EMBO/soda-nel huggingface dataset:
Non-canonical Wnt signaling promotes colon tumor growth, chemoresistance and tumor fibroblast activation (2023)
Figure 4

Figure 4. Tumor-sphere formation is prevented by non-canonical Wnt inhibition. A, Cell number was determined by the MTT assay for SW620 and HCT116 cells cultured in DMEM plus 1% FCS or 10% FCS (mean ± SD of three biological replicates).

B, Clonogenic assay carried out with HCT116 cells cultured in DMEM plus 1% or 10% FCS for 10 days. Colonies were stained with crystal violet and counted using ImageJ software. Results are presented as the mean ± SD of triplicate samples of three independent experiments. The micrograph shows the results of a representative experiment.

C, Representative micrographs of the effects of Ror2 depletion on HCT116 cells soft-agar colony formation (21 days) (left). Representative micrographs of SW620 cells are shown in Appendix Fig S7B. Results show the mean ± SD of the number of colonies in triplicate samples of three independent experiments (right).

D, E, Cells were seeded for tumor-sphere formation in tumor organoid complete medium. In D, representative micrographs of shCtl and shRor2 SW620 cells. The scale bar corresponds to 25 μm. In E, SW620 cells were supplemented with LGK (10 μM), starting the day of seeding. After four days, the wells were stained (top) and the number of colonies counted and represented (bottom) (mean ± SD of three biological replicates). A more detailed micrograph of the colonies is shown in Appendix Fig S7D.

F, RNA was isolated from SW620 control, stably-depleted of Ror2, treated with LGK974 for 16 h or transfected with siSnail1 and the expression of the stem cell gene markers LGR5, ASCL2, SMOC2 and the differentiation marker KRT20 was assessed by qRT-PCR. The figure shows the mean ± SD of at least three biological replicates (performed in triplicate) represented with respect to the corresponding control (dashed line).

Data information: *, p < 0.05; **, p < 0.01; ***, p < 0.001 (unpaired t-test).

Source data are available online for this figure.
Non-canonical Wnt signaling promotes colon tumor growth, chemoresistance and tumor fibroblast activation (2023)
Figure 5

Figure 5. Non-canonical Wnt controls tumorigenesis of colon cancer cells.

A, In vivo tumor growth of shCtl or shRor2 SW620 cells subcutaneously injected in NOD/SCID mice (105 cells/flank; eight tumors per condition) (mean ± SD).

B, C, Representative images of Fibronectin and Vimentin staining in consecutive sections from paraffin-embedded tumors obtained when tumors were initially detected (days 18 or 22). Staining with an anti-human antibody is also enclosed. The scale bar corresponds to 10 μm (B). Fibronectin and Vimentin staining were quantified as described in Methods (six tumors, three areas per tumor) and represented in C (mean ± SD).

D, Representative images of the invading front of the tumors. The scale bar corresponds to 4 μM.

E, Percentage of tumors showing muscle invasion.

F, shCtl and shRor2 SW620 and HCT116 cells were seeded in transwell chambers coated with Matrigel when appropriate. After 48 h, cells were removed from the upper surface of the membrane and cells present at the lower surface were fixed and stained with Crystal Violet. Results are presented as mean ± SD from three biological replicates.

Data information: *, p < 0.05; **, p < 0.01; (unpaired t-test).

Source data are available online for this figure.
Non-canonical Wnt signaling promotes colon tumor growth, chemoresistance and tumor fibroblast activation (2023)
Figure 6

Figure 6. Inhibition of non-canonical Wnt decreases the tumorigenic and metastatic potential of primary colon tumor cells.

A, B, LAKTP murine tumor (MTO) cells were seeded in the indicated culture conditions and supplemented with LGK (10 μM) or WntC59 (100 nM). Total protein extracts (A) or RNA (B) were obtained and analyzed after 16 of treatment. Quantification of the WB is also presented (A, right). Results are the mean ± SD of at least three biological replicates.

