Journal: Nature Communications

Article Title: Membrane potential drives the exit from pluripotency and cell fate commitment via calcium and mTOR

doi: 10.1038/s41467-022-34363-w

Figure Lengend Snippet: a Percentages of embryos with abnormal gastrulation after depletion of kcnh6 (MO, CRISPR) or Kcnh channel blockade with Ergtoxin, and rescue of kcnh6 depletion with medium conditions that hyperpolarize the V m (low K + , val valinomycin, sodium substitution with choline; treatments performed stages 8–12). Above: examples of embryos scored for the graph; posterior views (dorsal to the top) of stage 15 embryos after successful (control) or unsuccessful ( kcnh6 CRISPR = CR) gastrulation; arrowhead points to blastopore closure. Graph reports mean ± SEM; total embryo numbers (N) in the graph are from 3 independent experiments (except for Ergtoxin: 2 independent experiments with devitellinized embryos); p -values are ( kcnh6 MO vs Control MO) = 8.66e–010, ( kcnh6 MO+mRNA vs kcnh6 MO) = 2.59e-004, ( kcnh6 CRex4 vs Control) = 7.19e-018, ( kcnh6 CRex3 vs Control) = 5.79e-022, ( kcnh6 CR+low K + vs kcnh6 CR) = 1.64e-005, ( kcnh6 CR+val vs kcnh6 CR+DMSO) = 1.22e-002, ( kcnh6 CR+choline vs kcnh6 CR) = 2.34e-006, (ErgTx vs Control) = 6.57e-010; two-sided Fisher’s exact test. b Representative intracellular recording in the prospective ectoderm of a control stage 10 embryo; V m is measured relative to the medium (baseline); the dip in membrane potential indicates the electrode breaking into the cell. c The V m as measured by intercellular recordings in the prospective ectoderm of stage 10 Control MO and kcnh6 MO-injected embryos; graph reports mean ± SEM; p -value ( kcnh6 MO vs Control MO) is 1.07e-006 (unpaired two-tailed student’s t -test); each data point represents one cell; data from 10 cells/5 embryos/3 independent experiments. d Live animal pole images of GCaMP6/mCherry at stage 10. e Quantification of GCaMP6 fluorescence intensity normalized to mCherry in mCherry+ cells; graph shows mean ± SEM; data points represent single cells; data from N cells (in graph)/10 embryos/3 independent experiments; p = 1.14e-006; unpaired two-tailed student’s t -test. f Maximum area undergoing simultaneous Ca 2+ transients within a 20 s time lapse recording as a percentage of total animal pole area; the animal poles of 13 Control MO and 15 Kcnh6 MO embryos were recorded over 3 independent experiments; p = 1.04e-003; unpaired two-tailed student’s t -test. Key for asterisks: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001, ns nonsignificant with p > 0.05. Source data are provided as a Source Data file.

Article Snippet: Full-length human KCNH6 (NM_030779.3 cloned in pCS107), GCaMP6 (subcloned in pCSDest) and mCherry cDNAs (Addgene #34935; in pCS2 + ), were used to generate capped mRNAs in vitro by first linearizing with appropriate restriction enzymes and then transcribing with the mMessage machine kit (Ambion). mRNAs were injected at 3 pg (human KCNH6 ), 150 pg ( GCaMP6 ), and 150 pg ( mCherry ) per embryo.

Techniques: CRISPR, Control, Membrane, Injection, Two Tailed Test, Fluorescence

Journal: eLife

Article Title: The scaffolding protein flot2 promotes cytoneme-based transport of wnt3 in gastric cancer

