Journal: bioRxiv

Article Title: Intraflagellar transport complex B proteins regulate the Hippo effector Yap1 during cardiogenesis

doi: 10.1101/777128

Figure Lengend Snippet: (A) Schematic representation of the zebrafish heart at 55 hpf in frontal and lateral views. The dorsal pericardium is shown in grey, the myocardium in red, the avcPE in light green and the epicardial cells in dark green. V, ventricle; At, atrium. (B) Percentage of avcPE cluster number in iguana (n=43), ift88 (n=50) and elipsa/ift54 (n=38) mutants and their controls (n=24; n=43; n=40 respectively), between 50 and 57 hpf. (C) High-speed avcPE imaging (digital red mask to improve visualization) of iguana and ift88 mutants and their control. (D) Graphs show avcPE cell number quantified on iguana (n=20), ift88 (n=18 and 29) and elipsa (n=25) mutants in epi:GFP background and their controls (n=12; n=15; n=28 n=18 respectively). (t-test iguana p-value 0.001; ift88 p-value 0.0002 and p-value 0.015 respectively; elipsa p-value 0.0005). 3D projections of whole mount immunofluorescence of hearts using an anti-myosin heavy chain antibody (red) and GFP (green) expression. Ventral views, anterior is to the top. Arrowheads mark avcPE and asterisk shows lack of avcPE. (E) Percentage of avcPE cluster number on iguana-/-, ift88-/-, epi:GFP (n=9) and controls (n=23) at 55 hpf. (F) Graph shows avcPE cell number quantified in wild type (n=13) and double heterozygous controls (n=10) (ift88+/+; iguana+/+; epi:GFP and ift88 +/- ; iguana +/- ; epi:GFP) and double ift88; iguana mutants (n=9) (ift88-/-; iguana-/-; epi:GFP) (Kruskal-Wallis p-value 0.014). 3D projections of whole mount immunofluorescence of hearts using myosin heavy chain antibody (red) and GFP (green) expression. Ventral views, anterior is to the top. Arrowheads mark avcPE. V, ventricle; At, atrium. (G) Schematic representation of an E9.5 mouse embryo. Heart tube (HT) in dark grey and PE in white. Section of the PE represented inside the yellow box. (H) Left side graph shows quantification of PE volume (µm3) in Ift20 KO (n=4) and control mice (n=3) (control 5.35×10 6 µm 3 ±3.63×10 5 ; Ift20 KO 9.06×10 6 µm 3 ±1.46×10 6 ) (t-test p-value 0.008) and that of Ift88 KO (n=4) and control embryo (n=4) on the right (control 2.78×10 6 µm 3 ±1. 3×10 6 ; Ift88 KO 7.05×10 6 µm 3 ±1.2×10 6 ) (t-test p-value 0.003). In the lower panel, 3D projections of immunofluorescence whole-mount performed on control and Ift20 KO embryos. PE marked using anti-Wt1 antibody. White dotted shapes enclose the PE area. In all graphs, red bars indicate mean ± standard deviation.

Article Snippet: We performed IPs using GFP-Trap (ChromoTek) agarose beads in two conditions: Control IP (YAP1-Myc , pEGFP-C1 and HA-Amotl1( )) and IFT88 IP (YAP1-Myc, IFT88-GFP ( ) and HA-Amotl1).

Techniques: Imaging, Control, Immunofluorescence, Expressing, Standard Deviation

Journal: bioRxiv

Article Title: Intraflagellar transport complex B proteins regulate the Hippo effector Yap1 during cardiogenesis

doi: 10.1101/777128

Figure Lengend Snippet: (A) Confocal sections of whole mount immunofluorescence of iguana-/-, epi:GFP and control using anti-myosin heavy chain antibody (MHC) (magenta), anti-GFP (cyan) and DAPI (white) antibodies at 55 hpf. Asterisk shows lack of avcPE as there is only one rounded PE cell. (B) Confocal sections of whole mount immunofluorescence of ift88-/-, epi:GFP and control using anti-myosin heavy chain antibody (MHC) (magenta), anti-GFP antibody (cyan) and DAPI (white) at 50 hpf. (C) Confocal sections of whole mount immunofluorescence of elipsa-/-, epi:GFP and control using anti-myosin heavy chain antibody (MHC) (magenta), anti-GFP (cyan) and DAPI (white) antibodies at 55 hpf. (D) Confocal sections of whole mount immunofluorescence of ift88-/-, iguana-/-, epi:GFP and ift88+/+, iguana+/+, epi:GFP using anti-myosin heavy chain antibody (MHC) (magenta), anti-GFP (cyan) and DAPI (white) antibodies at 55 hpf. All images are ventral views, anterior is to the top. V, ventricle; At, atrium; PE, avcPE. Zoomed regions contained inside the red boxes are showed on the right side.

