Journal: Frontiers in Molecular Neuroscience

Article Title: Pou2f2 Regulates the Distribution of Dorsal Interneurons in the Mouse Developing Spinal Cord

doi: 10.3389/fnmol.2019.00263

Figure Lengend Snippet: Onecut (OC) factors moderate expression of Pou2f2 in the dorsal spinal cord. (A,B) In situ hybridization for Pou2f2 on transverse sections (lumbar level) of control or Hnf6/Oc2 −/− spinal cords at e11.5. (A) In control embryos, Pou2f2 is expressed at low levels in the dorsal part of the spinal cord and at higher levels in the intermediate zone. (B) In Hnf6/Oc2 −/− mutant embryos, Pou2f2 expression is upregulated and cells displaying high Pou2f2 levels are observed more dorsally than in control littermates. The pictures show part of right hemisections as indicated on the scheme to the left. (C) Measurement of Pou2f2 in situ hybridization signal intensity in control or Hnf6/Oc2 −/− at e11.5. Pou2f2 expression is upregulated in the absence of the OC factors in the dorsal spinal cord ( p ≤ 0.05). Mean values ± SEM, n = 3. * = p ≤ 0.05. Solid lines delineate the spinal cord. Scale bar = 100 μm.

Article Snippet: Primary antibodies against the following proteins were used: Brn3a (mouse 1:1,000; Santa Cruz #sc-8429), Dmrt3 (guinea pig; 1:1,000; kindly provided by K. Kullander #170), Foxd3 (guinea pig; 1:1,000; or rabbit; 1:1,000; kindly provided by T. Müller), Foxp1 (goat; 1:1,000; R&D Systems #AF4534), HNF6 [guinea pig; 1:2,000; (Espana and Clotman, ) or rabbit; 1:100; Santa Cruz #sc-13050 or sheep; 1:1,000 R&D Systems #AF6277], Isl1/2 (goat; 1:3,000; Neuromics #GT15051 or mouse; 1:6,000; DSHB #39.4D5), Lbx1 (guinea pig; 1:10,000 or rabbit; 1:5,000; kindly provided by T. Müller), Lhx1/5 (mouse; 1:1,000; DSHB #4F2), Lmx1b (guinea pig; 1:10,000 or rabbit; 1:2,000; kindly provided by T. Müller), OC2 [rat; 1:400; (Clotman et al., ) or sheep; 1:500; R&D Systems #AF6294], OC3 [guinea pig; 1:6,000; (Pierreux et al., ), Phox2a (rabbit; 1:500; kindly provided by J.-F. Brunet), Pou2f2 (rabbit; 1:2,000; Abcam #ab178679), Wt1 (rabbit; 1:2,000; Santa Cruz #sc-192)].

Techniques: Expressing, In Situ Hybridization, Control, Mutagenesis

Journal: Frontiers in Molecular Neuroscience

Article Title: Pou2f2 Regulates the Distribution of Dorsal Interneurons in the Mouse Developing Spinal Cord

doi: 10.3389/fnmol.2019.00263

Figure Lengend Snippet: The OC factors moderate Pou2f2 expression in dI2 interneurons. (A–F) Immunodetection of Pou2f2 (red) in Foxd3 + (green) or Foxd3 + (green) Brn3a + (blue) dI2 interneurons on transverse sections of thoracic spinal cord at e10.5 (A,B) , e11.5 (C,D) and e12.5 (E,F) . Pou2f2 is detected in post-mitotic Foxd3 + dI2 interneurons, particularly in the ventral part of the population, at e10.5 and e11.5 but is almost absent at e12.5. Pou2f2 signal is stronger in Foxd3 + dI2 interneurons in Hnf6/Oc2 −/− spinal cord at all studied developmental stages. (G–I) Relative quantification of Pou2f2 positive dI2 neurons at e10.5 (G) , e11.5 (H) and e12.5 (I) . The proportion of Pou2f2 + dI2 is not significantly different in Hnf6/Oc2 −/− spinal cords. The pictures show part of right hemisections as indicated on the schemes to the left. Solid lines delineate the spinal cord. Insets in (E,F) are magnified views of boxed ventral regions. Arrowheads in (F) point to triple-labeled cells. Mean values ± SEM, n = 3. Scale bars = 100 μm.