C, Cell number was analyzed by CellTiter-Go assay in MTO cells supplemented with WntC59 (100 nM) every three days (mean ± SD of three biological replicates).

D, MTO were seeded in the presence of WntC59 or recombinant R-ECD (0.2 μg/ml) and four days later the wells were stained and the number of cells represented respect the corresponding control (mean ± SD of three biological replicates).

E, Effect of LGK and WntC59 on the expression of the stem cell gene markers and the differentiation marker KRT20 was determined by qRT-PCR (mean ± SD of three biological replicates performed in triplicated).

F, G, In vivo tumor growth of MTO cells subcutaneously injected in NOD/SCID mice (F) (5x105 cells/flank; eight tumors per condition) and treated with WntC59 (10 mg/kg/day in the drinking water) or vehicle. Tumor weight of NOD/SCID mice at day 26 is presented in G. In F and G, the mean ± SD of the eight tumors is shown.

H, Representative images of Hematoxylin/Eosin (H/E), Fibronectin and Vimentin staining in sections from paraffin-embedded tumors obtained at day 26 in NOD/SCID mice. The scale bars correspond to 50 μm (H/E, Fibronectin) or 100 μm (Vimentin).

I, J, WB (I) and RNA analysis (J) of MTO cells infected with shRor2 or shCtl. Quantification of the WB is also presented (I, right). Results are the mean ± SD of three biological replicates (performed in triplicated in J).

K, C57BL/6J mice (eight tumors per condition) were injected subcutaneously with luciferase-expressing LAKTP MTO (shCtl or shRor2). The kinetics of in vivo luminescence are shown (mean ± SD of the values of eight tumors). Tumor weight at day 38 is presented in L.

M, Representative images of Fibronectin and Vimentin staining in sections from paraffin-embedded tumors obtained at day 38. The scale bars correspond to 50 μm.

N, O, C57BL6 mice were injected in the spleen with MTO cells and treated since day 1 with WntC59 or vehicle. In N, kinetics of in vivo luminescence (mean ± SD of the values in five mice); in O, the percentage of mice showing in vivo luminescence.

Data information: *, p < 0.05; **, p < 0.01; ***, p < 0.001 (unpaired t-test). The dashed line in A, B, E, I and J corresponds to the protein or RNA value in control cells. Source data are available online for this figure.
Non-canonical Wnt signaling promotes colon tumor growth, chemoresistance and tumor fibroblast activation (2023)
Figure 7

Figure 7. Wnt5a and Ror2 modulate cisplatin resistance of tumor cells.

A-C, Viability was assayed by MTT in the indicated cells treated with LGK when specified and supplemented with cisplatin for 24 h. In B, cisplatin resistance was assessed in shRor2 cells transfected with a Snail1 expression plasmid or the corresponding empty plasmid. The figure shows the mean ± SD of the results of three experiments.

D, Cells were treated with cisplatin for the indicated times. Total extracts were lysed and analyzed by WB.

E, HCT116 cells were treated with LGK (10 μM), recombinant Wnt5a (50 ng/ml) and control or DKK1 conditioned medium (obtained as described in Methods), combined with cisplatin (40 μM) for 16 h. Total extracts were obtained and analyzed by WB.

F, H, MTO cells, either not-transfected or infected with shCtl or shRor2 were treated with LGK (10 μM), WntC59 (100 nM) or the corresponding controls, supplemented with cisplatin when indicated and analyzed by WB. Quantification of the results of Snail1 analysis from F and H is presented in Figure EV3I.

G, I, MTO cell viability was determined as above (mean ± SD of the results of three-four experiments).

Data information: *, p < 0.05; **, p < 0.01; ***, p < 0.001 (unpaired t-test).

Source data are available online for this figure.

EXPANDED VIEW FIGURE LEGENDS
p97 and p47 promote membrane tethering during mitotic Golgi reassembly in cooperation with FTCD (2020)
Figure 1

Figure 1. Identification of FTCD as a p47-binding protein.

