Journal: PLoS ONE

Article Title: C9C5 positive mature oligodendrocytes are a source of Sonic Hedgehog in the mouse brain

doi: 10.1371/journal.pone.0229362

Figure Lengend Snippet: (A-B) Immunostaining of coronal brain sections from an adult mouse of the cerebral cortex and corpus callosum with the C9C5 antibody (yellow) and the oligodendroglial markers (A) CC1 (green) and Sox10 (red) or (B) CAII (green) and PDGFRα (red). White boxes (A, B) highlight magnifications of the cerebral cortex and the corpus callosum. (A) C9C5/CC1/Sox10 triple positive cells (white arrowhead) with stellate morphology are presented in merge and single channels with the nuclear marker DAPI. (B) C9C5 + (white arrowhead), CAII + (orange arrow), and PDGFRα + (white arrow) cells are presented in merge and single channels with the nuclear marker DAPI. Note that C9C5 positive cells are CAII and PDGFRα negative. Staining was replicated at least on three mice. Ctx, cerebral cortex; cc, corpus callosum.

Article Snippet: The primary antibodies were incubated overnight at 4°C: rabbit anti-SHHN (1/300, C9C5, #2207, Cell Signaling), mouse anti-GFAP (1/400, MAB360, Millipore), goat anti-Olig2 (1/400, AF2418, R&D Systems), mouse anti-S100β (1/500, S2532, Sigma), mouse anti-adenomatous polyposis coli (APC) (1/600, clone CC1, OP80, Millipore), chicken anti-βgalactosidase (1/200, ab9361, Abcam), goat anti-Sox10 (1/100, AF2864-SP, R&D Systems), rat anti-PDGFRα (1/300, 558774, BD Pharmingen), sheep anti-Carbonic Anhydrase II (CAII) (1/200, AHP206, BIORAD), rabbit anti-Iba1 (1/500, 019–19741, Fujifilm Wako), mouse anti-HuC/D (1/400, A-21271, Molecular Probes).

Techniques: Immunostaining, Marker, Negative Staining

Journal: PLoS ONE

Article Title: C9C5 positive mature oligodendrocytes are a source of Sonic Hedgehog in the mouse brain

doi: 10.1371/journal.pone.0229362

Figure Lengend Snippet: Quantification of C9C5 + cells in oligodendrocyte populations in the adult mouse brain.

Article Snippet: The primary antibodies were incubated overnight at 4°C: rabbit anti-SHHN (1/300, C9C5, #2207, Cell Signaling), mouse anti-GFAP (1/400, MAB360, Millipore), goat anti-Olig2 (1/400, AF2418, R&D Systems), mouse anti-S100β (1/500, S2532, Sigma), mouse anti-adenomatous polyposis coli (APC) (1/600, clone CC1, OP80, Millipore), chicken anti-βgalactosidase (1/200, ab9361, Abcam), goat anti-Sox10 (1/100, AF2864-SP, R&D Systems), rat anti-PDGFRα (1/300, 558774, BD Pharmingen), sheep anti-Carbonic Anhydrase II (CAII) (1/200, AHP206, BIORAD), rabbit anti-Iba1 (1/500, 019–19741, Fujifilm Wako), mouse anti-HuC/D (1/400, A-21271, Molecular Probes).

Techniques: Marker

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: VLA-4 mediated adhesion of melanoma cells on the blood–brain barrier is the critical cue for melanoma cell intercalation and barrier disruption

doi: 10.1177/0271678x18775887

Figure Lengend Snippet: Figure 2. CD49d binding to VCAM-1 mediates shear resistant arrest of mouse melanoma cells on the BBB. (a) Shear resistant arrest of mouse B16F10 melanoma cells on IL-1b stimulated pMBMECs after 29 (left) or 30 (right) seconds at 1.5 dyn/cm2. Melanoma cells were treated with the isotype control antibody (left) or with anti CD49d antibody (right). An additional movie file shows this in more detail (see Additional File 3). (b) Quantitative evaluation of (a). (c) Binding of mouse B16F10 melanoma cells to fibronectin (FN), ICAM-1, VCAM-1 or BSA under static conditions. Representative experiment performed in triplicates at least. (d) Shear resistant arrest of B78chOVA mouse melanoma cells on ICAM-1 or VCAM-1. (e) Shear resistant arrest of B78chOVA mouse melanoma cells on VCAM-1 after treatment with an isotype antibody or an antibody blocking CD49d (aCD49d). (b–e) Three or more experiments per condition.