Article Snippet: We performed IPs using GFP-Trap (ChromoTek) agarose beads in two conditions: Control IP (YAP1-Myc , pEGFP-C1 and HA-Amotl1( )) and IFT88 IP (YAP1-Myc, IFT88-GFP ( ) and HA-Amotl1).

Techniques: Immunofluorescence, Control

Journal: bioRxiv

Article Title: Intraflagellar transport complex B proteins regulate the Hippo effector Yap1 during cardiogenesis

doi: 10.1101/777128

Figure Lengend Snippet: (A-A’’) Graphs show epicardial cell numbers quantified in ift88, iguana and elipsa mutants in epi:GFP background. (A) At 55 hpf, ift88 mutants (n=15) showed increased epicardial cell numbers (t-test p value 0.04). 3D projections of whole mount immunofluorescence of hearts using anti-myosin heavy chain antibody (red) and GFP (green) expression. Ventral view, anterior is to the top. Arrows mark some epicardial cells; ( A’ ) iguana mutants (n=20) showed a tendency towards decreased epicardial cell numbers (t-test p value 0.055), while ( A’’ ) elipsa mutants (n=18) showed a tendency towards increased epicardial cell number (t-test p value 0.08). (B) Graph shows avcPE cell numbers quantified in yap1-/- (n=15) and control (n=21) embryos in tcf21:nsl-GFP background at 55 hpf. (t-test p-value 0.12) Control and yap1-/- immunofluorescence confocal sections labelled with anti-myosin heavy chain antibody (MHC) (red), GFP (green), DAPI (white). Ventral view, anterior is to the top. Yellow arrowheads point at the avcPE. (B’) Graph shows epicardial cell numbers quantified in yap1-/- (n=15) and control (n=21) embryos in tcf21:nsl-GFP background at 55 hpf. (Mann Whitney p-value 0.63) (C) For cilia quantification, we divided the dorsal pericardial wall in to three different regions: SV region, including the sinus venosus (pink); PE region, where the avcPE forms (yellow) and Deeper region (purple). These three regions were subdivided in to right and left halves, containing the ventricle or the atrium respectively. At 48 hpf (n=9 larvae), prior to PE formation, cilia protruding from the dorsal pericardium showed a heterogeneous distribution. Interestingly, the right half of the SV (6 ± 1) and the PE (17 ± 3) regions, where both PE clusters will form, presented higher cilia number than the rest of the regions. At 55 hpf, when the avcPE is formed, the cilia distribution was similar to that observed at 48 hpf (n= 6 larvae). (D) Confocal section of actb2:Mmu.Arl13b-GFP embryo (55 hpf). Yellow dotted circle encloses the avcPE. Yellow arrows point to cilia protruding from the ventral and dorsal pericardium. Yellow arrowheads point at immotile and bent cilia protruding from a few avcPE cells. (E) Confocal section of iguana; actb2:Mmu.Arl13b-GFP and control embryos (55 hpf). Yellow arrowhead points at cilium protruding from the avcPE. Yellow asterisk shows lack of cilia in the avcPE (enclosed in the white circle). Confocal section of ift88; actb2:Mmu.Arl13b-GFP and control embryos (55 hpf). In control embryo, yellow arrowhead points at cilium protruding from the ventral pericardium. In ift88 mutant embryo, yellow arrowheads point at cilia protruding from ventral and dorsal pericardium and the avcPE (enclosed in the white circle). (F) Coronal and sagittal sections acquired by light sheet microscopy to illustrate the methods used to measure PE volume (labeled with anti-Wt1 antibody). Red and yellow dotted shapes enclose the PE area. In all images ventral views, anterior is to the top. V, ventricle; At, atrium. In all graphs, red bars indicate mean ± standard deviation.

Article Snippet: We performed IPs using GFP-Trap (ChromoTek) agarose beads in two conditions: Control IP (YAP1-Myc , pEGFP-C1 and HA-Amotl1( )) and IFT88 IP (YAP1-Myc, IFT88-GFP ( ) and HA-Amotl1).