doi: 10.7554/elife.77376

Figure Lengend Snippet: Figure 1. Gastric epithelial normal and cancer cell lines utilise cytonemes to transport Wnt3 intercellularly. (A) Confocal images of normal gastric epithelial cell line (HFE-145) and gastric cancer (GC) cell lines (MKN-28, MKN-7, and AGS) expressing LifeAct-GFP to visualise actin-based structures. Yellow arrows indicate examples of filopodia. (B) Quantification of filopodia length in GC cell lines MKN-28, MKN-7, and AGS (n=7, 8, 25; n=number of cells). Significance is calculated by Student’s t-test. (C) Immunofluorescent images of HFE, MKN-28, MKN-7, and AGS, stained with antibodies against Wnt3 (green) and actin (Phalloidin-iFluor594, red). Scale bar 10 µm. High-magnification images indicate an example of a Wnt3-bearing cytonemes. Scale bar 2.5 µm. (D) Quantification of Wnt3-positive filopodia in gastric epithelial (HFE-145) and cancer (AGS) cells as a percentage of total filopodia (number of cells analysed = 6, 8, 6). Significance is calculated by Student’s t-test. (E) Immunohistochemistry (IHC) images of AGS cells overexpressing Wnt3 and stained with an antibody against Wnt3 (green) and actin (iFluor594, red). Scale bar 10µm. High-magnification images highlight cytonemes. Scale bar 2.5 µm. (F) IHC images of AGS cells treated with the Porcupine inhibitor IWP2 (100 µM, 48 hr) and stained with an antibody against Wnt3 (green) and actin (iFluor594, red). (G) Live confocal cell imaging of AGS cells expressing Wnt3-mCherry and LifeAct-GFP. Cytoneme-localised Wnt3-mCherry highlighted by yellow arrows. (H–J) IHC images of AGS cells stained with antibodies against (H) Myosin-X (MyoX) and (I) Evi/Wntless (red) and Wnt3 (red) and (J) Evi/Wntless (green Scale bars 10 µm). Phalloidin labels actin (FITC-Phalloidin, green; Phalloidin-iFluor350, blue).

Article Snippet: The following plasmids were used in transfections and/or microinjections: pCS2 + membrane- mCherry (Mattes et al., 2018), pCAG- mGFP membrane- bound GFP (Addgene 14757), pEGFP- N1 Flot2- GFP (Neumann- Giesen et al., 2004), pEGFP- N1 ∆N- Flot2- GFP (Neumann- Giesen et al., 2004), 7×TRE SuperTOPFlash- NLS- mCherry (Moro et al., 2012), JNK KTR- mCherry (Regot et al., 2014), pCS2 + LifeAct GFP, pCS2 + Rab5 GFP (Jim Smith Group), Rab7- eGFP (from Rüdiger Rudolf), pEGFP- N1 LAMP1- mTurq2 (Addgene #98828), IRSp534K- mCherry/GFP (Stanganello et al., 2015), pCS2 + Wnt8a- mCherry (Stanganello et al., 2015), pCS2 + Ror2- mCherry (cloned in with ClaI and XbaI), pEGFP- N3 mRor2-∆CRD- GFP (XhoI- XhoI mRor2 insert taken from pcDNA- mRor2, subcloned into SalI site of pEGFP- N3 vector), pCS2 + Ror2- eBFP2 (cloned by inserting eBFP2 into pCS2 + Ror2 plasmid using XbaI and SnaBI), pCS2 + Wnt3- mCherry (cloned from Addgene plasmid pcDNA3.2- Wnt3 (#35909) into pCS2±mCherry vector using ClaI and XbaI).

Techniques: Expressing, Staining, Immunohistochemistry, Imaging

Journal: eLife

Article Title: The scaffolding protein flot2 promotes cytoneme-based transport of wnt3 in gastric cancer

doi: 10.7554/elife.77376

Figure Lengend Snippet: Figure 2. Wnt3 cytonemes regulate paracrine Wnt/β-catenin signalling and proliferation. (A) Experimental protocol for measuring paracrine Wnt signalling activation. HFE cells expressing the SuperTOPFlash (STF) reporter, 7×TCF-NLS-mCherry, were cocultivated with AGS cells expressing indicated constructs. Fluorescence of STF mCherry reporter was measured after 48 hr and compared to untransfected control cells. (B) Representative