Article Snippet: CD49d immunostaining was performed with rabbit anti human CD49d (AHP1225, BioRad Laboratories Inc.) at a concentration of 30 mg/ml using citrate buffer for antigen retrieval.

Techniques: Binding Assay, Shear, Control, Blocking Assay

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: VLA-4 mediated adhesion of melanoma cells on the blood–brain barrier is the critical cue for melanoma cell intercalation and barrier disruption

doi: 10.1177/0271678x18775887

Figure Lengend Snippet: Figure 3. CD49d on human melanoma brain metastases in situ. Custom-made tissue microarray (TMA) from human melanoma brain metastasis (38 biopsies from 38 patients, 12 autopsies from multiple metastasis of 3 patients). Tissue blocks were grouped according to their high, low or negative staining for human CD49d protein expression. (a, b) Example of CD49d-high melanoma metastasis. In this example, all tumor cells are strongly immunoreactive. (c, d) Melanoma metastasis negative for CD49d. (e) Non- affected brain tissue. (f) Thymus, positive control. (a, c) Hematoxylin–eosin. (b, d–f) CD49d immunostaining. Scale bars: (a) 100 mm, valid also for b–d. (e) 20 mm, valid also for f.

Article Snippet: CD49d immunostaining was performed with rabbit anti human CD49d (AHP1225, BioRad Laboratories Inc.) at a concentration of 30 mg/ml using citrate buffer for antigen retrieval.

Techniques: In Situ, Microarray, Negative Staining, Expressing, Positive Control, Immunostaining

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: VLA-4 mediated adhesion of melanoma cells on the blood–brain barrier is the critical cue for melanoma cell intercalation and barrier disruption

doi: 10.1177/0271678x18775887

Figure Lengend Snippet: Figure 4. CD49d on human melanoma cells translates into increased adhesiveness to the BBB. (a) Flow cytometry of two human melanoma cell lines with either high CD49d (left, hMel-CD49dhi) or low CD49d (right, hMel-CD49dlo) expression. Black lines, isotype control. Red lines, anti CD49d. (b) Quantitative PCR comparing mRNA level of CD49d in hMel-CD49dhi (hi, set to 1.0) to hMel- CD49dlo (lo). (c) Shear resistant arrest of hMel-CD49dhi (hi) and hMel-CD49dlo (lo) on VCAM-1. (d) Shear resistant arrest of hMel- CD49dhi to VCAM-1, ICAM-1 or BSA. Where indicated, hMel-CD49dhi was treated with an antibody blocking CD49d (aCD49d) or isotype control (isotype). (e) Shear resistant arrest of hMel-CD49dhi melanoma cells on unstimulated (ctrl) or IL-1b stimulated pMBMECs. (f) Shear resistant arrest of hMel-CD49dhi melanoma cells on IL-1b stimulated pMBMECs after treatment with isotype control (isotype) or anti CD49d blocking antibody.

Article Snippet: CD49d immunostaining was performed with rabbit anti human CD49d (AHP1225, BioRad Laboratories Inc.) at a concentration of 30 mg/ml using citrate buffer for antigen retrieval.