Article Snippet: Primary antibodies against the following proteins were used: Brn3a (mouse 1:1,000; Santa Cruz #sc-8429), Dmrt3 (guinea pig; 1:1,000; kindly provided by K. Kullander #170), Foxd3 (guinea pig; 1:1,000; or rabbit; 1:1,000; kindly provided by T. Müller), Foxp1 (goat; 1:1,000; R&D Systems #AF4534), HNF6 [guinea pig; 1:2,000; (Espana and Clotman, ) or rabbit; 1:100; Santa Cruz #sc-13050 or sheep; 1:1,000 R&D Systems #AF6277], Isl1/2 (goat; 1:3,000; Neuromics #GT15051 or mouse; 1:6,000; DSHB #39.4D5), Lbx1 (guinea pig; 1:10,000 or rabbit; 1:5,000; kindly provided by T. Müller), Lhx1/5 (mouse; 1:1,000; DSHB #4F2), Lmx1b (guinea pig; 1:10,000 or rabbit; 1:2,000; kindly provided by T. Müller), OC2 [rat; 1:400; (Clotman et al., ) or sheep; 1:500; R&D Systems #AF6294], OC3 [guinea pig; 1:6,000; (Pierreux et al., ), Phox2a (rabbit; 1:500; kindly provided by J.-F. Brunet), Pou2f2 (rabbit; 1:2,000; Abcam #ab178679), Wt1 (rabbit; 1:2,000; Santa Cruz #sc-192)].

Techniques: Expressing, Immunodetection, Quantitative Proteomics, Labeling

Journal: Frontiers in Molecular Neuroscience

Article Title: Pou2f2 Regulates the Distribution of Dorsal Interneurons in the Mouse Developing Spinal Cord

doi: 10.3389/fnmol.2019.00263

Figure Lengend Snippet: The OC factors moderate Pou2f2 expression in dI3 interneurons. (A–F) Immunodetection of Pou2f2 (red) in Isl1 + (green) dI3 interneurons on transverse sections of thoracic spinal cord at e10.5 (A,B) , at e11.5 (C,D) and e12.5 (E,F) . Pou2f2 is detected in the ventral part of the post-mitotic Isl1 + dI3 interneuron population from e10.5. Pou2f2 signal is stronger in Isl1 + dI3 interneurons in Hnf6/Oc2 −/− spinal cord at all studied developmental stages. (G–I) Relative quantification of Pou2f2 positive dI3 neurons at e10.5 (G) , e11.5 (H) and e12.5 (I) . The proportion of Pou2f2 + dI3 is not significantly different in Hnf6/Oc2 −/− spinal cords. The pictures show part of right hemisections as indicated on the schemes to the left. Solid lines delineate the spinal cord. Mean values ± SEM, n = 3. Scale bars = 100 μm.

Article Snippet: Primary antibodies against the following proteins were used: Brn3a (mouse 1:1,000; Santa Cruz #sc-8429), Dmrt3 (guinea pig; 1:1,000; kindly provided by K. Kullander #170), Foxd3 (guinea pig; 1:1,000; or rabbit; 1:1,000; kindly provided by T. Müller), Foxp1 (goat; 1:1,000; R&D Systems #AF4534), HNF6 [guinea pig; 1:2,000; (Espana and Clotman, ) or rabbit; 1:100; Santa Cruz #sc-13050 or sheep; 1:1,000 R&D Systems #AF6277], Isl1/2 (goat; 1:3,000; Neuromics #GT15051 or mouse; 1:6,000; DSHB #39.4D5), Lbx1 (guinea pig; 1:10,000 or rabbit; 1:5,000; kindly provided by T. Müller), Lhx1/5 (mouse; 1:1,000; DSHB #4F2), Lmx1b (guinea pig; 1:10,000 or rabbit; 1:2,000; kindly provided by T. Müller), OC2 [rat; 1:400; (Clotman et al., ) or sheep; 1:500; R&D Systems #AF6294], OC3 [guinea pig; 1:6,000; (Pierreux et al., ), Phox2a (rabbit; 1:500; kindly provided by J.-F. Brunet), Pou2f2 (rabbit; 1:2,000; Abcam #ab178679), Wt1 (rabbit; 1:2,000; Santa Cruz #sc-192)].

