis of PCIF1 in HT29 and HCT116 cells depleted of PCIF1 using four different sgRNAs. GAPDH served as a loading control.

B-E. Matrigel invasion assay (B), fibronectin adhesion assay (C), colony formation assay (D), and wound healing migration assay (E) of the indicated control and PCIF1-depleted CRC cell lines. Representative images and quantification of cells are shown at the left and right, respectively, of each panel. Data are the mean ± SD of n=3 replicates/condition. *P < 0.05, **P < 0.01, ***P < 0.001 by Student's t-test.

F. Growth of the indicated HCT116 tumors after subcutaneous injection into athymic nude mice. Tumor volume was recorded on the indicated days. Data are the mean ± SEM of the number of mice/group indicated on each panel. n, the numbers of mice. *P < 0.05, **P < 0.01 by Student's t-test.

G. Survival analysis of mice injected bearing the indicated HCT116 tumors. Data are the mean ± SEM of the number of mice/group indicated on each panel. n, the numbers of mice. **P < 0.01, ***P < 0.001 by Student's t-test.
Role of PCIF1-mediated 5′-cap N6-methyladeonsine mRNA methylation in colorectal cancer and anti-PD-1 immunotherapy (2022)
Figure 3

Figure. 3 PCIF1 target genes identified by RNA-seq and m6Am-exo-seq.

A. LC-MS/MS identification and quantification of m6Am or m6A levels in mRNA from control and PCIF1-depleted HCT116 cells. Data are presented as the enrichment of methylated vs unmethylated mRNA. Data are the mean ± SD of n=3 biological replicates. **P < 0.01 by Student's t-test.

B. Metagene plots analysis of m6Am enrichment around the transcription start site (TSS) of all expressed genes in control and PCIF1-depleted HCT116 cells.

C. Venn diagram of PCIF1 peaks number, downstream genes and identified genes from m6Am-exo-seq and RNA-seq.

D. Meta-enrichment analysis summary for the 76 genes identified as PCIF1 targets in HCT116 cells.

E. Western blot analysis of FOS and PCIF1 in control and PCIF1-depleted HCT116 and HT29 cells. GAPDH served as a loading control.

F. Genome browser views of FOS with m6Am sites. Read coverage of input sample and IP sample are shown in blue and red, respectively, and green rectangle indicates the m6Am peaks located near the TSS.

G. m6Am-exo-qPCR analysis of m6Am enrichment in FOS mRNAs from the indicated HCT116 cells. GAPDH served as a negative control. Data are the mean ± SD of n=3 biological replicates. **P < 0.01 by Student's t-test.

H. qRT-PCR analysis of FOS mRNA stability in control and PCIF1-depleted HCT116 and HT29 cells treated with actinomycin D for the indicated times. Data are the mean ± SD of n=3 biological replicates. *P < 0.05, **P < 0.01 by Student's t-test.

I. Growth of tumors after subcutaneous injection of the indicated HCT116 cells into athymic nude mice. Tumor volumes were recorded on the indicated days. Data are the mean ± SEM of n=5. *P < 0.05 by Student's t-test.
Role of PCIF1-mediated 5′-cap N6-methyladeonsine mRNA methylation in colorectal cancer and anti-PD-1 immunotherapy (2022)
Figure 4

Figure. 4 Functional role of FOS in CRC.

A. Western blot analysis of FOS in control and FOS-depleted HCT116 and HT29 cells. GAPDH served as a loading control.

B-D. MTS cell proliferation assay (B), wound healing migration assay (C), and colony formation assay (D) of control and FOS-depleted HCT116 cells. Representative images and quantification of colonies are shown to the left and right, respectively. Data are the mean ± SD of n=3 biological replicates. *P < 0.05, **P < 0.01, ***P < 0.001 by Student's t-test.

E. Growth of control and FOS-depleted HCT116 tumors after subcutaneous injection into athymic nude mice. Tumor volume was recorded on the indicated days. Data are the mean ± SEM of n=10 mice/group. **P < 0.01 by Student's t-test.

F. Survival analysis of mice bearing control and FOS-depleted HCT116 tumors. Data are the mean ± SEM of n= 5 mice/group. *P < 0.05 by Student's t-test.

