rabbit anti-ph3 (phosphorylated at ser10)  (Merck KGaA)

Journal: bioRxiv

Article Title: The transcription factor Chronophage/BCL11A/B promotes intestinal stem cell proliferation and endocrine differentiation in the Drosophila intestine

doi: 10.1101/2024.08.05.606739

Figure Lengend Snippet: a-b’’’ cph-YFP CPTI-1740 ;esg-Gal4 ts ,UAS-mCherry >UAS-Notch RNAi shows Cph-YFP expression in Notch-RNAi induced tumours. Animals were dissected after 7 days of induction at 29°C and stained with the EE marker Prospero (Pros, greyscale). a-a’’’ shows the expression of Cph in cph-YFP CPTI-1740 ; esg-Gal4 ts > UAS-mCherry control guts. b-b’’’ Cph expression in esg-Gal4 ts > UAS-mCherry, UAS-Notch RNAi guts. Note the distinct overlap in expression of Cph and the ISC/EB-marker escargot ( esg ) in the tumours outlined in white. There are also tumour cells that are Pros + and Cph + (arrowhead). This experiment was carried out three independent times (compiled n-values are as follows : esg-Gal4 ts > UAS-mCherry n =27 , cph-YFP CPTI-1740 ;esg-Gal4 ts >UAS-Notch RNAi n= 80). c qRT-PCR of cph mRNA in esg-Gal4 ts >UAS-Notch RNAi 14 days after induction alongside esg-Gal4 ts > UAS-mCherry control flies. n =15 guts were dissected from each genotype and used for total RNA extraction and cDNA generation. Data show a 34-fold increase in cph expression in the esg-Gal4 ts > UAS-Notch RNAi flies compared to esg-Gal4 ts > UAS-mCherry control flies ( p =3.7e-4). d cph knockdown decreases number of mitotic cells in Notch RNAi guts. Quantification of mitosis in esg-Gal4 ts > UAS-mCherry control , cph RNAi , Notch RNAi and cph RNAi ;Notch RNAi flies. Females of the appropriate genotype were collected, induced at 29°C and dissected after 7 days. Guts were stained with anti-pH3S10 and counted along the length of the intestine. There was a significant decrease in the numbers of pH3 + cells in the cph RNAi ;Notch RNAi compared to the Notch RNAi guts ( p =2.0e-5). The bar chart was compiled from two biological repeats. cph RNAi ;Notch RNAi n =51, Notch RNAi n =23, Cph RNAi n = 14, esg-Gal4 ts > UAS-mCherry n = 15. e Percentage of the portion of gut taken up by tumours in cph RNAi ;Notch RNAi guts is significantly reduced compared to Notch RNAi ( p 1.3e-6). f Loss of cph significantly extends lifespan of Notch RNAi tumour-bearing flies ( p = 4.2e-5). Data in Kaplan-Meier curve is representative of 3 independent repeats using two independent cph RNAi lines (see Supplementary Figure 4).

Article Snippet: The following antibodies were used: rabbit anti-phosphorylated Histone H3 (PH3) (1:500; Cell Signalling Technology Cat# 9701), chicken anti-GFP (1:1000; ThermoFisher A10262), mouse anti-Nubbin/Pdm1 (1:10; DSHB Cat# Nub 2D4O), mouse anti-Prospero (1:50; DSHB Cat# MR1A) and mouse anti-Delta (1:100; DSHB Cat# c594.9b).