Techniques: Expressing, Immunodetection, Quantitative Proteomics

Journal: Frontiers in Molecular Neuroscience

Article Title: Pou2f2 Regulates the Distribution of Dorsal Interneurons in the Mouse Developing Spinal Cord

doi: 10.3389/fnmol.2019.00263

Figure Lengend Snippet: The OC factors moderate Pou2f2 expression in dI5 interneurons and Dmrt3 + dI6 interneuron subset. (A–D) Immunodetection of Pou2f2 (red) in Lmx1b + (green) dI5 neurons on transverse sections of thoracic spinal cord at e10.5 (A,B) and at e11.5 (C,D) . Pou2f2 is detected in most of Lmx1b + dI5 interneurons at e10.5 and is then restricted to the ventral part of the population. Pou2f2 signal is stronger in Lmx1b + dI5 interneurons in Hnf6/Oc2 −/− spinal cord at all studied developmental stages. (E,F) The percentage of Pou2f2 positive dI5 neurons was quantified at e10.5 (E) and e11.5 (F) , and is not significantly different in Hnf6/Oc2 −/− spinal cords. (G–I) Immunodetection and relative quantification of Pou2f2 in Dmrt3 + (green) dI6 subset at e12.5. The proportion of dI6 Dmrt3 + Pou2f2 + is significantly increased at thoracic level in Hnf6/Oc2 −/− mutant embryos ( p ≤ 0.05). Again, Pou2f2 signal is stronger in the absence of OC factors. The pictures show part of right hemisections as indicated on the schemes to the left. Solid lines delineate the spinal cord. Mean values ± SEM, n = 3. * = p ≤ 0.05. Scale bars = 100 μm.

Article Snippet: Primary antibodies against the following proteins were used: Brn3a (mouse 1:1,000; Santa Cruz #sc-8429), Dmrt3 (guinea pig; 1:1,000; kindly provided by K. Kullander #170), Foxd3 (guinea pig; 1:1,000; or rabbit; 1:1,000; kindly provided by T. Müller), Foxp1 (goat; 1:1,000; R&D Systems #AF4534), HNF6 [guinea pig; 1:2,000; (Espana and Clotman, ) or rabbit; 1:100; Santa Cruz #sc-13050 or sheep; 1:1,000 R&D Systems #AF6277], Isl1/2 (goat; 1:3,000; Neuromics #GT15051 or mouse; 1:6,000; DSHB #39.4D5), Lbx1 (guinea pig; 1:10,000 or rabbit; 1:5,000; kindly provided by T. Müller), Lhx1/5 (mouse; 1:1,000; DSHB #4F2), Lmx1b (guinea pig; 1:10,000 or rabbit; 1:2,000; kindly provided by T. Müller), OC2 [rat; 1:400; (Clotman et al., ) or sheep; 1:500; R&D Systems #AF6294], OC3 [guinea pig; 1:6,000; (Pierreux et al., ), Phox2a (rabbit; 1:500; kindly provided by J.-F. Brunet), Pou2f2 (rabbit; 1:2,000; Abcam #ab178679), Wt1 (rabbit; 1:2,000; Santa Cruz #sc-192)].