Techniques: Immunofluorescence, Expressing, Control, MANN-WHITNEY, Mutagenesis, Microscopy, Labeling, Standard Deviation

Journal: bioRxiv

Article Title: Intraflagellar transport complex B proteins regulate the Hippo effector Yap1 during cardiogenesis

doi: 10.1101/777128

Figure Lengend Snippet: (A,B) Ift20 and Ift88 KO mice show left-right patterning defects including heart looping defects at E9.5. (C) Control and Ift20 KO cryosections imaged by confocal microscopy after labelling with anti-TBX18 (white), anti-Arl13b (red) and anti-γ-tubulin (yellow) antibodies and Hoechst (blue) at E9.5. Zoomed region (enclosed in yellow box) shows the lack of Arl13b signal in Ift20 KO mice. Individual channels are shown for Arl13b (red), γ-tubulin (yellow). (C’) Control and Ift88 KO cryosections labelled with anti-TBX18 (white), anti-Arl13b (red) and anti-γ-tubulin (yellow) antibodies and Hoechst (blue) at E9.5. Zoomed region (enclosed in yellow box) shows the decrease of Arl13b signal in Ift88 KO mice. Individual channels are shown for Arl13b (red), γ-tubulin (yellow). (C’’) Graph shows the percentage of ciliated PE cells in Ift20 KO (n=3, Ift88 KO (n=3) and control (n=3) mice. The percentage of ciliated PE cells is severely reduced in Ift20 KO (n=3) and Ift88 KO (n=3) when compared to control (n=3) mice. (t-test Ift20 p-value 0.024; t-test Ift88 p-value 0.025) (D) Control and Ift88 KO cryosections labelled with WT1 (red), p-smad 1/5/9 (yellow) and Hoechst (blue) at E9.5. Individual channel is displayed for p-smad 1/5/9 as ice LUT to facilitate the visualization of signal intensity (green is the minimum and red is the maximum).Graph shows that the percentage of p-smad 1/5/9 positive PE cells is similar in Ift88 KO (n=3 embryos: 1477 nuclei analyzed) and controls (n=3 embryos: 1514 nuclei analyzed) (Chi-square test of homogeneity =0.15225, p-value 0.6964 on 1 degree of freedom).

Article Snippet: We performed IPs using GFP-Trap (ChromoTek) agarose beads in two conditions: Control IP (YAP1-Myc , pEGFP-C1 and HA-Amotl1( )) and IFT88 IP (YAP1-Myc, IFT88-GFP ( ) and HA-Amotl1).

Techniques: Control, Confocal Microscopy

Journal: bioRxiv

Article Title: Intraflagellar transport complex B proteins regulate the Hippo effector Yap1 during cardiogenesis

doi: 10.1101/777128

Figure Lengend Snippet: (A) Graphs show total myocardial and atrial-myocardial cell number quantified in ift88 (n=16) mutants and controls (n=11) at 50 hpf. (t-test total myocardium p-value 0.0034; atrial myocardium p-value <0.0001). (A’) 3D projections of whole mount immunofluorescence of hearts using myosin heavy chain antibody (MHC) (red). Ventral views, anterior is to the top. (B) Graphs show total myocardial and atrial-myocardial cell number quantified in elipsa (n=14) mutants and controls (n=15) at 55 hpf. (t-test total myocardium p-value 0.0026; atrial myocardium p-value 0.017). (C) Graphs show total myocardial and atrial-myocardial cell number quantified in yap1 (n=14) mutants and controls (n=17) at 55 hpf. (t-test total myocardium p-value 0.0001; atrial myocardium p-value 0.0009). (D) Graph shows number of p-smad1/5 positive cells in the myocardium and dorsal pericardium (DP) quantified in ift88 mutants (n=8) and controls (n=7) at 50 hpf. Myocardium (t-test p-value 0.009) and DP (t-test p-value 0.036). (D’) 3D projections of whole mount immunofluorescence of hearts using myosin heavy chain antibody (MHC) (red), epi:GFP (green) and p-smad1/5 (yellow) antibody. Arrows mark dorsal pericardial cells positive for epi:GFP and p-smad1/5. Ventral views, anterior is to the top. (E) Graph shows number of p-smad1/5 positive cells in the myocardium and dorsal pericardium (DP) quantified in elipsa mutants (n=14) and controls (n=10) at 55 hpf. Myocardium (t-test p-value < 0.0001) and DP (t-test p-value 0.0004). (F) Graph shows number of p-smad1/5 positive cells in the myocardium and dorsal pericardium (DP) quantified in yap1 mutants (n=12) and controls (n=11) at 55 hpf. Myocardium (t-test p-value < 0.0001) and DP (t-test p-value 0.599). (G) Graph shows that the percentage of p-smad 1/5/9 positive PE cells is higher in Ift20 KO (n=4 embryos: 1862 nuclei analyzed) compared to control (n=3 embryos: 1414 nuclei analyzed) mice (Chi-square test of homogeneity =51.593, p-value 6.829e-13 on 1 degree of freedom). (G’) Control and Ift20 KO immunofluorescence confocal cryosections labelled with WT1 (red), p-smad 1/5/9 (yellow) and Hoechst (blue) at E9.5. Yellow dotted lines enclose the PE area. Individual channels are displayed for p-smad 1/5/9 (signal is shown as ice LUT to facilitate the visualization of signal intensity, where green is the minimum and red is the maximum), Hoechst (white) and WT1 (white). In all graphs, red bars indicate mean ± standard deviation. V, ventricle; At, atrium. p-smad, p-smad1/5 in panel D ’ and p-smad1/5/9 in panel G ’ .