Article Snippet: The following plasmids were used in transfections and/or microinjections: pCS2 + membrane- mCherry (Mattes et al., 2018), pCAG- mGFP membrane- bound GFP (Addgene 14757), pEGFP- N1 Flot2- GFP (Neumann- Giesen et al., 2004), pEGFP- N1 ∆N- Flot2- GFP (Neumann- Giesen et al., 2004), 7×TRE SuperTOPFlash- NLS- mCherry (Moro et al., 2012), JNK KTR- mCherry (Regot et al., 2014), pCS2 + LifeAct GFP, pCS2 + Rab5 GFP (Jim Smith Group), Rab7- eGFP (from Rüdiger Rudolf), pEGFP- N1 LAMP1- mTurq2 (Addgene #98828), IRSp534K- mCherry/GFP (Stanganello et al., 2015), pCS2 + Wnt8a- mCherry (Stanganello et al., 2015), pCS2 + Ror2- mCherry (cloned in with ClaI and XbaI), pEGFP- N3 mRor2-∆CRD- GFP (XhoI- XhoI mRor2 insert taken from pcDNA- mRor2, subcloned into SalI site of pEGFP- N3 vector), pCS2 + Ror2- eBFP2 (cloned by inserting eBFP2 into pCS2 + Ror2 plasmid using XbaI and SnaBI), pCS2 + Wnt3- mCherry (cloned from Addgene plasmid pcDNA3.2- Wnt3 (#35909) into pCS2±mCherry vector using ClaI and XbaI).

Techniques: Activation Assay, Expressing, Construct, Fluorescence, Control

Journal: eLife

Article Title: The scaffolding protein flot2 promotes cytoneme-based transport of wnt3 in gastric cancer

doi: 10.7554/elife.77376

Figure Lengend Snippet: Figure 3. Flotillin-2 is over-expressed and promotes filopodia formation and elongation in gastric cancer cells. (A) Flot2 protein levels in HFE-145 and AGS cells as quantified by Western blot after normalising to beta-actin levels (n=3) and by RT-qPCR after normalising to Glyceraldehyde-3- Phosphate Dehydrogenase (GAPDH) as a housekeeping gene (n=4). Relative protein and mRNA levels are compared to HFE-145. Error bars represent SEM. Significance is calculated by Student’s t-test. (B) Representative images of HFE and AGS cells expressing membrane-mCherry and indicated Flotillin-2 (Flot2) constructs or siRNA after 48 hr. Scale bars 10 µm. (C–D) Filopodia quantifications of HFE and AGS cells transfected with indicated Flot2 plasmids or siRNA. Significance calculated by Student’s t-test with Bonferroni correction for multiple comparisons. Average cumulative filopodia length (C), average filopodia number per cell (D). (n per condition [HFE]=22, 19, 25, 23, 24). (n per condition [AGS]=25, 21, 25, 25, 25; n=number of cells measured). (E) Distribution of filopodia, categorised by length as a percentage of total filopodia per HFE or AGS cell 48 hr post-transfection with indicated Flot2 plasmids or siRNA. A Pearson’s χ2 test was performed to test for significance between control (ctrl) group (expected) and experimental groups (observed) with 5 degrees of freedom (df) and a p-value <0.05. The specific χ2 values are as follows, HFE: ctrl siRNA 0.86, Flot2 0.001, dnFlot2 <0.001, Flot2 siRNA <0.001, and for AGS: ctrl siRNA 0.65, Flot2 0.007, dnFlot2 <0.001, and Flot2 siRNA <0.001. Asterisks mark significant differences.

Article Snippet: The following plasmids were used in transfections and/or microinjections: pCS2 + membrane- mCherry (Mattes et al., 2018), pCAG- mGFP membrane- bound GFP (Addgene 14757), pEGFP- N1 Flot2- GFP (Neumann- Giesen et al., 2004), pEGFP- N1 ∆N- Flot2- GFP (Neumann- Giesen et al., 2004), 7×TRE SuperTOPFlash- NLS- mCherry (Moro et al., 2012), JNK KTR- mCherry (Regot et al., 2014), pCS2 + LifeAct GFP, pCS2 + Rab5 GFP (Jim Smith Group), Rab7- eGFP (from Rüdiger Rudolf), pEGFP- N1 LAMP1- mTurq2 (Addgene #98828), IRSp534K- mCherry/GFP (Stanganello et al., 2015), pCS2 + Wnt8a- mCherry (Stanganello et al., 2015), pCS2 + Ror2- mCherry (cloned in with ClaI and XbaI), pEGFP- N3 mRor2-∆CRD- GFP (XhoI- XhoI mRor2 insert taken from pcDNA- mRor2, subcloned into SalI site of pEGFP- N3 vector), pCS2 + Ror2- eBFP2 (cloned by inserting eBFP2 into pCS2 + Ror2 plasmid using XbaI and SnaBI), pCS2 + Wnt3- mCherry (cloned from Addgene plasmid pcDNA3.2- Wnt3 (#35909) into pCS2±mCherry vector using ClaI and XbaI).