Techniques: Expressing, Immunodetection, Quantitative Proteomics, Mutagenesis

Journal: Cancer research

Article Title: Chemotherapy-induced extracellular vesicle miRNAs promote breast cancer stemness by targeting ONECUT2

doi: 10.1158/0008-5472.CAN-18-4055

Figure Lengend Snippet: (A) MDA231, MCF-7, and BT474 cells were transfected with siRNA against ONECUT2 (GeneSolution/GS including equal mixture of 4 preselected siRNAs, or individual siRNA #1 and #2), or a control siRNA, or with PBS. After 48 h, cells were collected for sphere formation assay. (B) ALDEFLUOR assay of MDA231 cells transfected with siRNA as indicated for 72 h. (C) RT-qPCR-determined RNA levels of indicated genes in various BC cells at 48 h following transfection with indicated siRNA. (D) Western blots showing the expression levels of indicated proteins in BC cells at 48 h following transfection of ONECUT2 siRNA-GS or control siRNA. (E) MDA231 cells were transfected with indicated siRNA and seeded at an equal number on day 0. DTX (10 nM) or DOXO (500 nM) was added on day 1, and replenished every 24 h. On day 3, cell viability (left) and cell number (right) were determined by MTS assay and cell counting, respectively, and compared to the PBS treatment group. (F) RT-qPCR of indicated genes in MDA231 cells at 72 h after transfection with indicated siRNA. *P<0.05, **P<0.01, ***P<0.001 compared to control siRNA or as indicated. Numbers below Western images indicate quantification after normalization to GAPDH with the first lane set as 1.

Article Snippet: The ONECUT2 overexpression plasmid (#RC211951) was purchased from OriGene (Rockville, MD); stable transfection of MDA231 cells was achieved by G418 selection to generate the MDA231/ONECUT2 cells.

Techniques: Transfection, Tube Formation Assay, Quantitative RT-PCR, Western Blot, Expressing, MTS Assay, Cell Counting

Journal: Cancer research

Article Title: Chemotherapy-induced extracellular vesicle miRNAs promote breast cancer stemness by targeting ONECUT2

doi: 10.1158/0008-5472.CAN-18-4055

Figure Lengend Snippet: (A,B) MDA231 cells stably expressing a mammalian expression plasmid of human ONECUT2 cDNA or the empty vector were exposed to PBS or EVs from PBS/DTX/DOXO-treated MDA231 cells for 48 h before being analyzed by sphere formation assay (A) and Western blots (B). (C) RT-qPCR-determined RNA levels of indicated genes in MDA231 cells stably expressing ONECUT2 or vector. (D) MDA231, MCF-7, and BT474 cells transfected with the ONECUT2 expression plasmid or empty vector were analyzed by Western blots. (E) MDA231 cells stably expressing ONECUT2 or vector were seeded at an equal number on day 0. DTX (10 nM) or DOXO (500 nM) was added after 6 h, and replenished every 24 h. Cell viability (left) was measured by MTS assay every 24 h and cell number (right) was determined by cell counting on day 3. Data were normalized to the PBS control group. (F) RT-qPCR of indicated genes in MDA231 cells stably expressing ONECUT2 or vector. *P<0.05, **P<0.01, ***P<0.001. Numbers below Western images indicate quantification after normalization to GAPDH with the first lane set as 1.

Article Snippet: The ONECUT2 overexpression plasmid (#RC211951) was purchased from OriGene (Rockville, MD); stable transfection of MDA231 cells was achieved by G418 selection to generate the MDA231/ONECUT2 cells.

Techniques: Stable Transfection, Expressing, Plasmid Preparation, Tube Formation Assay, Western Blot, Quantitative RT-PCR, Transfection, MTS Assay, Cell Counting