Techniques: Expressing, Staining, Marker, Control, Quantitative RT-PCR, RNA Extraction, Knockdown

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Small ribonucleoprotein particle protein SmD3 governs the homeostasis of germline stem cells and the crosstalk between the spliceosome and ribosome signals in Drosophila .

doi: 10.1096/fj.201802536RR

Figure Lengend Snippet: Figure 2. Knockdown of SmD3 affects the homeostasis of proliferation and apoptosis in Drosophila S2 cells. A) Relative SmD3 mRNA levels in NC (siRNA) and SmD3 siRNA cells to validate the knockdown efficiency. B) Ratio of PH3 positive cells in NC (siRNA) and SmD3 siRNA-555 S2 cells. C) Ratio of TUNEL-positive cells in NC (siRNA) and SmD3 siRNA-555 S2 cells. D) Immunostaining of NC (siRNA) and SmD3 siRNA-555 S2 cells using anti-PH3 (red) and DNA (blue). E) Immunostaining of NC (siRNA) and SmD3 siRNA-555 S2 cells using TUNEL (red) and DNA (blue). F) Flow cytometry testing of NC (siRNA) and SmD3 siRNA-555 S2 cells. G) CCK-8 assay for NC (siRNA) and SmD3 siRNA-555 S2 cells. Comp-Alexa Fluor 647-A., compartment of Annexin V Alexa Fluor 647; Comp-PI-A., compartment of propidium lodide; n.s., not significant; OD, optical density; Q, quadrant. Scale bars, 30 mm. *P , 0.05, **P , 0.01, ***P , 0.001.

Article Snippet: The antibodies usedwere as follows: mouse anti-Eya [1:20; Developmental Studies Hybridoma Bank (DSHB), Iowa City, IA, USA]; rat anti–Drosophila E-cadherin (DE-cadherin) (1:20; DSHB); mouse anti–fasciclin III (FasIII) (1:50; DSHB); rabbit anti-Vasa (1:1000; a gift from C. Tong); rat anti–zinc finger homeodomain 1 (Zfh1) (1:2000; a gift from C. Tong); rabbit anti–phosphorylated histone 3 (PH3) (53348, 1:1000; Cell Signaling Technology, Danvers, MA, USA); and rabbit antiHA (3724, 1:1000; Cell Signaling Technology).

Techniques: Knockdown, TUNEL Assay, Immunostaining, Flow Cytometry, CCK-8 Assay

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Small ribonucleoprotein particle protein SmD3 governs the homeostasis of germline stem cells and the crosstalk between the spliceosome and ribosome signals in Drosophila .

doi: 10.1096/fj.201802536RR

Figure Lengend Snippet: Figure 3. Overexpression of SmD3 increases proliferation ability and cell death in Drosophila S2 cells. A) Immunostaining of blank control (Ctrl), vector, and SmD3-overexpressing S2 cells using anti-PH3 (red) and DNA (blue). B) Ratio of PH3-positive cells in Ctrl, vector, and SmD3-overexpressing S2 cells. C) Immunostaining of Ctrl, vector, and SmD3-overexpressing S2 cells using TUNEL (red) and DNA (blue). D) Ratio of TUNEL-positive cells in Ctrl, vector, and SmD3-overexpressing S2 cells. E) Flow cytometry of Ctrl, vector, and SmD3-overexpressing S2 cells. The ratio of viable cells dramatically decreased, whereas that of apoptotic and necrotic cells significantly increased. F) CCK-8 assay of Ctrl, vector, and SmD3-overexpressing S2 cells. Comp-Alexa Fluor 647-A., compartment of Annexin V Alexa Fluor 647; Comp-PI-A., compartment of propidium lodide; n.s., not significant; OD, optical density; Q, quadrant. Scale bars, 30 mm. *P , 0.05, **P , 0.01, ***P , 0.001.

Article Snippet: The antibodies usedwere as follows: mouse anti-Eya [1:20; Developmental Studies Hybridoma Bank (DSHB), Iowa City, IA, USA]; rat anti–Drosophila E-cadherin (DE-cadherin) (1:20; DSHB); mouse anti–fasciclin III (FasIII) (1:50; DSHB); rabbit anti-Vasa (1:1000; a gift from C. Tong); rat anti–zinc finger homeodomain 1 (Zfh1) (1:2000; a gift from C. Tong); rabbit anti–phosphorylated histone 3 (PH3) (53348, 1:1000; Cell Signaling Technology, Danvers, MA, USA); and rabbit antiHA (3724, 1:1000; Cell Signaling Technology).