Techniques: Western Blot, Quantitative RT-PCR, Expressing, Membrane, Construct, Transfection, Control

Journal: eLife

Article Title: The scaffolding protein flot2 promotes cytoneme-based transport of wnt3 in gastric cancer

doi: 10.7554/elife.77376

Figure Lengend Snippet: Figure 6. Flotillin-2 promotes cytoneme formation and Wnt8a signalling in zebrafish development. (A) Experimental setting to generate small clones expressing indicated constructs in the zebrafish embryo. (B, C) Confocal images of zebrafish epiblast cells injected with Flot2-GFP / memCherry and Flot2-GFP / Wnt8a-mCherry and imaged at 8 hpf. Scale bars represent 10 µm (D) Representative images of zebrafish epiblast cells injected with memCherry indicated constructs. Scale bar 10 µm. (E), (F), Quantification of filopodia from epiblast cells injected. Significance is calculated by

Article Snippet: The following plasmids were used in transfections and/or microinjections: pCS2 + membrane- mCherry (Mattes et al., 2018), pCAG- mGFP membrane- bound GFP (Addgene 14757), pEGFP- N1 Flot2- GFP (Neumann- Giesen et al., 2004), pEGFP- N1 ∆N- Flot2- GFP (Neumann- Giesen et al., 2004), 7×TRE SuperTOPFlash- NLS- mCherry (Moro et al., 2012), JNK KTR- mCherry (Regot et al., 2014), pCS2 + LifeAct GFP, pCS2 + Rab5 GFP (Jim Smith Group), Rab7- eGFP (from Rüdiger Rudolf), pEGFP- N1 LAMP1- mTurq2 (Addgene #98828), IRSp534K- mCherry/GFP (Stanganello et al., 2015), pCS2 + Wnt8a- mCherry (Stanganello et al., 2015), pCS2 + Ror2- mCherry (cloned in with ClaI and XbaI), pEGFP- N3 mRor2-∆CRD- GFP (XhoI- XhoI mRor2 insert taken from pcDNA- mRor2, subcloned into SalI site of pEGFP- N3 vector), pCS2 + Ror2- eBFP2 (cloned by inserting eBFP2 into pCS2 + Ror2 plasmid using XbaI and SnaBI), pCS2 + Wnt3- mCherry (cloned from Addgene plasmid pcDNA3.2- Wnt3 (#35909) into pCS2±mCherry vector using ClaI and XbaI).

Techniques: Clone Assay, Expressing, Construct, Injection

Journal: eLife

Article Title: The scaffolding protein flot2 promotes cytoneme-based transport of wnt3 in gastric cancer

doi: 10.7554/elife.77376

Figure Lengend Snippet: Figure 1. Gastric epithelial normal and cancer cell lines utilise cytonemes to transport Wnt3 intercellularly. (A) Confocal images of normal gastric epithelial cell line (HFE-145) and gastric cancer (GC) cell lines (MKN-28, MKN-7, and AGS) expressing LifeAct-GFP to visualise actin-based structures. Yellow arrows indicate examples of filopodia. (B) Quantification of filopodia length in GC cell lines MKN-28, MKN-7, and AGS (n=7, 8, 25; n=number of cells). Significance is calculated by Student’s t-test. (C) Immunofluorescent images of HFE, MKN-28, MKN-7, and AGS, stained with antibodies against Wnt3 (green) and actin (Phalloidin-iFluor594, red). Scale bar 10 µm. High-magnification images indicate an example of a Wnt3-bearing cytonemes. Scale bar 2.5 µm. (D) Quantification of Wnt3-positive filopodia in gastric epithelial (HFE-145) and cancer (AGS) cells as a percentage of total filopodia (number of cells analysed = 6, 8, 6). Significance is calculated by Student’s t-test. (E) Immunohistochemistry (IHC) images of AGS cells overexpressing Wnt3 and stained with an antibody against Wnt3 (green) and actin (iFluor594, red). Scale bar 10µm. High-magnification images highlight cytonemes. Scale bar 2.5 µm. (F) IHC images of AGS cells treated with the Porcupine inhibitor IWP2 (100 µM, 48 hr) and stained with an antibody against Wnt3 (green) and actin (iFluor594, red). (G) Live confocal cell imaging of AGS cells expressing Wnt3-mCherry and LifeAct-GFP. Cytoneme-localised Wnt3-mCherry highlighted by yellow arrows. (H–J) IHC images of AGS cells stained with antibodies against (H) Myosin-X (MyoX) and (I) Evi/Wntless (red) and Wnt3 (red) and (J) Evi/Wntless (green Scale bars 10 µm). Phalloidin labels actin (FITC-Phalloidin, green; Phalloidin-iFluor350, blue).