Techniques: Flow Cytometry, Expressing, Control, Real-time Polymerase Chain Reaction, Shear, Blocking Assay

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: VLA-4 mediated adhesion of melanoma cells on the blood–brain barrier is the critical cue for melanoma cell intercalation and barrier disruption

doi: 10.1177/0271678x18775887

Figure Lengend Snippet: Figure 5. In vitro live cell imaging demonstrated the ultimate role of VLA-4/VCAM-1 for melanoma cell intercalation into the BBB. (a) Three consecutive microscopic images of two B78chOVA melanoma cells that intercalate into a monolayer of pMBMECs. Two intercalation events are depicted (arrowhead). 0 min, at the start of intercalation. 15 min, in the process of intercalation 15 min after start of intercalation. 30 min, intercalation is completed 30 min after start of intercalation. Overlay of the phase contrast channel (all cells) and the red fluorescent channel (B78chOVA). Images were taken with a 10 objective, scale bar 20 mm. An additional movie file shows this in more detail (Supplementary Movie 4). (b) Disruption of LifeAct-GFP pMBMECs by B78chOVA melanoma cells after 3 h. Some gaps in the pMBMECs are exemplarily marked with arrows. Ctrl, unstimulated pMBMECs. TNF-a, stimulated pMBMECs. B78chOVA and pMBMECs were left untreated (isotype) or were treated with anti CD49d or anti-VCAM-1 (aV1/aCD49d), respectively. Representative images 210 min after addition of B78chOVA melanoma cells to the pMBMECs are presented. Additional movie file shows this in more detail (Supplementary Movie 5). (c) Intercalation of B78chOVA melanoma cells into the pMBMEC layer over time. Numbers of gaps per FOV were counted at the time points indicated. One experiment performed in triplicates. (d) Statistical evaluation of B78chOVA melanoma cell intercalation at time point 210 min.

Article Snippet: CD49d immunostaining was performed with rabbit anti human CD49d (AHP1225, BioRad Laboratories Inc.) at a concentration of 30 mg/ml using citrate buffer for antigen retrieval.

Techniques: In Vitro, Live Cell Imaging, Disruption

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: VLA-4 mediated adhesion of melanoma cells on the blood–brain barrier is the critical cue for melanoma cell intercalation and barrier disruption

doi: 10.1177/0271678x18775887

Figure Lengend Snippet: Figure 6. VLA-4/VCAM-1-mediated adhesion causes BBB barrier disruption by melanoma cells. (a) Relative TEER of pMBMECs without (ctrl) or with 2,000, 10,000 or 50,000 B16F10 melanoma cells over 12 h. The mean TEER of all samples at t ¼ 0 (79.3 4.64 V/ cm2) was set to 100%. (b) Relative TEER of pMBMECs without B16F10 melanoma cells (ctrl), with 50,000 B16F10 melanoma cells (B16F10), with melanoma-conditioned medium (cond medium) or with 50,000 CD4þ T cells (T cells) after 3 h of measurement. Values are expressed in percent to TEER at t ¼ 0, which was set to 100%. (c) Relative TEER of pMBMECs with B16F10 melanoma cells that remained untreated (w/o Ab), treated with the blocking anti CD49d antibody (aCD49d) or with the blocking anti CD11a antibody after 3 h of co-culture. TEER of pMBMECs co-cultured with untreated B16F10 was set to 1.0; the respective mean raw value was 63.6 10.69 V/cm2.

Article Snippet: CD49d immunostaining was performed with rabbit anti human CD49d (AHP1225, BioRad Laboratories Inc.) at a concentration of 30 mg/ml using citrate buffer for antigen retrieval.

Techniques: Disruption, Blocking Assay, Co-Culture Assay, Cell Culture

Journal: Nature Communications

Article Title: Nuclear transporter Importin-13 plays a key role in the oxidative stress transcriptional response