Techniques: Over Expression, Immunostaining, Control, Plasmid Preparation, TUNEL Assay, Flow Cytometry, CCK-8 Assay

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Small ribonucleoprotein particle protein SmD3 governs the homeostasis of germline stem cells and the crosstalk between the spliceosome and ribosome signals in Drosophila .

doi: 10.1096/fj.201802536RR

Figure Lengend Snippet: Figure 4. SmD3 rescue assay in Drosophila S2 cells. A) Relative SmD3 mRNA level in NC (siRNA)+vector, NC (siRNA)+SmD3, SmD3 siRNA+vector, and SmD3 siRNA+SmD3 S2 cells. B) Ratio of PH3-positive cells in NC (siRNA)+vector, NC (siRNA)+SmD3, SmD3 siRNA+vector, and SmD3 siRNA+SmD3 S2 cells. C) Immunostaining of NC (siRNA)+vector, NC (siRNA)+SmD3, SmD3 siRNA+vector, and SmD3 siRNA+SmD3 S2 cells using anti-PH3 (red) and DNA (blue). D) Ratio of TUNEL-positive cells in NC (siRNA)+vector, NC (siRNA)+SmD3, SmD3 siRNA+vector, and SmD3 siRNA+SmD3 S2 cells. E) Immunostaining of NC (siRNA)+vector, NC (siRNA)+SmD3, SmD3 siRNA+vector, and SmD3 siRNA+SmD3 S2 cells using TUNEL (red) and DNA (blue). F) Flow cytometry test of NC (siRNA)+vector, NC (siRNA)+SmD3, SmD3 siRNA+vector, and SmD3 siRNA+SmD3 S2 cells. The ratio of viable cells dramatically decreased, whereas the ratios of apoptotic and necrotic cells significantly increased, in SmD3 siRNA+SmD3 S2 cells. Immunostaining signals of certain areas were enlarged views in big frames. Comp- Alexa Fluor 647-A., compartment of Annexin V Alexa Fluor 647; Comp-PI-A., compartment of propidium lodide; n.s., not significant; OD, optical density; Q, quadrant. Scale bars, 30 mm. *P , 0.05, **P , 0.01, ***P , 0.001.

Article Snippet: The antibodies usedwere as follows: mouse anti-Eya [1:20; Developmental Studies Hybridoma Bank (DSHB), Iowa City, IA, USA]; rat anti–Drosophila E-cadherin (DE-cadherin) (1:20; DSHB); mouse anti–fasciclin III (FasIII) (1:50; DSHB); rabbit anti-Vasa (1:1000; a gift from C. Tong); rat anti–zinc finger homeodomain 1 (Zfh1) (1:2000; a gift from C. Tong); rabbit anti–phosphorylated histone 3 (PH3) (53348, 1:1000; Cell Signaling Technology, Danvers, MA, USA); and rabbit antiHA (3724, 1:1000; Cell Signaling Technology).

Techniques: Rescue Assay, Plasmid Preparation, Immunostaining, TUNEL Assay, Flow Cytometry

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Small ribonucleoprotein particle protein SmD3 governs the homeostasis of germline stem cells and the crosstalk between the spliceosome and ribosome signals in Drosophila .