Journal: Cancer research

Article Title: Chemotherapy-induced extracellular vesicle miRNAs promote breast cancer stemness by targeting ONECUT2

doi: 10.1158/0008-5472.CAN-18-4055

Figure Lengend Snippet: (A) A schematic representative showing putative binding sites of miR-9–5p, miR-203a-3p, and miR-195–5p in the 3’UTR of human ONECUT2 and the regions cloned into the luciferase reporter plasmid constructs. (B) MDA231 cells were transfected with psiCHECK2 reporter plasmids containing indicated ONECUT2 3’UTR region or with the psiCHECK2 vector (2 µg DNA per 2×105 cells). After 12 h, transfected cells were exposed to PBS or EVs from PBS/DTX (4 nM)/DOXO (125 nM)-treated MDA231 cells for 48 h before luciferase activities were measured. Ratio between Renilla luciferase and firefly luciferase activities (Rluc/Fluc) is shown. (C) MDA231 cells were co-transfected with indicated psiCHECK2 reporter plasmids (2 µg DNA per 2×105 cells) and miRNA mimics (individually or with a 1:1:1 mixture of miR-9–5p, miR-203a-3p, and miR-195–5p mimics for a total of 25 pmol). Luciferase activities were analyzed at 48 h. *P<0.05, **P<0.01, ***P<0.001.

Article Snippet: The ONECUT2 overexpression plasmid (#RC211951) was purchased from OriGene (Rockville, MD); stable transfection of MDA231 cells was achieved by G418 selection to generate the MDA231/ONECUT2 cells.

Techniques: Binding Assay, Clone Assay, Luciferase, Plasmid Preparation, Construct, Transfection

Journal: Cancer research

Article Title: Chemotherapy-induced extracellular vesicle miRNAs promote breast cancer stemness by targeting ONECUT2

doi: 10.1158/0008-5472.CAN-18-4055

Figure Lengend Snippet: (A) Left: Western blots showing modified expression of Rab27a and Onecut2 in indicated cell lines. Right: nanoparticle tracking analysis of EVs from an equal number of producing cells showing reduced EV secretion by MDA231/Rab27aKD cells. (B-E) Xenograft tumors were established in NSG mice by injecting 2×105 of indicated cells into the #4 mammary fat pad. When tumor size reached ~300 mm3, mice were treated weekly with DTX (15 mg/kg) for 3 weeks. (B) Tumor onset and volume. The time of DTX treatments were indicated by arrows. (C) Western blots of indicated proteins using tumors collected before and after DTX treatment. Numbers below Western images indicate quantification after normalization to GAPDH with the first lane set as 1. (D) RT-qPCR analysis of indicated genes using tumors collected before and after DTX treatment. (E) EVs were prepared from the sera of indicated mice before and after the 3-week DTX treatment. Levels of miRNAs were determined by RT-qPCR using a cel-miR-39–3p spike-in control for normalization. (F) Twelve pairs of pre- and post-NT human breast tumors were analyzed by IHC to determine the ONECUT2 expression levels in tumor cells. Wilcoxon test was performed. *P<0.05, **P<0.01, ***P<0.001.

Article Snippet: The ONECUT2 overexpression plasmid (#RC211951) was purchased from OriGene (Rockville, MD); stable transfection of MDA231 cells was achieved by G418 selection to generate the MDA231/ONECUT2 cells.

Techniques: Western Blot, Modification, Expressing, Quantitative RT-PCR

Journal: Nature medicine

Article Title: ONECUT2 is a Targetable Master Regulator of Lethal Prostate Cancer that Suppresses the Androgen Axis

doi: 10.1038/s41591-018-0241-1

Figure Lengend Snippet: (a) The normalized ChIP-seq signal across merged OC2 replicates (N=2 biologically independent experiments), and individual samples of AR, H3K27ac and H3K4me1 in the peak set representing AR-enriched regions, OC2-enriched regions, and co-bound regions is shown. The number of peaks represented in each heatmap are: OC2-enriched regions (4,927 peaks), co-bound regions (2,151 peaks), and AR-enriched regions (10,726 peaks). The upper panel represents the average ChIP signal across each peak set.

Article Snippet: OC2 (#SR306292) and REST (#SR304036) siRNA pools and Universal scrambled negative control siRNA duplexes (Origene) were used according to manufacturers’ instructions.