doi: 10.1038/s41467-021-26125-x

Figure Lengend Snippet: a–c HeLa cells were transfected with non-targeting (NT) or IPO13 targeting siRNA 72 h prior to treatment with 125 μM H 2 O 2 for 1 h. Cells were transfected to express GFP-KLF4 and processed for CLSM imaging and/or Western analysis. Cells were stained with Hoechst to highlight nuclei. a Total cell extracts were subjected to Western analysis using rabbit-anti-IPO13 (Protein Tech), with mouse-anti-actin (Abcam) as a control, and imaged using the ChemiDoc Gel Imaging System (Biorad). b Representative images of cells transfected to express GFP-KLF4. Scale bar = 10 μm. c Quantitative analysis of ectopic KLF4 protein localisation was carried out using the ImageJ software on images such as those in ( a ) to determine the nuclear to cytoplasmic ratio (Fn/c). Data represent the mean ± SEM for 44 cells (NT Untreated (UT)), 41 cells (NT+H 2 O 2 Treated), 35 cells (IPO13 siRNA UT), and 37 cells (IPO13 siRNA + H 2 O 2 treated) respectively for the nuclear (Fn) and cytoplasmic fluorescence (Fc) above background fluorescence. p values (two-tailed student’s t -test) top to bottom: p < 0.0001, p = 0.1789, p = 0.9899, and p < 0.0001. d – e HeLa cells were co-transfected to express either DSRED or DSRED-tagged IPO13 with GFP-KLF4 and treated for 1 h ± 125 μM H 2 O 2 prior to CLSM imaging. Cells were stained with Hoechst to highlight nuclei. d Representative images of cells co-expressing DSRED or DSRED-IPO13 with GFP-KLF4. Scale bar = 10 μm e . Quantitative analysis of ectopic KLF4 protein localisation performed as per ( c ). Data represent the mean ± SEM for 28 cells (DSRED UT), 38 cells (DSRED + H 2 O 2 Treated), 27 cells (DSRED-IPO13 + UT) and 44 cells (DSRED-IPO13 + H 2 O 2 Treated). p values (two-tailed student’s t -test) top to bottom: p < 0.0001, p < 0.0001, p < 0.0001, and p < 0.0001. f – g HeLa cells were transfected with siRNA as in a – c . prior to treatment with 125 μM H 2 O 2 for 1 h. Cells immediately processed for CLSM imaging and immunostained with anti-SP1. f Representative images of cells immunostained with anti-SP1. Scale bar = 10 μm g . Quantitative analysis of endogenous SP1 localisation carried out as per ( c ). Data represent the mean ± SEM for 39 cells (NT UT), 47 cells (NT + H 2 O 2 Treated), 41 cells (IPO13 siRNA UT), and 43 cells (IPO13 siRNA + H 2 O 2 Treated). p values (two-tailed student’s t-test) top to bottom: p < 0.0001, p < 0.0001, p < 0.0001, and p = 0.2018. h – i HeLa cells were co-transfected to express either GFP or GFP-tagged IPO13 and treated for 1 h ± 125 μM H 2 O 2 prior to immunostaining using anti-SP1 antibody. h Representative images of cells immunostained with anti-SP1. Scale bar = 10 μm. i Quantitative analysis of endogenous SP1 localisation carried out as in ( c ). Data represent the mean ± SEM for 37 cells (GFP UT), 32 cells (GFP + H 2 O 2 Treated), 29 cells (GFP-IPO13 UT), and 38 cells (GFP-IPO13 + H 2 O 2 Treated). p values (two-tailed student’s t -test) top to bottom: p < 0.0001, p < 0.0001, p < 0.0001 and p < 0.0001. Source data are provided as a Source Data file.

Article Snippet: Input/IP samples were probed by Western blotting using mouse-anti-GFP (Roche) or rabbit anti-IMP13 (Protein Tech) antibodies and then imaged using the ChemiDoc Gel Imaging System (Biorad). b , c , e , f Densitometric analysis was performed on images such as those in a and d for binding of DSRED-IPO13 to GFP-KLF4 ( a ) or GFP-SP1 ( d ) under H 2 O 2 treated conditions and untreated (UT) conditions with and without the addition of GTP γ S. The amount of co-immunoprecipitated IPO13 was normalized to the amount of protein available for co-immunoprecipitation (as seen in the input panels).