Article Snippet: We performed IPs using GFP-Trap (ChromoTek) agarose beads in two conditions: Control IP (YAP1-Myc , pEGFP-C1 and HA-Amotl1( )) and IFT88 IP (YAP1-Myc, IFT88-GFP ( ) and HA-Amotl1).

Techniques: Immunofluorescence, Control, Standard Deviation

Journal: bioRxiv

Article Title: Intraflagellar transport complex B proteins regulate the Hippo effector Yap1 during cardiogenesis

doi: 10.1101/777128

Figure Lengend Snippet: (A) The top two graphs show total myocardial and atrial-myocardial cell numbers quantified in ift88 (n=13) mutants and controls (n=14) at 55 hpf. (t-test total myocardium p-value 0.016; atrial myocardium p-value 0.003). The bottom two graphs show total myocardial and atrial-myocardial cell number quantified in iguana (n=24) mutants and controls (n=18) at 55 hpf. (t-test total myocardium p-value 0.2; atrial myocardium p-value 0.68). (B) Whole mount bmp4 in situ hybridization performed on control and ift88 mutant embryos at 48 hpf and at 55 hpf on ift88, elipsa and iguana mutants and their controls. Yellow arrows point to bmp4 overexpression, while white asterisks mark reduced or absent expression. Ventral views, anterior is to the top. V, ventricle; At, atrium. (C) Graphs show number of p-smad1/5 positive cells in the atrial myocardium quantified in ift88 (at 50 hpf n=8; at 55 hpf n=7), elipsa (n=14) mutants and their controls (n=7; n=5; n=10 respectively). At 50 hpf, ift88 mutants show p-smad 1/5 increased cell number on the atrial myocardium (t-test p value 0.0017). Similar data were obtained at 55 hpf (t-test p value 0.0008). At 55 hpf, elipsa mutants also show p-smad 1/5 increased cell number in the atrial myocardium (t-test p value 0.0003). At 55 hpf, ift88 mutants show p-smad 1/5 increased cell numbers in the myocardium (t-test p value 0.005). (D) 3D projections of whole mount immunofluorescence of hearts using anti-myosin heavy chain antibody (MHC) (red), epi:GFP (green) and anti-p-smad1/5 (yellow) antibody. Arrows mark avcPE cells positive for epi:GFP and p-smad1/5. Zoomed confocal sections show avcPE in ift88 mutant and control embryos. Individual channels are displayed for p-smad 1/5 and GFP. Ventral views, anterior is to the top. In all graphs, red bars indicate mean ±standard deviation.

Article Snippet: We performed IPs using GFP-Trap (ChromoTek) agarose beads in two conditions: Control IP (YAP1-Myc , pEGFP-C1 and HA-Amotl1( )) and IFT88 IP (YAP1-Myc, IFT88-GFP ( ) and HA-Amotl1).

Techniques: In Situ Hybridization, Control, Mutagenesis, Over Expression, Expressing, Immunofluorescence, Standard Deviation

Journal: bioRxiv

Article Title: Intraflagellar transport complex B proteins regulate the Hippo effector Yap1 during cardiogenesis

