Journal: Cell proliferation

Article Title: Transcription factor PBX4 regulates limb development and haematopoiesis in mice.

doi: 10.1111/cpr.13580

Figure Lengend Snippet: FIGURE 1 PBX4 is specifically expressed in mouse testis. (A) Schematic diagrams of Pbx4 gene structure and PBX4 protein domains. Blue and white rectangles indicate protein-coded exons and UTR regions, respectively. (B) Left panel: detection of Pbx4 expression by RT-PCRs in various tissues of adult mice. Right panel: quantitative RT-PCR results of Pbx4 expression in different testicular cells. eST, elongating spermatids; pacSC, pachytene spermatocytes; plpSC, preleptotene spermatocytes; rST, round spermatids; Sertoli, Sertoli cells; SG-A, type A spermatogonia; SG-B, type B spermatogonia. The expression levels are normalised by that of SG-A, n ≥3. (C) Schematic diagram of PBX4-FLAG knockin (KI) strategy. LHA: left homologous arm; RHA: right homologous arm. (D) Western blot analyses of PBX4 in multiple mouse organs using α-FLAG. * labels the absence of ACTB signal in heart and rectus femoris samples due to low expression levels. Equal amounts of total protein in all samples were loaded based on quantification using the BCA kit. (E) Immunohistochemical (IHC) staining of PBX4-FLAG in testicular sections using α-FLAG. pacSC, pachytene spermatocytes; rST, round spermatids. Scale bar, 20 μm.

Article Snippet: For PBX4-FLAG KI mice, donor DNA fragment containing the left and right homologous arms flanking the 3 FLAG coding sequence was cloned into a plasmid modified from a knockin targeting construct (#59721, addgene) and amplified in bacteria.

Techniques: Expressing, Quantitative RT-PCR, Knock-In, Western Blot, Immunohistochemical staining, Immunohistochemistry

Journal: The Journal of Neuroscience

Article Title: A Smaug2-Based Translational Repression Complex Determines the Balance between Precursor Maintenance versus Differentiation during Mammalian Neurogenesis

doi: 10.1523/JNEUROSCI.2172-15.2015

Figure Lengend Snippet: nanos1 mRNA is a Smaug2 target in embryonic cortical precursors. A, RT-PCR for nanos1, nanos2, and nanos3 mRNAs in murine cortices from E11 to birth (P0). nanos1 mRNA expression was detected using two different primer sets. PCR products were sequenced to confirm specificity. +ve, Sample with known expression of target mRNA and used as a positive control for the reaction; -ve, sample generated in the absence of reverse transcriptase. B, Schematic of SREs in the nanos1 mRNA transcript. Yellow arrow labeled CDS represents the protein-coding region. C, Western blot analysis for Nanos1 in E11.5 to 2-month-old cortices. The blot was reprobed for ERK1/2 as a loading control. D, Western blot of HEK-293T cells transfected with a Flag-tagged mouse Smaug2 construct and immunoprecipitated with anti-Smaug2 or with control nonspecific rabbit IgG, probed with antibodies for Smaug2. As a control, 10% of the input homogenate was loaded. E, Western blot (top) of E12.5 cortical lysates immunoprecipitated with the same Smaug2 antibody as in D or with control, nonspecific rabbit IgG and probed with anti-Smaug2. As a positive control, 10% of the input homogenate was loaded. Similar immunoprecipitates were generated in parallel, mRNA was extracted, and the samples were analyzed for nanos1, nanos2, and nanos3 mRNAs using RT-PCR (second to bottom panels). F, Confocal images of FISH for nanos1 (left), nanos2 (center), and nanos3 (right) mRNAs (black granules) in coronal sections of the E12.5 cortex. v, Ventricle. Scale bar, 10 μm. G, Higher-magnification confocal images of the VZ/SVZ of an E13.5 cortical section showing FISH for nanos1 mRNA (red) and immunostaining for Smaug2 (green). Top, Merge. Boxed regions are shown at higher magnification in the right panels, which also show colocalization of Smaug2 and nanos1 mRNA on the z-axis (XZ and YZ), as indicated by the hatched white lines. Scale bar, 10 μm. H, Confocal images of the E12.5 cortex showing FISH for nanos1 mRNA (magenta) and immunostaining for Smaug2 (green). The VZ/SVZ is divided into five bins of identical width, as denoted by the hatched white lines, and boxed regions within some of these bins are shown at higher magnification in the right panels. Arrows indicate foci with colocalized nanos1 mRNA and Smaug2. Arrowheads indicate foci with only nanos1 mRNA. v, Ventricle. Scale bar, 10 μm. I, J, Quantification of sections similar to that shown in H for the distribution of total nanos1 mRNA-positive foci (I) and the relative proportion of nanos1 mRNA-positive foci that colocalize with Smaug2 in each bin (J). *p < 0.05. **p < 0.01. ***p < 0.001. n = 3. K, L, Quantification of sections similar to those shown in H for the proportion of nanos1, nanos2, or nanos3 mRNA foci that colocalize with Smaug2 across the entire E12.5 VZ/SVZ (K) or only in Bin1 (L), the apical-most region of the VZ. *p < 0.05. ***p < 0.001. n = 3. Statistics were performed with ANOVA and Tukey's post hoc multiple comparisons test. Error bars indicate SEM.