Techniques: Transfection, Imaging, Western Blot, Staining, Control, Software, Fluorescence, Two Tailed Test, Expressing, Immunostaining

Journal: Nature Communications

Article Title: Nuclear transporter Importin-13 plays a key role in the oxidative stress transcriptional response

doi: 10.1038/s41467-021-26125-x

Figure Lengend Snippet: HeLa cells transfected to co-express GFP or GFP-KLF4 ( a – c ) or -SP1 ( d – f ) with DSRED-IPO13. At 16 h post-transfection, cells were treated with or without 125 μM H 2 O 2 for 1 h before lysis and incubation with or without GTP γ S and immunoprecipitation (IP) using GFP-Trap (Chromotek). Input/IP samples were probed by Western blotting using mouse-anti-GFP (Roche) or rabbit anti-IMP13 (Protein Tech) antibodies and then imaged using the ChemiDoc Gel Imaging System (Biorad). b , c , e , f Densitometric analysis was performed on images such as those in a and d for binding of DSRED-IPO13 to GFP-KLF4 ( a ) or GFP-SP1 ( d ) under H 2 O 2 treated conditions and untreated (UT) conditions with and without the addition of GTP γ S. The amount of co-immunoprecipitated IPO13 was normalized to the amount of protein available for co-immunoprecipitation (as seen in the input panels). This value was normalized to the amount of immunoprecipitated GFP-KLF4 or –SP1. Pooled results representing the mean ± SEM (error bars) for IPO13 bound under H 2 O 2 treated conditions relative to UT conditions (no GTP γ S treatment - No Add.) ( b n = 2 independent experiments, e n = 4 independent experiments) or with GTP γ S treatment under UT and treated conditions ( c n = 2 independent experiments, f n = 4 independent experiments) are shown. p values (two-tailed student’s t test) left to right: p = 0.0422 and p = 0.0375. Source data are provided as a Source Data file.

Article Snippet: Input/IP samples were probed by Western blotting using mouse-anti-GFP (Roche) or rabbit anti-IMP13 (Protein Tech) antibodies and then imaged using the ChemiDoc Gel Imaging System (Biorad). b , c , e , f Densitometric analysis was performed on images such as those in a and d for binding of DSRED-IPO13 to GFP-KLF4 ( a ) or GFP-SP1 ( d ) under H 2 O 2 treated conditions and untreated (UT) conditions with and without the addition of GTP γ S. The amount of co-immunoprecipitated IPO13 was normalized to the amount of protein available for co-immunoprecipitation (as seen in the input panels).

Techniques: Transfection, Lysis, Incubation, Immunoprecipitation, Western Blot, Imaging, Binding Assay, Two Tailed Test

Journal: Cell

Article Title: Mammalian oocytes store proteins for the early embryo on cytoplasmic lattices.

doi: 10.1016/j.cell.2023.10.003

Figure Lengend Snippet: Figure 5. Essential oocyte proteins are stored on cytoplasmic lattices (A, D, G, J, and M) Quantification of relative protein abundance of proteins shown in (B), (E), (H), (K), and (N), respectively; (J) includes all significantly changed YHWA/14-3-3 proteins, and (M) includes all significantly changed tubulin proteins. (B, E, H, K, and N) Representative ExM micrographs of wild-type oocytes labeled for PADI6 (magenta) and UHRF1, SKP1, SPIN1, pan-YWHA/14-3-3, or a-tubulin (green), respectively. Insets are magnifications of outlined regions. Scale bars, 2 mm. Scale bars of insets, 1 mm. All scale bares are corrected for the expansion factor. All images were acquired using a laser scanning confocal microscope in Airyscan mode. (C, F, I, L, and O) Quantification of candidate protein enrichment on cytoplasmic lattices. Fold enrichment was measured by dividing the candidate protein mean fluorescence intensity within a cytoplasmic lattice segmentation mask by the cytoplasmic signal outside. Vertical lines mark the mean. Whiskers represent minimum and maximum values. See also Figures S6 and S7.

Article Snippet: Oocytes were immunostained with an anti-a-tubulin antibody (Biorad, #MCA78G) and glued to a coverslip using CellTak (Corning, #354240) for the pre-ExM image acquisition.