doi: 10.1101/777128

Figure Lengend Snippet: (A) Graphs show number of Yap1-positive cells in the myocardium and dorsal pericardium (DP) quantified in ift88 (n=13), elipsa (n=6) and their controls (n=12 and n=7 respectively). ift88 mutants show increased Yap1-positive myocardial cell numbers (t-test p value 0.03) and a tendency towards higher Yap1 positive DP cell numbers (t-test p value 0.07). elipsa mutants show higher Yap1-positive myocardial cell numbers (t-test p value 0.036). (A’) Control and ift88-/-, epi:GFP immunofluorescence confocal sections labelled with anti-myosin heavy chain antibody (MHC) (red), GFP (green),-Yap1 (white) and DAPI (blue) at 55 hpf. Ventral view, anterior is to the top. Individual channel is displayed for Yap1 (signal is shown as ice LUT to facilitate the visualization of signal intensity, where green is the minimum and red is the maximum). Yellow arrows mark nuclear Yap1-positive atrial myocardial cells. White arrowheads mark the avcPE. (B) Graph shows avcPE cell number quantified in control (n=9), ift88-/-, epi:GFP (n=8) and Verteporfin (5µM)-treated ift88-/-, epi:GFP (n=16) and control (n=20) embryos (55hpf). Control embryos treated with Verteporfin showed smaller avcPE compared to untreated controls (t-test p-value 0.04). Verteporfin-treated ift88-/-; epi:GFP embryos presented lower avcPE cell numbers than non-treated ift88-/-; epi:GFP embryos (t-test p-value 0.027). ift88-/-; epi:GFP embryos showed bigger avcPE compared to untreated (t-test p-value 0.01) and treated controls (t-test p-value 0.0001). (C) Graph shows number of p-smad 1/5-positive cells in the myocardium quantified in control (n=7), ift88-/-, epi:GFP (n=5) and Verteporfin (20µM)-treated ift88-/-, epi:GFP (n=5) and control (n=6) embryos from 31 hpf to 55 hpf. Control embryos treated with Verteporfin showed decreased p-smad 1/5-positive cell numbers compared to untreated controls (t-test p-value 0.0219). Verteporfin-treated ift88-/-; epi:GFP embryos presented less p-smad 1/5-positive cells than non-treated ift88-/-; epi:GFP embryos (t-test p-value 0.0173). ift88-/-; epi:GFP embryos showed more p-smad 1/5-positive cells compared to untreated (t-test p-value <0.0001) and treated controls (t-test p-value <0.0001). (D) Percentage of avcPE cluster number in amotl2a+/+ (n=26) and amotl2a-/- (n=38) embryos between 50 and 57 hpf. (E) Whole mount bmp4 in situ hybridization in amotl2a+/+ (n=18/25) and amotl2a-/- (n=13/17) embryos (55hpf). Yellow arrow shows bmp4 overexpression. Ventral views, anterior is to the top. (F) Graph shows number of p-smad 1/5-positive cells in the myocardium quantified in Verteporfin (20µM)-treated amotl2a-/- (n=19) embryos from 31 to 55 hpf and untreated amotl2a-/- (n=12) embryos. Treated embryos showed decreased p-smad 1/5-positive cell numbers compared to untreated ones (t-test p-value 0.0148). (F’) Graph shows avcPE cell number quantified in Verteporfin (20µM)-treated amotl2a-/- (n=13) embryos from 31 to 55 hpf and untreated amotl2a-/- (n=10) embryos. Treated embryos showed decreased avcPE cell numbers compared to untreated ones (t-test p-value 0.0059). (G) Whole mount bmp4 in situ hybridization in XAV939 (10µM)-treated amotl2a-/- (n=16) from 31 to 55 hpf and untreated embryos (n=11). Treated embryos showed either decreased (n=8/16) or absent (n=7/16) bmp4 expression at the atrioventricular canal myocardium and the venous pole. Ventral views, anterior is to the top. (H) Graphs show percentages of YAP1-positive PE cells and double YAP1-AMOTL1-positive PE cells in Ift20 KO (n=5 embryos: 1196 nuclei analyzed) and control (n=4 embryos: 929 nuclei analyzed) mice at E9.5. The percentage of nuclear YAP1-positive PE cells (Chi-square test of homogeneity =25.354, p-value 4,77E-07 on 1 degree of freedom) and nuclear YAP1-AMOTL1-positive cells (Chi-square test of homogeneity =12,025, p-value 5,25E-04 on 1 degree of freedom) were higher in Ift20 KO than in control mice. (H’) Control and Ift20 KO immunofluorescence confocal cryosections labelled with TBX18 (red), YAP1 (yellow) and Hoechst (blue) at E9.5. White dotted lines enclose the PE area. (H’’) Zoomed region shows the difference between nuclear YAP1-positive cells (yellow arrows) and YAP1-negative cells (yellow asterisks). Hoechst signal (blue) highlights cell nuclei. YAP1 signal is shown as fire LUT to facilitate the visualization of signal intensity, where blue is the minimum and yellow is the maximum. In all graphs, red bars indicate mean ± standard deviation.

Article Snippet: We performed IPs using GFP-Trap (ChromoTek) agarose beads in two conditions: Control IP (YAP1-Myc , pEGFP-C1 and HA-Amotl1( )) and IFT88 IP (YAP1-Myc, IFT88-GFP ( ) and HA-Amotl1).