(A) Isolation of FTCD from rat liver extract. The p47(1-170) fragment, which does not contain the p97-binding site, was immobilized on beads and incubated with rat liver extract. Bound proteins were fractionated by SDS-PAGE, and stained with CBB.

(B) FTCD binds directly to p47. GST-tagged p47 (0.54 μg) or GST-tagged p37 (0.45 μg) was incubated with His-tagged FTCD (0.80 μg), and then isolated on glutathione beads. The blots were probed with antibodies to FTCD and GST.

(C) FTCD and p47 form a complex in cells. HepG2 cells were solubilized and incubated with anti-p47 antibodies. The immunoprecipitates were fractionated by SDS-PAGE, followed by Western blotting with antibodies to p47 and FTCD.

(D) FTCD, p47 and p97 form a complex in cells. HepG2 cells were solubilized and incubated with anti-FTCD antibodies. The immunoprecipitates were fractionated by SDS-PAGE, followed by Western blotting with antibodies to FTCD, VCIP135, p97, p47 and syntaxin5.

(E) p47 binds to the CD domain of FTCD. GST-tagged p47 (0.50 μg) was incubated with either His-tagged FTCD(1-326) (FT, 0.88 μg) or His-tagged FTCD(327-541) (CD, 0.63 μg). The blots were probed with antibodies to His-tag and GST.

(F) p47(E86K, E88K) does not bind to FTCD. GST-tagged p47wt/mutant (0.50 μg) was incubated with the indicated His-tagged proteins (p97, 1.0 μg; FTCD, 0.75 μg). The blots were probed with antibodies to p97, FTCD and GST.

(G) FTCD(R382A), which lacks binding ability to polyglutamates, does not bind to p47. GST-tagged p47 (0.35 μg) was incubated with His-tagged FTCDwt/mutant (0.53 μg). The blots were probed with antibodies to His-tag and GST.

(H) p47 binds to FTCD in Golgi membranes. Salt-washed Golgi membranes were first incubated with either GST-tagged p47wt, p47(E86K, E88K) or p47(F253S). The isolated membranes were solubilized, and then GST-tagged p47wt/mutants and their binding proteins were recovered with glutathione-beads. The bound proteins were assayed by Western blotting with antibodies to FTCD and GST.
p97 and p47 promote membrane tethering during mitotic Golgi reassembly in cooperation with FTCD (2020)
Figure 2

Figure 2. FTCD binds to p97 as well as to p47.

(A) FTCD directly binds to p97. GST-tagged p97 (2.1 μg) was incubated with His-tagged FTCD (0.80 μg), and then isolated on glutathione beads. The blots were probed with antibodies to FTCD and GST.

(B) Nucleotide dependency of the binding of FTCD with p97. FTCD (0.80 μg) and GST-tagged p97 (2.1 μg) were incubated in the presence of the indicated nucleotide (1 mM). The blots were probed with antibodies to FTCD and GST.

(C) FTCD binds to the C-terminal region of p97. GST-tagged p97 fragments were incubated with either FTCD or p47. The blots were probed with antibodies to FTCD and p47.

(D) p97 also binds to the CD domain of FTCD. GST-tagged p97 (2 μg) was incubated with either His-tagged FTCD(1-326) (FT, 0.88 μg) or His-tagged FTCD(327-541) (CD, 0.62 μg). The blots were probed with antibodies to His-tag and GST.

(E) p97(V722K, E724K) shows very low binding affinity to FTCD. GST-tagged p97wt/mutant ( 2.0 μg) was incubated with the indicated His-tagged proteins (FTCD, 2.0 μg; p47, 0.5 μg). The blots were probed with antibodies to p47, FTCD and GST.

(F) FTCD(R382A), which lacks binding ability to polyglutamates, shows a very low binding affinity to p97. GST-tagged p97 (1.4 μg) was incubated with His-tagged FTCDwt/mutant (0.53 μg). The blots were probed with antibodies to His-tag and GST.