Techniques: Quantitative Proteomics, Labeling, Microscopy, Protein Enrichment

Journal: bioRxiv

Article Title: HTT silencing delays onset and slows progression of Huntington’s disease like phenotype: Monitoring with a novel neurovascular biomarker

doi: 10.1101/2020.11.17.386631

Figure Lengend Snippet: ( a ) Schematics of the designed HTT-gRNA (T1 and T3) and AAV vectors. ITR, inverted terminal repeat; HA, human influenza hemagglutinin; NLS, nuclear localization sequence; KASH, Klarsicht, ANC-1, Syne Homology; WPRE, woodchuck hepatitis virus post-transcriptional regulatory element; RFP, red fluorescent protein. ( b ) Timeline for AAV injections, HTT level measures and longitudinal CBVa assessment. ( c ) RFP fluorescence showing the transduction of AAV-HTT-gRNAs in the striatum. Scale bar = 900 μm (top image) and 40 μm (bottom image). ( c ) The images of Western blotting with indicated antibodies to HTT, NeuN, and β-actin. ( e ) Quantification results of mHTT (MW1), total HTT (combining upper and lower bands in the MCA2050 + MCA2051 blot), wtHTT (lower and major band in the blot with MAB 2166 antibody), and NeuN in the striatum injected with AAV-HTT gRNA + Cas9 (HTT gRNA, right striatum-R) or AAV-control gRNA + Cas9 (Con gRNA, left striatum-L). n=3, * p < 0.05 versus the values of con gRNA group by Student’s t -tests. ( f ) Representative CBVa maps in mouse brains from indicated genotypes and treatment groups. Top row shows the raw images, and the red ROIs indicate the quantified brain region. Bottom row shows the representative CBVa maps in the mice at the indicated genotypes and treatment at 3 months of age. The scale bars are shown on the right and warmer color represents higher CBVa values. ( g ) Quantification of longitudinal CBVa changes in the striatum of zQ175 HD mice and WT littermates with indicated treatment at indicated ages. Mean ± SEM, n = 6-7. * p < 0.05, comparison between zQ175 mice injected with HTT gRNAs (+ spCas9) versus zQ175 mice injected with control RNA (+ spCas9) by Two-way ANOVA with Bonferroni post hoc analysis.

Article Snippet: The membrane was blotted with the following primary antibodies: HTT (MAB 2166, 1:1000, MilliporeSigma, USA), HTT (MCA2050, 1:1000, BioRad, USA), HTT (MCA2050, 1:1000, BioRad, USA), mHTT (MW1, Anti-poly-Q, 1:1000, Millipore Sigma, USA) and mouse anti-β-actin (Sigma, mouse monoclonal antibody, 1:5000).

Techniques: Sequencing, Fluorescence, Transduction, Western Blot, Injection

Journal: bioRxiv

Article Title: HTT silencing delays onset and slows progression of Huntington’s disease like phenotype: Monitoring with a novel neurovascular biomarker

doi: 10.1101/2020.11.17.386631

Figure Lengend Snippet: ( a ) Timeline for AAV injections and outcome measures. (b) Longitudinal striatal volume data were quantified from structural MRI scans from indicated groups at indicated ages. ( c ) Mice were tested on a tapered beam and time crossing the beam (Traverse time) was recorded from different groups at indicated ages. ( d ) Mice were tested on a 5 mm beam and time crossing the beam (Traverse time) was recorded from different groups at indicated ages. All data in (b) through (d) are Mean ± SEM, n = 6-9. * p < 0.05, comparison between zQ175 mice injected with HTTg RNAs (+ spCas9) versus zQ175 mice injected with control RNA (+ spCas9) by Two-way ANOVA with Bonferroni post hoc analysis. ( e ) Mutant HTT (mHTT) aggregates were labeled by immunostaining with EM48 antibody in the striatum of zQ175 mice at 9 months of age. EM48-positive mHTT aggregates (green), pan neuronal marker NeuN (red), and nucleus (blue, DAPI) were indicated. Scale bar = 20 μm. ( f ) Numbers of mHTT aggregates (per mm 2 ) were quantified in the striatum. Mean ± SEM, n = 4, ** p < 0.01 versus the values of con gRNA group by standard Student’s t -tests.