doi: 10.1096/fj.201802536RR

Figure Lengend Snippet: Figure 8. Effects of SmD3 on proliferation and apoptosis by RpL18. A) Relative RpL18 and SmD3 mRNA levels in vector and RpL18-overexpressing S2 cells. B) Relative RpL18 and SmD3 mRNA levels in vector (96 h) and RpL18+SmD3 S2 cells. C ) Ratio of PH3-posi- tive cells in vector (96 h) and RpL18+SmD3 S2 cells. D) Immu- nostaining of vector (96 h) and RpL18+SmD3 S2 cells using anti- PH3 (red) and DNA (blue). E) Ratio of TUNEL-positive cells in vector (96 h) and RpL18+SmD3 S2 cells. F) Immunostaining of vector (96 h) and RpL18+SmD3 S2 cells using TUNEL (red) and DNA (blue). G) Flow cytome- try test of vector (96 h) and RpL18+SmD3 S2 cells. The ratio of viable cells dramatically decreased, and the ratio of apo- ptotic and necrotic cells signif- icantly increased. Comp-Alexa Fluor 647-A., compartment of Annexin V Alexa Fluor 647; Comp-PI-A., compartment of propidium lodide; n.s., not sig- nificant; OD, optical density; Q, quadrant. Scale bars, 30 mm. *P , 0.05, **P , 0.01, ***P , 0.001.

Article Snippet: The antibodies usedwere as follows: mouse anti-Eya [1:20; Developmental Studies Hybridoma Bank (DSHB), Iowa City, IA, USA]; rat anti–Drosophila E-cadherin (DE-cadherin) (1:20; DSHB); mouse anti–fasciclin III (FasIII) (1:50; DSHB); rabbit anti-Vasa (1:1000; a gift from C. Tong); rat anti–zinc finger homeodomain 1 (Zfh1) (1:2000; a gift from C. Tong); rabbit anti–phosphorylated histone 3 (PH3) (53348, 1:1000; Cell Signaling Technology, Danvers, MA, USA); and rabbit antiHA (3724, 1:1000; Cell Signaling Technology).

Techniques: Plasmid Preparation, TUNEL Assay, Immunostaining

Journal: Disease Models & Mechanisms

Article Title: A novel zebrafish intestinal tumor model reveals a role for cyp7a1 -dependent tumor–liver crosstalk in causing adverse effects on the host

doi: 10.1242/dmm.032383

Figure Lengend Snippet: pInt-Gal4 -driven kras G12D expression leads to abnormal proliferation of intestinal cells. (A,B) Representative images of tumor-bearing larvae [ Tg(pInt-Gal4) +/Tg ; Tg(5×UAS:EGFP-P2A-kras G12D ) +/Tg ] and the sibling controls [ Tg(pInt-Gal4) +/Tg ; Tg(UAS:EGFP) +/Tg ] at 5 dpf. Bright-field (A) and EGFP (B) images are shown. Scale bar: 500 µm. (C) qPCR analysis for the EGFP-P2A-kras G12D transgene in the sibling controls and tumor-bearing larvae. The scores are normalized to expression of rpl13a . The data harbors three biological replicates. Error bars represent means±s.e.m. (D-I) Representative images of DAPI staining for transversal sections of the posterior intestine of tumor-bearing larvae and the sibling controls at 5 dpf. DAPI (D,E) and EGFP (F,G) images are shown. In the merged images (H,I), DAPI and EGFP signals are shown in blue and green, respectively. Scale bar: 100 µm. (J) The number of EGFP- and DAPI-positive intestinal cells. The number of nuclei was manually counted from a single section per individual larva. The data harbors 7 and 11 biological replicates from tumor-bearing larvae and the sibling controls, respectively. Error bars represent means±s.e.m. Statistical significance was tested using Student's t -test (unpaired, one-tailed). (K-R) Representative images of fluorescent immunohistochemistry for phosphorylated histone H3 (pH3) in transversal sections of the posterior intestine of tumor-bearing larvae and the sibling controls at 5 dpf. pH3 (K,L), DAPI (M,N) and EGFP (O,P) images are shown. White arrow indicates intestinal cells positive for pH3, EGFP and DAPI. In the merged images (Q,R), pH3, DAPI and EGFP signals are shown in red, blue and green, respectively. Scale bar: 100 µm. (S) The number of intestinal cells positive for pH3, EGFP and DAPI. The number of pH3-, EGFP- and DAPI-positive cells was counted from a single section per individual larva. The data harbors 8 and 6 biological replicates from tumor-bearing larvae and the sibling controls, respectively. Error bars represent means±s.e.m. Data are representative of at least two independent experiments.