Techniques: Control, Immunofluorescence, In Situ Hybridization, Over Expression, Expressing, Standard Deviation

Journal: bioRxiv

Article Title: Intraflagellar transport complex B proteins regulate the Hippo effector Yap1 during cardiogenesis

doi: 10.1101/777128

Figure Lengend Snippet: (A) The top two graphs show number of Yap1-positive cells in the atrial myocardium quantified in ift88-/-, epi:GFP (n=13) and elipsa-/-, epi:GFP (n=6) mutants and their controls (n=11 and n=7 respectively) at 55 hpf. Mutants show increased Yap1-positive cell numbers (t-test ift88 p value 0.024 and elipsa p value 0.042). Bottom graph shows number of Yap1-positive cells in the myocardium and dorsal pericardium (D.P.) quantified in iguana-/-, epi:GFP (n=16) mutants and their controls (n=17) at 55 hpf (t-test myocardium p value 0.875 and D.P. p value 0.312). (B) Control and iguana-/-, epi:GFP immunofluorescence confocal sections labelled with anti-myosin heavy chain antibody (MHC) (red), GFP (green), anti-Yap1 antibody (white) and DAPI (blue) at 55 hpf. Individual channel is displayed for Yap1 (signal is shown as ice LUT to facilitate visualization of signal intensity, where green is the minimum and red is the maximum) and DAPI (white). Ventral view, anterior is to the top. (B’) Zoomed region (yellow box in panel B ) shows Yap1 and DAPI channels to illustrate the method used to quantify Yap1-positive (Yap1 signal in the nucleus: yellow arrow) and –negative (yellow asterisks) cells. (C) Whole mount bmp4 in situ hybridization in untreated elipsa mutant (n=18) and control (n=29) embryos and treated with XAV939 (10µM) (elipsa mutant, n=10 and control, n=17) or Verteporfin (20µM) (elipsa mutant, n=35 and control, n=12) from 31 to 55 hpf. Treated embryos showed either decreased or absent bmp4 expression at the atrioventricular canal myocardium and the venous pole. Ventral views, anterior is to the top. (D) Graphs show the percentages of AMOTL1-positive PE cells, double AMOTL1-TBX18-positive PE cells and triple YAP1-AMOTL1-TBX18-positive PE cells in Ift20 KO (n=5 embryos: 1196 nuclei analyzed) and control (n=4 embryos: 929 nuclei analyzed) mice at E9.5. The percentage of nuclear AMOTL1-positive cells (Chi-square test of homogeneity = 14,748, p-value 1,23E-04 on 1 degree of freedom), nuclear AMOTL1-TBX18-positive cells (Chi-square test of homogeneity = 12,506, p-value 4,06E-04 on 1 degree of freedom) and nuclear YAP1-AMOTL1-TBX18-positive cells (Chi-square test of homogeneity = 6,9059, p-value 8,59E-03 on 1 degree of freedom) were higher in Ift20 KO than in control mice. Control (n=4) and Ift20 KO (n=5) sections labelled with TBX18 (red), Amotl1 (yellow) and Hoechst (blue). Individual AMOTL1 channel shows the difference between nuclear AMOTL1-positive cells (white arrow) and AMOTL1-negative cells (white asterisk). AMOTL1 signal is shown as fire LUT to facilitate visualization of the signal intensity, where blue is the minimum and yellow is the maximum. In all graphs, red bars indicate mean ±standard deviation. V, ventricle; At, atrium.

Article Snippet: We performed IPs using GFP-Trap (ChromoTek) agarose beads in two conditions: Control IP (YAP1-Myc , pEGFP-C1 and HA-Amotl1( )) and IFT88 IP (YAP1-Myc, IFT88-GFP ( ) and HA-Amotl1).

Techniques: Control, Immunofluorescence, In Situ Hybridization, Mutagenesis, Expressing, Standard Deviation