Article Snippet: The membrane was blotted with the following primary antibodies: HTT (MAB 2166, 1:1000, MilliporeSigma, USA), HTT (MCA2050, 1:1000, BioRad, USA), HTT (MCA2050, 1:1000, BioRad, USA), mHTT (MW1, Anti-poly-Q, 1:1000, Millipore Sigma, USA) and mouse anti-β-actin (Sigma, mouse monoclonal antibody, 1:5000).

Techniques: Injection, Mutagenesis, Labeling, Immunostaining, Marker

Journal: bioRxiv

Article Title: The Bric-à-Brac transcription factors are necessary for formation of functional germline stem cell niches through control of dpp expression in the Drosophila melanogaster ovary

doi: 10.1101/689323

Figure Lengend Snippet: bab1 and bab2 functions in niche cells are not necessary for expression of several CC specification markers. (A-F) Whole mount immunostaining of the medial region of prepupal ovaries (projections of adjacent confocal sections). Nuclei are labelled with DAPI (grey). Anterior is up. Scale bars: 10 µm. (A-A’’’,C-C’’,E-E’’) correspond to the control ovaries. One hhG>GFP niche is encircled in each panel (pink dotted line). (B-B’’’,D-D’’,F-F’’) correspond to bab1 and bab2 RNAi-mediated knockdown ovaries. The entire cluster of hhG+ cells depleted of Bab1 and Bab2 is encircled in each panel (pink dotted line). (A,C,E) The green and yellow brackets indicate Terminal Filament (TF) cells and Cap Cells (CCs), respectively, and the green arrowheads point to transition cells. (B,D,E) The green and yellow brackets indicate the anterior- and posterior-most hhG+ cells, respectively. (A-A’’’) In the control ovaries, the two CCs markers, nuclear P1444-lacZ (Royal LUT) and nuclear Traffic Jam (red), are detected in CCs (yellow bracket) and in the transition cell (green arrowhead). Nuclear P1444-lacZ is also detected at a weaker level in some TF cells. The Germ Cells (GCs), marked with Vasa protein (red and cytoplasmic), in direct contact with niche cells (white arrowheads) show nuclear pMad (green) and are thus considered as Germinal Stem Cells (GSCs). (B-B’’’) Posterior-most medial hhG+ cells depleted of Bab1 and Bab2, express both CC markers (Tj and P1444-lacZ ) and are adjacent to GCs which do not present pMad+ (B’’’, arrowheads) and thus are not considered to be GSCs. (C-C”) In control ovaries, Delta (Royal LUT) accumulates at the plasma membrane and in cytoplasmic vesicles in TF cells, is detected in vesicle around the transition cell (green arrowhead) and sometimes in CCs (arrow). (D-D”) Upon depletion of Bab1 and Bab2 in hhG+ cells, Delta is not present at the plasma membrane, but is found in some vesicles (arrows). (E-E”,F-F”) In both the control (E-E”) and upon depletion of Bab1 and Bab2 in hhG+ cells (F-F”), the Notch pathway transcriptional reporter E(spl)mβ-CD2 is expressed since CD2 (Royal LUT) accumulates at the plasma membrane of TF cells / anterior-most hhG+ cells (green brackets) and of CCs / posterior-most hhG+ cells (yellow brackets), also accumulating Tj.

Article Snippet: The following primary antibodies were used: rabbit anti-Smad3 (1:200, ab52903, Abcam); rabbit anti-Bab1 (1:4000, gift from T. Williams); rabbit anti-GFP (1:200, FP-37151B, Interchim); rat anti-Bab2 (1:4000, gift from J-L. Couderc); rat IgM anti-Vasa (1:500, Developmental Studies Hybridoma Bank-DSHB); mouse anti-En/Inv recognizing both paralogs (1:200, 4D9, DSHB); guinea-pig anti GFP (1:200, 132 002, Synaptic System); mouse anti-β-Gal (1:400, 40-1a, DSHB,); guinea pig anti-Traffic Jam (1:5000, gift from D. Godt); mouse anti-Delta (1:200, C594.9B, DSHB), mouse anti-rat CD2 (1:100, MCA154GA, Biorad); mouse anti hts (1:200, DSHB).

Techniques: Expressing, Immunostaining