Article Snippet: The following plasmids were used in transfections and/or microinjections: pCS2 + membrane- mCherry (Mattes et al., 2018), pCAG- mGFP membrane- bound GFP (Addgene 14757), pEGFP- N1 Flot2- GFP (Neumann- Giesen et al., 2004), pEGFP- N1 ∆N- Flot2- GFP (Neumann- Giesen et al., 2004), 7×TRE SuperTOPFlash- NLS- mCherry (Moro et al., 2012), JNK KTR- mCherry (Regot et al., 2014), pCS2 + LifeAct GFP, pCS2 + Rab5 GFP (Jim Smith Group), Rab7- eGFP (from Rüdiger Rudolf), pEGFP- N1 LAMP1- mTurq2 (Addgene #98828), IRSp534K- mCherry/GFP (Stanganello et al., 2015), pCS2 + Wnt8a- mCherry (Stanganello et al., 2015), pCS2 + Ror2- mCherry (cloned in with ClaI and XbaI), pEGFP- N3 mRor2-∆CRD- GFP (XhoI- XhoI mRor2 insert taken from pcDNA- mRor2, subcloned into SalI site of pEGFP- N3 vector), pCS2 + Ror2- eBFP2 (cloned by inserting eBFP2 into pCS2 + Ror2 plasmid using XbaI and SnaBI), pCS2 + Wnt3- mCherry (cloned from Addgene plasmid pcDNA3.2- Wnt3 (#35909) into pCS2±mCherry vector using ClaI and XbaI).

Techniques: Expressing, Staining, Immunohistochemistry, Imaging

Journal: eLife

Article Title: The scaffolding protein flot2 promotes cytoneme-based transport of wnt3 in gastric cancer

doi: 10.7554/elife.77376

Figure Lengend Snippet: Figure 2. Wnt3 cytonemes regulate paracrine Wnt/β-catenin signalling and proliferation. (A) Experimental protocol for measuring paracrine Wnt signalling activation. HFE cells expressing the SuperTOPFlash (STF) reporter, 7×TCF-NLS-mCherry, were cocultivated with AGS cells expressing indicated constructs. Fluorescence of STF mCherry reporter was measured after 48 hr and compared to untransfected control cells. (B) Representative

Article Snippet: The following plasmids were used in transfections and/or microinjections: pCS2 + membrane- mCherry (Mattes et al., 2018), pCAG- mGFP membrane- bound GFP (Addgene 14757), pEGFP- N1 Flot2- GFP (Neumann- Giesen et al., 2004), pEGFP- N1 ∆N- Flot2- GFP (Neumann- Giesen et al., 2004), 7×TRE SuperTOPFlash- NLS- mCherry (Moro et al., 2012), JNK KTR- mCherry (Regot et al., 2014), pCS2 + LifeAct GFP, pCS2 + Rab5 GFP (Jim Smith Group), Rab7- eGFP (from Rüdiger Rudolf), pEGFP- N1 LAMP1- mTurq2 (Addgene #98828), IRSp534K- mCherry/GFP (Stanganello et al., 2015), pCS2 + Wnt8a- mCherry (Stanganello et al., 2015), pCS2 + Ror2- mCherry (cloned in with ClaI and XbaI), pEGFP- N3 mRor2-∆CRD- GFP (XhoI- XhoI mRor2 insert taken from pcDNA- mRor2, subcloned into SalI site of pEGFP- N3 vector), pCS2 + Ror2- eBFP2 (cloned by inserting eBFP2 into pCS2 + Ror2 plasmid using XbaI and SnaBI), pCS2 + Wnt3- mCherry (cloned from Addgene plasmid pcDNA3.2- Wnt3 (#35909) into pCS2±mCherry vector using ClaI and XbaI).