(G) p97 binds to FTCD in Golgi membranes. Salt-washed Golgi membranes were incubated with either GST-tagged p97wt or p97(V722k, E724K). The isolated membranes were solubilized, and then GST-tagged p97wt/mutants and their binding proteins were recovered with glutathione-beads. The bound proteins were assayed by Western blotting with antibodies to FTCD and GST.
p97 and p47 promote membrane tethering during mitotic Golgi reassembly in cooperation with FTCD (2020)
Figure 3

Figure 3. FTCD is necessary for the Golgi biogenesis.

(A) Immunofluorescence staining of FTCD and GM130, a Golgi marker. HepG2 cells were fixed, permeabilized, stained with a monoclonal antibody to FTCD, a polyclonal antibody to GM130 and DAPI, and observed by confocal microscopy. Scale bar = 10 μm.

(B) Depletion of FTCD by FTCD siRNA duplexes. HepG2 cells were either mock transfected with water or transfected with two distinct siRNA duplexes specific to FTCD. After incubation for 48 hrs, the cells were analyzed by Western blotting with antibodies to FTCD and α-tubulin.

(C) The Golgi in cells which were depleted of either FTCD or p47. HepG2 cells, which were treated with either FTCD siRNAs, p47 siRNA or mock for 48 hrs, were fixed and stained with a monoclonal antibody to GM130. Scale bar = 10 μm.

(D) The results of quantification of (C). The values are from five sets of independent experiments. Results are expressed as the mean±SD (n=5), with 100 cells counted in each group of each set. Asterisks indicate a significant difference at P<0.01 compared with the mock-treated cells (Bonferroni method).

(E) Representative EM images of the Golgi in HepG2 cells treated with either mock or FTCD siRNAs for 42 hrs. Scale bar = 0.5 μm.

(F) The results of quantification of (E). Golgi membranes in HepG2 cells were classified into cisternae, vesicles and tubules, and counted as previously described (Shorter & Warren, 1999). The results are shown as the mean±SD of five sets of independent experiments, with 15-20 cells counted in each group in each independent experiment. An asterisk indicates a significant difference (P<0.05) compared with the mock-treated cells in each category (Bonferroni method).
p97 and p47 promote membrane tethering during mitotic Golgi reassembly in cooperation with FTCD (2020)
Figure 4

Figure 4. FTCD is necessary for Golgi reassembly at the end of mitosis.

(A) The localization of FTCD in mitotic cells. Mitotic HepG2 cells were collected by flushing and cultured on coated coverslips for 10-40 mins. The cells were fixed, stained with a monoclonal antibody to FTCD and polyclonal antibodies to GM130 and α-tubulin, and observed by confocal microscopy. Panels d, h, and l indicate the images of α-tubulin and the other panels show the distribution of FTCD or/and GM130. Scale bar = 10 μm. Arrowheads indicate the colocalization of FTCD and GM130.

(B) The effects of FTCD siRNA treatment on Golgi reassembly at the end of mitosis. HepG2 cells were treated with either FTCD siRNA (FTCD siRNA 2 duplex) or mock for 48 hrs before the collection of mitotic cells. The collected mitotic cells were cultured for 35 mins and then fixed. The cells were stained with a polyclonal antibody to GM130 and a monoclonal antibody to α-tubulin, and observed by confocal microscopy. Panels a-p display representative images. Scale bar = 10 μm.

(C) Rescue experiments in FTCD siRNA-treated HepG2 cells. The indicated HA-tagged FTCDs, which were insensitive to FTCD siRNA, were expressed 25 hrs after the treatment of cells with FTCD siRNA2. The cells were further cultured for 23 hrs and then used for the collection of mitotic cells. The collected mitotic cells were cultured for 35 mins and fixed. The cells were stained with a monoclonal antibody to α-tubulin (panels g and h) and polyclonal antibodies to GM130 and HA (panels a-f). Scale bar = 10 μm.