Article Snippet: The primers used in qRT-PCR were nanos1 , forward 5′- CTACACCACACACATCCTCAAGG-3′ and reverse 5′- GCACTTTGGAGAGCGGGCAATA-3′ (OriGene); and nanos2 , and nanos3 (OriGene, same as above).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Positive Control, Generated, Labeling, Western Blot, Transfection, Construct, Immunoprecipitation, Immunostaining

Journal: The Journal of Neuroscience

Article Title: A Smaug2-Based Translational Repression Complex Determines the Balance between Precursor Maintenance versus Differentiation during Mammalian Neurogenesis

doi: 10.1523/JNEUROSCI.2172-15.2015

Figure Lengend Snippet: Nanos1 is necessary and sufficient to promote neurogenesis in vivo. A, Western blots of HEK-293T cell lysates cotransfected with murine Nanos1 or Flag-tagged murine Nanos2 or Nanos3 expression constructs and a control shRNA (Con) or a Nanos1 shRNA (shNos1) and probed with anti-Nanos1 or anti-Flag, as indicated. The blots were reprobed with ERK1/2 as a loading control. B–H, E13/E14 murine cortices were coelectroporated with a nuclear EGFP construct, and either a control (con) or Nanos1 shRNA (shNos1) and coronal sections were analyzed 3 d later at E16/E17. B, Images of electroporated sections immunostained for EGFP (green). v, Ventricle. Scale bar, 10 μm. C, Quantification of sections similar to those in B for the percentage of EGFP-positive cells located in the different cortical regions. **p < 0.01. n = 3 embryos each, at least 3 sections per embryo. D, Confocal micrographs of the VZ/SVZ (three top rows) or CP (bottom row) of electroporated sections immunostained for EGFP (green) and Pax6, Ki67, Tbr2, or Satb2 (all red). Arrows indicate double-labeled cells. v, Ventricle. Scale bar, 10 μm. E–H, Quantification of sections similar to those in D for the percentage of EGFP-positive cells that expressed Pax6 (E), Ki67 (F), Tbr2 (G), or Satb2 (H). **p < 0.01. ***p < 0.001. n = 3 embryos each, at least 3 sections per embryo. I–K, E13/E14 cortices were coelectroporated with a nuclear EGFP construct and a control (con) or Nanos1 shRNA (shNos1) ± an shRNA-resistant human Nanos1 expression vector (resc) and coronal sections were analyzed 3 d later at E16/E17. I, Images of electroporated sections immunostained for EGFP (green). v, Ventricle. Scale bar, 10 μm. J, K, Sections similar to those in I were immunostained for EGFP and Pax6 or Satb2 and the proportion of EGFP-positive cells that were also positive for the marker was quantified. **p < 0.01. ***p < 0.001. n = 3 embryos each, at least 3 sections per embryo. L–R, E13/E14 cortices were coelectroporated with a nuclear EGFP construct and either a control (con) or murine Nanos1 (Nos1-OE) expression vector, and coronal sections were analyzed 3 d later at E16/E17. L, Images of electroporated sections immunostained for EGFP (green). v, Ventricle. Scale bar, 10 μm. M, Quantification of sections as in L for the percentage of EGFP-positive cells located in the different cortical regions. *p < 0.05. ns, Nonsignificant. n = 3 embryos each, at least 3 sections per embryo. N, Confocal images of the VZ/SVZ (top three rows) or CP (bottom row) of electroporated sections similar to those in L immunostained for EGFP (green) and Pax6, Ki67, Tbr2, or Satb2 (all red). Arrows indicate double-labeled cells. Arrowheads indicate EGFP-positive, marker-negative cells. v, Ventricle. Scale bar, 10 μm. O–R, Quantification of sections as in N for the percentage of EGFP-positive cells that were also positive for Pax6 (O), Ki67 (P), Tbr2 (Q), or Satb2 (R). *p < 0.05. **p < 0.01. ***p < 0.001. n = 3 embryos each, at least 3 sections per embryo. J, K, Statistics were performed with ANOVA and Tukey's post hoc multiple comparisons test. All other panels, Statistics were performed with Student's t test. Error bars indicate SEM.

Article Snippet: The primers used in qRT-PCR were nanos1 , forward 5′- CTACACCACACACATCCTCAAGG-3′ and reverse 5′- GCACTTTGGAGAGCGGGCAATA-3′ (OriGene); and nanos2 , and nanos3 (OriGene, same as above).