Article Snippet: Sections were rehydrated with PBS at room temperature for 30 min, and then permeabilized and blocked with 5% normal goat serum in PBS supplemented with 0.5% Triton X-100 (0.5% PBT) for 1 h. Sections were then incubated with the following primary antibodies diluted in 5% normal goat serum in 0.5% PBT at 4°C overnight: rabbit anti-phosphorylated-Histone H3 (Ser10) (pH3) (EMD Millipore, 06-570; 1:100 dilution) and rabbit anti-E-cadherin (Cdh1) (Gene Tex, GTX125890; 1:100 dilution).

Techniques: Expressing, Staining, One-tailed Test, Immunohistochemistry

Journal: bioRxiv

Article Title: A novel zebrafish intestinal tumor model reveals a role for cyp7a1 -dependent tumor-liver crosstalk in tumor's adverse effects on host

doi: 10.1101/199349

Figure Lengend Snippet: (A)-(B) Representative images of tumor-bearing fish ( Tg(pInt-Gal4) +/Tg ; Tg(5×UAS:EGFP-P2A-kras G12D ) +/Tg ) and the sibling control ( Tg(pInt-Gal4) +/Tg ; Tg(UAS:EGFP) +/Tg ) at 5 dpf. Bright field (A) and EGFP (B) images are shown. Scale bar indicates 500 μm. (C) qPCR for EGFP-P2A-kras G12D expression in the sibling controls and tumor-bearing fish.The scores are normalized to expression of rpl13a . The data harbors three biological replicates. Error bars represent ± s.e.m. (D)-(I) Representative images of DAPI staining in intestine sections of tumor-bearing fish and the sibling controls at 5 dpf. DAPI (D, E) and EGFP (F, G) images are shown. In the merged images (H, I), DAPI and EGFP signals are shown in blue and green, respectively. Scale bar indicates 100 μm. (J) The number of EGFP and DAPI positive intestinal cells. The number of nuclei was manually counted from single section per individual fish. The data harbors 7 and 11 biological replicates from tumor-bearing fish and the sibling controls, respectively. Error bars represent ± s.e.m. Statistical significance was tested using student’s t -test (unpaired, one-tailed).(K)-(R) Representative images of fluorescent immunohistochemistry for phosphorylated histone H3 (pH3) in intestine sections of tumor-bearing fish and the sibling controls at 5 dpf. pH3 (K, L), DAPI (M, N) and EGFP (O, P) images are shown. White arrows indicate intestinal cells positive for pH3, EGFP, and DAPI. In the merged images (Q, R), pH3, DAPI and EGFP signals are shown in red, blue and green, respectively. Scale bar indicates 100 μm. (S) The number of intestinal cells positive for pH3, EGFP, and DAPI. The number of pH3, EGFP, and DAPI positive cells was counted from single section per individual fish. The data harbors 8 and 6 biological replicates from tumor-bearing fish and the sibling controls, respectively. Error bars represent ± s.e.m.

Article Snippet: Sections were rehydrated by PBS at room temperature for 30 min, and then permeabilized and blocked with 5% normal goat serum in PBS supplemented with 0.5% TritonX-100 (0.5% PBT) for 1 h. Sections were then incubated with the following primary antibodies diluted in 5% normal goat serum in 0.5% PBT at 4°C for overnight: rabbit anti-phosphorylated-Histone H3 (Ser10) (pH3) (EMD Millipore, 06-570; 1:100 dilution) and rabbit anti-E-cadherin (cdh1) (Gene Tex, GTX125890; 1:100 dilution).

Techniques: Control, Expressing, Staining, One-tailed Test, Immunohistochemistry