(D) The cells at cytokinesis in (B) and (C) were analyzed. The results are shown as the mean±SD of five sets of independent experiments, with 100 cells counted in each group in each independent experiment. Asterisks indicate a significant difference (P<0.01) compared with the mock-treated cells (Bonferroni method).
p97 and p47 promote membrane tethering during mitotic Golgi reassembly in cooperation with FTCD (2020)
Figure 5

Figure 5. p47 and p97 require their association with FTCD for their localization to the Golgi at the end of mitosis.

(A) p47 requires its association with FTCD for its localization to the Golgi at the end of mitosis. HepG2 cells were treated with FTCD siRNA and then the indicated HA-tagged FTCDs were expressed as described in Figure 4C. The mitotic cells were collected, cultured for 35 mins, and then fixed. The cells were stained with polyclonal antibodies to p47 and HA and a monoclonal antibody to GM130, and observed by confocal microscopy. Panels a-l show the distribution of p47 or/and GM130 and panels m-o show HA-FTCD expression. Scale bar = 10 μm.

(B) Insets in (A). p47 (red) and the GM130 (a Golgi marker, green) were visualized. Scale bar = 5 μm.

(C) The results of quantification of (A). The results are shown as the mean±SD of five sets of independent experiments, with 100 cells counted in each group in each independent experiments. Asterisks indicate a significant difference at P<0.01 compared with the mock-treated cells (Bonferroni method).

(D) p97 requires its association with FTCD for its localization to the Golgi at the end of mitosis. The cells were treated in the same way as (A) and stained with polyclonal antibodies to p97 and HA and a monoclonal antibody to GM130. Panels a-l show the distribution of p97 or/and GM130 and panels m-o show HA-FTCD expression. Scale bar = 10 μm.

(E) Insets in (D). p97 (red) and the GM130 (a Golgi marker, green) were visualized.

Scale bar = 5 μm.

(F) The results of quantification of (D). The results are shown as the mean±SD of five sets of independent experiments, with 100 cells counted in each group in each independent experiments. Asterisks indicate a significant difference at P<0.01 compared with the mock-treated cells (Bonferroni method).
p97 and p47 promote membrane tethering during mitotic Golgi reassembly in cooperation with FTCD (2020)
Figure 6

Figure 6. FTCD is necessary for p97/p47-mediated Golgi membrane fusion.

(A) Anti-FTCD antibodies inhibit p97/p47-mediated Golgi membrane fusion. Golgi fragments were mixed with the indicated components (p97, 60 μg/ml; p47, 30 μg/ml; p37, 24 μg/ml; p115, 40 μg/ml) together with either anti-FTCD or anti-p115 antibodies. Results are expressed as mean+SD of five sets of independent experiments; 0% and 100% represent the buffer only (25.9% in cisternal membranes) and p97/p47 with no antibodies ('+ none', 48.8%), respectively. Asterisks indicate a significant difference at P<0.01 compared with the no antibody groups ('+none') (Bonferroni method).

(B) p47(E86K, E88K), which lacks binding affinity to FTCD, does not function in p97/p47-mediated Golgi membrane fusion. Golgi fragments were incubated with p97 (60 μg/ml) and p47wt/mutant (30 μg/ml). Data are shown as the mean+SD of five sets of independent experiments; 0% and 100% represent the buffer only (27.3% in cisternal membranes) and p97+p47wt ('+p47wt', 50.9%), respectively. Asterisks indicate a significant difference at P<0.01 compared with the group in which p47wt as well as p97 were added ('+p47wt') (Bonferroni method).

(C) p97(V722K,E724K), which lacks binding affinity to FTCD, does not function in p97/p47-mediated Golgi membrane fusion. Golgi fragments were incubated with the following components: p97wt/mutant, 60 μg/ml; p47, 30 μg/ml; p37, 24 μg/ml; p115, 40 μg/ml. Data are shown as the mean+SD of five sets of independent experiments; 0% and 100% represent the buffer only (29.5% in cisternal membranes), and p47+p97wt ('+p97wt'. 57.5%), respectively. An asterisk indicates a significant difference at P<0.01 compared with the group in which p97wt as well as p47 were added ('+p97wt') (Bonferroni method).