Techniques: In Vivo, Western Blot, Expressing, Construct, shRNA, Labeling, Plasmid Preparation, Marker

Journal: The Journal of Neuroscience

Article Title: A Smaug2-Based Translational Repression Complex Determines the Balance between Precursor Maintenance versus Differentiation during Mammalian Neurogenesis

doi: 10.1523/JNEUROSCI.2172-15.2015

Figure Lengend Snippet: Smaug2 and nanos1 mRNA are associated with 4E-T in a P-Body-like granule in Pax6-positive apical precursors. A, Western blot analysis for Smaug2 (Smg2) and 4E-T in lysates of E12.5 cortical precursors cultured for 3 d and immunoprecipitated with anti-Smaug2 or with control, nonspecific rabbit IgG. As a positive control, 10% of the input homogenate was loaded. B, Western blot analysis for Smaug2 and 4E-T in lysates of E12.5 cortical precursors cultured for 3 d and immunoprecipitated with anti-4E-T or with control, nonspecific mouse IgG. As a positive control, 10% of the input homogenate was loaded. C, Confocal images of E12.5 cortical precursors cultured for 3 d and immunostained for Smaug2 (green) and 4E-T (magenta). Cultures were also counterstained with Hoechst (blue). Top, Boxed areas are shown at higher magnification in the bottom panels. Arrows indicate granules that are positive for both Smaug2 and 4E-T. Scale bar, 5 μm. D, Confocal images of E12.5 3 d cortical precursor cultures after the PLA with Smaug2 and 4E-T antibodies. Cultures were also counterstained with Hoechst (blue). Left, Boxed areas are shown at higher magnification to the right. Scale bar, 10 μm. E, Confocal images of E12.5 cortical precursors cultured for 3 d and immunostained for Smaug2 (red) and Dcp1 (green). Cultures were also counterstained with Hoechst (blue). Top, Boxed areas are shown at higher magnification in the bottom panels. Arrows indicate granules that are double labeled for Smaug2 and Dcp1. Scale bar, 10 μm. F, Confocal images of cortical precursor cultures after PLA with Smaug2 and Dcp1 antibodies. Cultures were also counterstained with Hoechst (blue). Left, Boxed areas are shown at higher magnification on the right. Scale bar, 10 μm. G, RT-PCR analysis for nanos1 mRNA in 4E-T immunoprecipitates (4E-T IP) from the E12.5 cortex. As a control, similar lysates were immunoprecipitated with a control, nonspecific mouse IgG (IgG). H, qRT-PCR analysis for nanos1 mRNA enrichment in multiple independent 4E-T immunoprecipitates from the E12.5 cortex, in comparison with control IgG immunoprecipitates. I, Confocal images of E12.5 cortical precursors cultured for 3 d and analyzed by FISH for nanos1 mRNA (red or magenta) and immunostaining for 4E-T or Smaug2 (both green). Cultures were also counterstained with Hoechst (blue). Arrows and arrowheads indicate nanos1 mRNA-positive foci that are or are not positive for the relevant protein, respectively. Scale bar, 5 μm. J, Quantification of cultures as in I for the percentage of total nanos1 mRNA-positive foci that also colocalized with Smaug2 (Smg2) or 4E-T alone, or with both together. *p < 0.05. **p < 0.01. ***p < 0.01. n = 3. K, Confocal images of the E12.5 cortical VZ immunostained with 4E-T (green) and subjected to FISH (magenta) with a nanos1 mRNA probe shown at low magnification (left) and high magnification (right). Cell nuclei were counterstained with Hoechst (blue). Bottom, Merge. Left, Boxed regions are shown at high magnification to the right. Arrows indicate foci positive for both nanos1 mRNA and 4E-T. Arrowheads indicate nanos1 mRNA foci that are negative for 4E-T. v, Ventricle. Scale bar, 10 μm. L, Quantification of sections similar to that shown in K for the relative proportion of nanos1 mRNA-positive foci that colocalized with 4E-T in each bin of the VZ/SVZ, as defined in Figure 4H. **p < 0.01. n = 3. M, N, Quantification of sections similar to those shown in K for the proportion of nanos1, nanos2, or nanos3 mRNA foci that colocalized with Smaug2 across the entire E12.5 VZ/SVZ (M) or only in Bin1 (N), the apical-most region of the VZ. *p < 0.05. n = 3. Statistics were performed with ANOVA and Tukey's post hoc multiple comparisons test. Error bars indicate SEM.

Article Snippet: The primers used in qRT-PCR were nanos1 , forward 5′- CTACACCACACACATCCTCAAGG-3′ and reverse 5′- GCACTTTGGAGAGCGGGCAATA-3′ (OriGene); and nanos2 , and nanos3 (OriGene, same as above).

Techniques: Western Blot, Cell Culture, Immunoprecipitation, Positive Control, Labeling, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Immunostaining