Journal: bioRxiv

Article Title: Intraflagellar transport complex B proteins regulate the Hippo effector Yap1 during cardiogenesis

doi: 10.1101/777128

Figure Lengend Snippet: (A) Co-IP experiment using HeLa cells transfected with IFT88-GFP, HA-Amotl1 and YAP1-Myc. (l.e. = long exposure. s.e. = short exposure). Co-IP experiment using HEK293 cells transfected with Flag-Amotl1 and IFT20-GFP. Endogenous levels of Yap1 are monitored. (B) Schematic representation of the IFT88 auxin-inducible degron (AID) system (C) Western blot analysis of IFT88 AID DLD-1 cells after 2 hours auxin treatment. (C’) Western blot analysis of IFT88 and YAP1 degradation after auxin treatment (0 h, 0.5h and 2 h). (D) Graph shows the increase in normalized YAP1 nuclear signal in cells treated with auxin (6h) (Mann-Whitney p-value <0.0001). (controls: 2 replicates, n=204 cells; Auxin 2h: 2 replicates, n=261 cells). (E) Graph shows the increase in YAP/WWTR1 (TAZ) nuclear signal in IFT88-siRNA (48h) treated MDCK cells (n=5 replicates, average cell number analyzed for each condition = 382, t-test p-value 0.004). Box and whiskers (5-95 percentile). Outliers are represented as red dots (NT-siRNA) or blue squares (IFT88-siRNA). (E’) Immunofluorescence confocal images (z-projection) of MDCK cells treated with NT – or IFT88-siRNA (48h). DAPI (blue) and YAP/WWTR1 (TAZ) (white inverted LUT). (F) Graph shows the increase in YAP/WWTR1 (TAZ) nuclear signal in IFT88-siRNA (48h) treated HeLa cells (n=5 replicates, average cell number analyzed for each condition = 252, t-test p-value 0.02). Box and whiskers (5-95 percentile). Outliers are represented as red dots (NT-siRNA) or blue squares (IFT88-siRNA). (F’) Immunofluorescence confocal images (z-projection) of HeLa cells treated with NT – or IFT88-siRNA (48h). DAPI (blue) and YAP/WWTR1 (TAZ) (white inverted LUT).

Article Snippet: We performed IPs using GFP-Trap (ChromoTek) agarose beads in two conditions: Control IP (YAP1-Myc , pEGFP-C1 and HA-Amotl1( )) and IFT88 IP (YAP1-Myc, IFT88-GFP ( ) and HA-Amotl1).

Techniques: Co-Immunoprecipitation Assay, Transfection, Western Blot, MANN-WHITNEY, Immunofluorescence

Journal: bioRxiv

Article Title: Intraflagellar transport complex B proteins regulate the Hippo effector Yap1 during cardiogenesis

doi: 10.1101/777128

Figure Lengend Snippet: (A) Immunofluorescence microscopy images (maximum projection) of MDCK cells treated with NT- or IFT88-siRNA (72h). DAPI (white), γ-tubulin (yellow) and IFT88 (red). White arrows highlight IFT88-positive centrosomes facilitating the visualization of IFT88 signal depletion upon IFT88-siRNA treatment. (A’) Zoom of dividing cells (green boxes) treated with NT-siRNA and IFT88-siRNA respectively. Centrosomes show reduced IFT88 (red) and γ-tubulin (yellow) signal after IFT88 depletion. DAPI (white). IFT88 channel is shown in fire LUT where blue is the minimum and yellow is the maximum to facilitate the visualization of the intensity reduction after the treatment. (B) Western blot analysis of HeLa and MDCK cells after 48h NT- and IFT88-siRNA treatments respectively. (C) Graphs show the increase in nuclear YAP/WWTR1 (TAZ) signal in IFT88-siRNA treated cells (blue), compared to NT-siRNA controls (red) at 24, 48 and 72h. Box and whiskers (5-95 percentile). (MDCK: 24h: n=1 replicate, average cell number analyzed for each condition = 42, t-test p-value 0.02; 48h: n=1 replicate, average cell number analyzed for each condition = 52, t-test p-value <0.0001; 72h: n=1 replicate, average cell number analyzed for each condition = 126, t-test p-value 0.01) (HeLa: 24h: n=1 replicate, average cell number analyzed for each condition = 12, t-test p-value 0.001; 48h: n=1 replicate, average cell number analyzed for each condition = 12, t-test p-value 0.0002; 72h: n=1 replicate, average cell number analyzed for each condition = 32, t-test p-value 0.047).

Article Snippet: We performed IPs using GFP-Trap (ChromoTek) agarose beads in two conditions: Control IP (YAP1-Myc , pEGFP-C1 and HA-Amotl1( )) and IFT88 IP (YAP1-Myc, IFT88-GFP ( ) and HA-Amotl1).

Techniques: Immunofluorescence, Microscopy, Western Blot

Journal: iScience

Article Title: Revisiting phosphoregulation of Cdc25C during M-phase induction

doi: 10.1016/j.isci.2024.111603

Figure Lengend Snippet:

Article Snippet: anti-myc for immunoprecipitation (IP) , Cell Signaling Technology , Cat# 2276; RRID: AB_331783.