Techniques: Activation Assay, Expressing, Construct, Fluorescence, Control

Journal: eLife

Article Title: The scaffolding protein flot2 promotes cytoneme-based transport of wnt3 in gastric cancer

doi: 10.7554/elife.77376

Figure Lengend Snippet: Figure 3. Flotillin-2 is over-expressed and promotes filopodia formation and elongation in gastric cancer cells. (A) Flot2 protein levels in HFE-145 and AGS cells as quantified by Western blot after normalising to beta-actin levels (n=3) and by RT-qPCR after normalising to Glyceraldehyde-3- Phosphate Dehydrogenase (GAPDH) as a housekeeping gene (n=4). Relative protein and mRNA levels are compared to HFE-145. Error bars represent SEM. Significance is calculated by Student’s t-test. (B) Representative images of HFE and AGS cells expressing membrane-mCherry and indicated Flotillin-2 (Flot2) constructs or siRNA after 48 hr. Scale bars 10 µm. (C–D) Filopodia quantifications of HFE and AGS cells transfected with indicated Flot2 plasmids or siRNA. Significance calculated by Student’s t-test with Bonferroni correction for multiple comparisons. Average cumulative filopodia length (C), average filopodia number per cell (D). (n per condition [HFE]=22, 19, 25, 23, 24). (n per condition [AGS]=25, 21, 25, 25, 25; n=number of cells measured). (E) Distribution of filopodia, categorised by length as a percentage of total filopodia per HFE or AGS cell 48 hr post-transfection with indicated Flot2 plasmids or siRNA. A Pearson’s χ2 test was performed to test for significance between control (ctrl) group (expected) and experimental groups (observed) with 5 degrees of freedom (df) and a p-value <0.05. The specific χ2 values are as follows, HFE: ctrl siRNA 0.86, Flot2 0.001, dnFlot2 <0.001, Flot2 siRNA <0.001, and for AGS: ctrl siRNA 0.65, Flot2 0.007, dnFlot2 <0.001, and Flot2 siRNA <0.001. Asterisks mark significant differences.

Article Snippet: The following plasmids were used in transfections and/or microinjections: pCS2 + membrane- mCherry (Mattes et al., 2018), pCAG- mGFP membrane- bound GFP (Addgene 14757), pEGFP- N1 Flot2- GFP (Neumann- Giesen et al., 2004), pEGFP- N1 ∆N- Flot2- GFP (Neumann- Giesen et al., 2004), 7×TRE SuperTOPFlash- NLS- mCherry (Moro et al., 2012), JNK KTR- mCherry (Regot et al., 2014), pCS2 + LifeAct GFP, pCS2 + Rab5 GFP (Jim Smith Group), Rab7- eGFP (from Rüdiger Rudolf), pEGFP- N1 LAMP1- mTurq2 (Addgene #98828), IRSp534K- mCherry/GFP (Stanganello et al., 2015), pCS2 + Wnt8a- mCherry (Stanganello et al., 2015), pCS2 + Ror2- mCherry (cloned in with ClaI and XbaI), pEGFP- N3 mRor2-∆CRD- GFP (XhoI- XhoI mRor2 insert taken from pcDNA- mRor2, subcloned into SalI site of pEGFP- N3 vector), pCS2 + Ror2- eBFP2 (cloned by inserting eBFP2 into pCS2 + Ror2 plasmid using XbaI and SnaBI), pCS2 + Wnt3- mCherry (cloned from Addgene plasmid pcDNA3.2- Wnt3 (#35909) into pCS2±mCherry vector using ClaI and XbaI).