Techniques: ChIP-sequencing

Journal: Nature medicine

Article Title: ONECUT2 is a Targetable Master Regulator of Lethal Prostate Cancer that Suppresses the Androgen Axis

doi: 10.1038/s41591-018-0241-1

Figure Lengend Snippet: (a) Multiplex IF staining with anti-OC2 and anti-AR antibodies in 6 cases of high-grade PC. Heatmap and dendrogram of nuclear staining intensities of AR and OC2, using the Euclidean distance and complete linkage method in an unsupervised cluster analysis, identifies two cell populations: AR high/OC2 low and AR low/OC2 high. Scatter plot of individual nuclei separated by intensity levels of AR and OC2. Cluster 1 (C1, green) represents high AR/low OC2 cells (N=1,109) and cluster 2 (C2, purple) represents low AR/high OC2 cells (N=199). Boxplots of intensity levels of nuclear AR and OC2 in C1 (left) or C2 (right). The boxes show the 25th-75th percentile range and the center line is the median. Whiskers show 1.5 times the IQR from the 25th or 75th percentile values. Left boxplot: P=2.1×10−191, right boxplot: P=2.3×10−66, Wilcoxon two-tailed rank-sum test.

Article Snippet: OC2 (#SR306292) and REST (#SR304036) siRNA pools and Universal scrambled negative control siRNA duplexes (Origene) were used according to manufacturers’ instructions.

Techniques: Multiplex Assay, Staining, Two Tailed Test

Journal: Nature medicine

Article Title: ONECUT2 is a Targetable Master Regulator of Lethal Prostate Cancer that Suppresses the Androgen Axis

doi: 10.1038/s41591-018-0241-1

Figure Lengend Snippet: (a) NE activation score in mCRPC tumors with high (N=65) and low (N=65) OC2 expression (top and bottom quartiles) from the DISC cohort. The boxes show the 25th-75th percentile range and the center line is the median. Whiskers show 1.5 times the IQR from the 25th or 75th percentile values. Data points beyond the whiskers are displayed using dots. Wilcoxon two-tailed rank-sum test, P=4.6×10−5.

Article Snippet: OC2 (#SR306292) and REST (#SR304036) siRNA pools and Universal scrambled negative control siRNA duplexes (Origene) were used according to manufacturers’ instructions.

Techniques: Activation Assay, Expressing, Two Tailed Test

Journal: Nature medicine

Article Title: ONECUT2 is a Targetable Master Regulator of Lethal Prostate Cancer that Suppresses the Androgen Axis

doi: 10.1038/s41591-018-0241-1

Figure Lengend Snippet: (a) Cell survival 4 days after start of OC2 depletion. The mean + S.D. from three independent experiments is shown. Unpaired two-tailed Student´s t-test, C4–2: sh1★★P= 7.58×10−5, sh2★★P= 7.48×10−5; LNCaP: sh1★★P=1.57×10−6, sh2★★P=2.03×10−6, 22Rv1: sh1★★P=5.42×10−6, sh2★★P= 0.0002.

Article Snippet: OC2 (#SR306292) and REST (#SR304036) siRNA pools and Universal scrambled negative control siRNA duplexes (Origene) were used according to manufacturers’ instructions.

Techniques: Two Tailed Test

Journal: Nature medicine

Article Title: ONECUT2 is a Targetable Master Regulator of Lethal Prostate Cancer that Suppresses the Androgen Axis

doi: 10.1038/s41591-018-0241-1

Figure Lengend Snippet: (a) Relative OC2 mRNA levels in 6 prostate cell lines determined by RT-qPCR (left). ACTB and GAPDH expression levels were used for normalization. Data show mean + S.D. from triplicates. Results are representative of two independent experiments. The IC50 values for compound CSRM617 shown are the mean from two independent experiments (right).

Article Snippet: OC2 (#SR306292) and REST (#SR304036) siRNA pools and Universal scrambled negative control siRNA duplexes (Origene) were used according to manufacturers’ instructions.

Techniques: Quantitative RT-PCR, Expressing