Techniques: Western Blot, Immunoprecipitation, Sequencing, Modification, Mass Spectrometry, Recombinant, Protease Inhibitor, Mutagenesis, Purification, Expressing

Journal: Pathogens

Article Title: The SIRT1-Mediated p53 Deacetylation Pathway Modulates Apoptosis and Promotes Viral Replication in MVC-Infected Cells

doi: 10.3390/pathogens15030242

Figure Lengend Snippet: Immunofluorescence analysis of SIRT1 expression and its interaction with p53. ( A ) Subcellular localization of SIRT1 and MVC-VP2. WRD cells were subjected to mock infection or MVC infection at an MOI of 2, followed by immunostaining with anti-SIRT1 antibody (green), anti-VP2 antibody (red, as an indicator of MVC infection), and 4′,6-diamidino-2-phenylindole (DAPI, blue, for nuclear counterstaining) at 24 and 48 hpi. ( B , C ) Verification of the interaction between SIRT1 and p53 was performed by means of co-immunoprecipitation (Co-IP) assay. ( B ) COS-1 cells were transfected with pCMV-HA-SIRT1 and pCMV-Myc-p53 expression plasmids for 48 h. Total cellular lysates were prepared and subjected to immunoprecipitation (IP) with 3 μg anti-HA antibody or 3 μg rabbit IgG (isotype control). The immunoprecipitates and input lysates were analyzed by means of Western blotting (WB) using anti-Myc antibody. ( C ) Reciprocal Co-IP assay: total lysates from transfected COS-1 cells were immunoprecipitated with 3 μg anti-Myc antibody or 3 μg mouse IgG (isotype control), followed by WB detection with anti-HA antibody. ( D ) Colocalization of SIRT1 and p53 in transfected COS-1 cells. COS-1 cells were co-transfected with pCMV-HA-SIRT1 and pCMV-Myc-p53 plasmids for 48 h; the cells were fixed, permeabilized, and subjected to immunofluorescence (IF) staining. HA-SIRT1 was probed with anti-HA antibody and visualized with Alexa Fluor 594-conjugated secondary antibody (red), Myc-p53 with anti-Myc antibody, and Alexa Fluor 488-conjugated secondary antibody (green), and cell nuclei were counterstained with DAPI (blue). Images were acquired using a confocal laser scanning microscope (CLSM). ( E ) Immunofluorescence staining showing the overlap of endogenous SIRT1 and p53. WRD cells were infected with MVC for 48 h, then fixed, permeabilized, and subjected to IF staining with anti-SIRT1 antibody (red fluorescence) and anti-p53 antibody (green fluorescence); nuclei were counterstained with DAPI (blue). Colocalization signals were observed using a CLSM. Scale bars: 10 μm. Values represent the mean ± SD from three independent experiments. Comparisons were performed using Student’s t -tests. * p < 0.05. For colocalization analysis, Pearson’s correlation coefficient and overlap coefficient were calculated (shown on the right of each panel). ns: not significant.

Article Snippet: Primary antibodies employed for Western blotting are listed below: anti-acetylated p53 (K382) (rabbit pAb, MAB13552; WB: 1:500) was obtained from R&D Systems (Minneapolis, MN, USA); anti-SIRT1 (rabbit pAb, 13161-1-AP; WB: 1:500), anti-p53 (rabbit pAb, 10442-1-AP; WB: 1:1000), anti-GAPDH (rabbit pAb, 10494-1-AP, WB: 1:10,000), anti-cleaved caspase 3 (rabbit pAb, 25128-1-AP; WB: 1:500), anti-bcl 2 (rabbit pAb, 26593-1-AP; WB: 1:400), Bax (rabbit pAb, 50599-2-Ig; WB: 1:1000), p21 (rabbit pAb, 10355-1-AP; WB: 1:500), anti-HA (rabbit pAb, 51064-2-AP; WB: 1:1000; IP: 3 μg), anti-Myc (mouse mAb, 60003-2-Ig; WB: 1:1000; IP: 3 μg), and anti-Flag (mouse mAb, 66008-3-Ig; WB: 1:1000) were obtained from Proteintech (Wuhan, China); anti-NS1(rabbit pAb, 18929-1; WB: 1:1000) and anti-VP2 (rabbit pAb, 20351-1; WB: 1:1500) were generated in collaboration with Abmart Company (Shanghai, China).

Techniques: Immunofluorescence, Expressing, Infection, Immunostaining, Co-Immunoprecipitation Assay, Transfection, Immunoprecipitation, Control, Western Blot, Staining, Laser-Scanning Microscopy, Fluorescence