Techniques: Western Blot, Quantitative RT-PCR, Expressing, Membrane, Construct, Transfection, Control

Journal: eLife

Article Title: The scaffolding protein flot2 promotes cytoneme-based transport of wnt3 in gastric cancer

doi: 10.7554/elife.77376

Figure Lengend Snippet: Figure 4. Flotillin-2 marks Wnt3 cytonemes and influences paracrine Wnt/β-catenin signalling and proliferation. (A) Immunohistochemistry (IHC) analysis showing endogenous localisation of Flot2 (green) in AGS cells. TRITC phalloidin was used to visualise actin. Arrows indicate the localisation of Flot2 to filopodia. Scale bars 5 µm. High-magnification images indicate an example of a Flot2-bearing cytonemes. Scale bars 2.5 µm. (B), IHC analysis shows that Flot2 co-localises with Wnt3 on cytonemes. (C) Confocal images showing the subcellular localisation of Flot2-GFP in AGS cells. Arrows indicate

Article Snippet: The following plasmids were used in transfections and/or microinjections: pCS2 + membrane- mCherry (Mattes et al., 2018), pCAG- mGFP membrane- bound GFP (Addgene 14757), pEGFP- N1 Flot2- GFP (Neumann- Giesen et al., 2004), pEGFP- N1 ∆N- Flot2- GFP (Neumann- Giesen et al., 2004), 7×TRE SuperTOPFlash- NLS- mCherry (Moro et al., 2012), JNK KTR- mCherry (Regot et al., 2014), pCS2 + LifeAct GFP, pCS2 + Rab5 GFP (Jim Smith Group), Rab7- eGFP (from Rüdiger Rudolf), pEGFP- N1 LAMP1- mTurq2 (Addgene #98828), IRSp534K- mCherry/GFP (Stanganello et al., 2015), pCS2 + Wnt8a- mCherry (Stanganello et al., 2015), pCS2 + Ror2- mCherry (cloned in with ClaI and XbaI), pEGFP- N3 mRor2-∆CRD- GFP (XhoI- XhoI mRor2 insert taken from pcDNA- mRor2, subcloned into SalI site of pEGFP- N3 vector), pCS2 + Ror2- eBFP2 (cloned by inserting eBFP2 into pCS2 + Ror2 plasmid using XbaI and SnaBI), pCS2 + Wnt3- mCherry (cloned from Addgene plasmid pcDNA3.2- Wnt3 (#35909) into pCS2±mCherry vector using ClaI and XbaI).

Techniques: Immunohistochemistry

Journal: eLife

Article Title: The scaffolding protein flot2 promotes cytoneme-based transport of wnt3 in gastric cancer

doi: 10.7554/elife.77376

Figure Lengend Snippet: Figure 6. Flotillin-2 promotes cytoneme formation and Wnt8a signalling in zebrafish development. (A) Experimental setting to generate small clones expressing indicated constructs in the zebrafish embryo. (B, C) Confocal images of zebrafish epiblast cells injected with Flot2-GFP / memCherry and Flot2-GFP / Wnt8a-mCherry and imaged at 8 hpf. Scale bars represent 10 µm (D) Representative images of zebrafish epiblast cells injected with memCherry indicated constructs. Scale bar 10 µm. (E), (F), Quantification of filopodia from epiblast cells injected. Significance is calculated by

Article Snippet: The following plasmids were used in transfections and/or microinjections: pCS2 + membrane- mCherry (Mattes et al., 2018), pCAG- mGFP membrane- bound GFP (Addgene 14757), pEGFP- N1 Flot2- GFP (Neumann- Giesen et al., 2004), pEGFP- N1 ∆N- Flot2- GFP (Neumann- Giesen et al., 2004), 7×TRE SuperTOPFlash- NLS- mCherry (Moro et al., 2012), JNK KTR- mCherry (Regot et al., 2014), pCS2 + LifeAct GFP, pCS2 + Rab5 GFP (Jim Smith Group), Rab7- eGFP (from Rüdiger Rudolf), pEGFP- N1 LAMP1- mTurq2 (Addgene #98828), IRSp534K- mCherry/GFP (Stanganello et al., 2015), pCS2 + Wnt8a- mCherry (Stanganello et al., 2015), pCS2 + Ror2- mCherry (cloned in with ClaI and XbaI), pEGFP- N3 mRor2-∆CRD- GFP (XhoI- XhoI mRor2 insert taken from pcDNA- mRor2, subcloned into SalI site of pEGFP- N3 vector), pCS2 + Ror2- eBFP2 (cloned by inserting eBFP2 into pCS2 + Ror2 plasmid using XbaI and SnaBI), pCS2 + Wnt3- mCherry (cloned from Addgene plasmid pcDNA3.2- Wnt3 (#35909) into pCS2±mCherry vector using ClaI and XbaI).

Techniques: Clone Assay, Expressing, Construct, Injection