G. Positive correlation between FOS and PCIF1 protein levels in tumors from 29 patients with CRC (R2 =0.6902, P < 0.001).
Role of PCIF1-mediated 5′-cap N6-methyladeonsine mRNA methylation in colorectal cancer and anti-PD-1 immunotherapy (2022)
Figure 7

Figure. 7 On-target efficacy of LNP-siRNA in vitro and in vivo

A. Structures of siRNAs utilized in this study.

B. mRNA expression of PCIF1 was quantified by qRT-PCR in the HCT116 cells transfected with gradient amount of control and PCIF1 siRNAs. Data are the mean ± SD of n=3 biological replicates. *P < 0.05, **P < 0.01, ***P < 0.001 by Student's t-test.

C. Western blot analysis of PCIF1 in HCT116 cells with control and PCIF1 siRNAs. GAPDH served as a loading control.

D. Cell viability assays were performed in in the HCT116 cells transfected with 100pmol control and PCIF1 siRNAs. Data are the mean ± SD of n=3 biological replicates. ***P < 0.001 by Student's t-test.

E. Size of LNP-siRNA formulations were analyzed by Zetasizer. Data are the mean ± SD of n=3 biological replicates.

F. Tumor growth of HCT116 cells treated with 1mg/kg control and PCIF1 LNP-siRNAs starting on day 9 and mice were intratumorally treated twice a week. Data are the mean ± SEM of n=5 mice/group. *P < 0.05, **P < 0.01 by Student's t-test.

G. Immunoblots of PCIF1 and FOS were carried out from the indicated tumors in four replicates with GAPDH as a loading control.

H. Schematic of the proposed function of PCIF1-mediated m6Am modification in CRC.

Expanded View Figures Legends
The novel BET-CBP/p300 dual inhibitor NEO2734 is active in SPOP mutant and wild-type prostate cancer (2019)
Figure 1

Figure 1. Identification of heterozygous and homozygous SPOP Q165P mutations and BET protein expression in PCa patient samples

A, B. A missense mutation in SPOP gene (Q165P, T >G) was identified in a PCa patient. Exome sequencing revealed that the primary tumor contained a heterozygous Q165P mutation (A) whereas the liver metastasis harbored a homozygous Q165P mutation (B).

C. A schematic shows a series of SPOP mutations including those detected in a large cohort of prostate adenocarcinomas (1,013 cases from MSKCC/DFCI (Armenia et al, 2018) and 499 cases from TCGA dataset (Cancer Genome Atlas Research, 2015)) and Q165P mutation identified in this report.

D, E. IHC analysis of BRD4 protein expression in SPOP wild type (WT) and mutated (MUT) PCa patient samples. The representative images of BRD4 IHC staining in both SPOP WT and MUT PCa patients are shown in (D) and the quantified IHC data are shown in (E). Scale bars: 100 µm for 20 x fields; 50 µm for 40 x fields. The red dot in (E) indicates SPOP Q165P sample. All data shown are means ± SEM. The P value was calculated by the unpaired two-tailed Student's t-test; ** P < 0.01. See Appendix Table S4 for the detailed comparison, P values and sample number (n).

F, G. IHC analysis of AR protein expression in SPOP WT and mutated PCa patient samples. The representative images of AR IHC staining in both SPOP WT and mutant PCa patients are shown in (F) and the quantified IHC data are shown in (G). Scale bars: 100 µm for 20 x fields; 50 µm for 40 x fields. The green dot indicates SPOP Q165P sample. All data shown are means ± SEM. The P value was calculated by the unpaired two-tailed Student's t-test; *** P < 0.01. See Appendix Table S4 for the detailed comparison, P values and sample number (n).
The novel BET-CBP/p300 dual inhibitor NEO2734 is active in SPOP mutant and wild-type prostate cancer (2019)
Figure 2

Figure 2. Q165P mutation partially impairs SPOP interaction with BRD4 and undermines BRD4 ubiquitination

A, B. LNCaP cells were transfected with the indicated plasmids for 48 h and harvested for western blot analysis with the indicated antibodies to detect the interaction between BRD4 and SPOP (A) and BRD4 ubiquitination (B).

C. C4-2 cells were stably infected with the lenti-virus expressing empty vector (EV) or SPOP mutants and harvested for western blot analysis with the indicated antibodies.

D. C4-2 xenograft tumors expressing EV or F133V and SPOP WT and Q165P PDX tumors were harvested for western blot analysis with the indicated antibodies. T1: tumor 1; T2: tumor 2.

E. Representative IHC images of PTEN protein in SPOP WT and Q165P PDX tumors. Scale bars: 100 µm for 20 x fields; 50 µm for 40 x fields.