(D) Panels a-e show representative EM images. Scale bar = 0.5 μm.
p97 and p47 promote membrane tethering during mitotic Golgi reassembly in cooperation with FTCD (2020)
Figure 9

Figure 9. FTCD-mediated tether is formed at the end of mitosis.

(A) FTCD located in mitochondria causes mitochondria aggregation during mitosis. Mitotic cells were collected by flushing from HeLa Tet-off cells in which either FTCDwt-HA-MAO or FTCD(R382A)-HA-MAO had been induced for 24 hrs. The mitotic cells were further cultured for 4 hrs to enable complete cytokinesis. Cells in which FTCDwt-HA-MAO was induced for 28 hrs without synchronization were used as the asynchronous control group. Cells were fixed and visualized with a monoclonal antibody to mitochondria and a polyclonal antibody to HA. Panels a-f display representative images. Scale bar = 10 μm.

(B) The results of quantification of (A). Results are shown as the mean±SD of seven sets of independent experiments, with 100 cells counted in each group in each independent experiment. An asterisk indicates a significant difference at P<0.01 compared with the others (Bonferroni method).

(C) A single living cell expressing FTCDwt-HA-MAO was tracked during mitosis. Mitotic cells were collected by flushing from the HeLa Tet-off cells inducibly expressing FTCDwt-HA-MAO, and cultured in the absence or presence of DOX. The cells were stained with MitoTracker at the 22 hr time point and confocal images of 30-40 cells were randomly taken at the 23 hr time point. From the 23 hr to 27 hr time point, cells were tracked in the bright field every 30 mins. At the 27 hr time point, confocal images were taken of the 21-38 cells that were successfully tracked. Panels a-j display representative images. Asterisks indicate the tracked cells. Scale bar = 10 μm.

(D) The results of quantification of (C). The experiment was repeated five times and each line shows the average percentage of an independent experiment. The percentages of cells that underwent mitosis are as follows: 41.5 % in the DOX(-) group and 54.7 % in the DOX(+) group.

(E) FTCD located in mitochondria causes mitochondria aggregation at the end of mitosis. Mitotic cells were collected from the HeLa Tet-off cells inducibly expressing FTCDwt-HA-MAO and cultured in the absence or presence of DOX as in (C). Cells were stained with MitoTracker at the 23hr time point and observed by confocal microscopy from the 24 hr to 26 hr time point. Panels a-t display representative images. Scale bar = 10 μm.

(F) The results of quantification of (E). Cells were classified by their cell cycle phases into the following three categories: prometaphase/metaphase, anaphase/telophase and cytokinesis. Results are shown as the mean±SD of three sets of independent experiments, with 10-14 cells counted in each group in each independent experiment. Asterisks indicate a significant difference at P<0.01 compared with the DOX(+) group and with the prometaphase/metaphase category (Bonferroni method).

EXPANDED VIEW FIGURE LEGENDS
TMEM16A expression in cholinergic neurons of medial habenula mediates anxiety-related behaviors (2019)
Figure 2

Fig 2. Specific expression of TMEM16A in the mHb neurons. A. A summary bar graph showing the quantitative real-time PCR data for TMEM16A, TMEM16B, and BEST1 with cDNA samples prepared from the mHb and hippocampus of adult mouse brains (N=3, n=3 each, unpaired Student t-test, data are presented as mean values ± s.e.m.). B. RNAscope ISH showed an apparent expression of TMEM16A mRNA co-localized with ChAT and Tac1 signals in the mHb compared to the ones in lHb. Enlarged images showed that signals of TMEM16A and Tac1 in the dorsal mHb or ChAT in the ventral mHb reside together at the single cell level. C. Immunohistochemical staining of 40-µm coronal brain sections with anti-TMEM16A antibody. In the mHb, TMEM16A signals colocalize with the cholinergic marker ChAT (arrows). High magnifications of the indicated areas are shown in the merged panel.