EMBO/soda-nel huggingface dataset:
Phosphoproteomics reveals conserved exercise-stimulated signaling and AMPK regulation of store-operated calcium entry (2019)
Figure 3

Figure 3. STIM1 is a novel exercise-regulated AMPK substrate.

(A) Schematic of STIM1 domain architecture is shown, with the AMPK consensus motifs for S257 and S521 (Gwinn et al, 2008).

(B) Western blots for STIM1 phosphorylated at S257 or S521 in (left) contracted (+) or sham (-) rat muscle and (right) muscle from mice that underwent a treadmill running protocol (+) or remained at rest (-). Total STIM1 and 14-3-3 were used as loading controls. 14-3-3 data for mouse running was re-used from Figure 1B.

(C) Western blots for phosphorylated ACC (S79) and STIM1 (S257 or S521) in L6 rat myoblasts with siRNA-mediated knockdown of endogenous STIM1 (siSTIM1) or transfected with scramble control siRNA (Control). Cells were treated with the AMPK activator A-769662 (+) or vehicle (-) for 30 min. Total STIM1 was used to demonstrate STIM1 knockdown efficiency. Total ACC and 14-3-3 were used as loading controls.

(D) Western blots for phosphorylated ACC (S79) and STIM1 (S257 or S521) in wild type (WT) or AMPK β1/β2 knockout (AMPKβ KO) mouse endothelial fibroblasts were isolated and treated with A-769662 (+) or vehicle (-) for 30 min. AMPKβ was used to demonstrate knockout, and AMPKɑ was used to demonstrate specificity for AMPKβ. Total ACC and STIM1 were used as loading controls.

(E) Western blots for total ACC and STIM1 and phosphorylated STIM1 (S257 and S521) and ACC (S79) in samples from an in vitro kinase assay to assess the ability of AMPK to phosphorylate YFP-STIM1.

(F) Representative Western blots for phosphorylated ACC (S79) and STIM1 (S257 and S521) in response to a 15 min treatment with increasing doses of thapsigargin (.01, .1, 1 or 10 µM) or .5 mM AICAR. DMSO was used as a vehicle control. 14-3-3 was used as a loading control. n=3 independent experiments.

(G) Western blots for phosphorylated ACC (S79) and STIM1 (S257 or S521) in wild type (WT) or AMPK KO mouse endothelial fibroblasts were isolated and treated with 2 µM thapsigargin (+) or vehicle (-) for 15 min.
Phosphoproteomics reveals conserved exercise-stimulated signaling and AMPK regulation of store-operated calcium entry (2019)
Figure 4

Figure 4. Phosphorylation of STIM1 affects its conformation.

Time correlated single photon counting was used to estimate the fluorescence lifetime of mClover2.

(A) The mClover2:tagRFP-T fret pair was validated in HEK E cells with the fluorophores joined by either a long rigid or short flexible linker.

(B) STIM1 OASF expressed in live L6 myoblasts before (-) or after (+) treatment with A-769662.

(C) L6 myoblasts with S257 mutated to A or E.

Data information: Mean lifetimes were estimated by fitting a single exponential decay to the TCSPC data and are presented as median ± min/max. 3 representative cells are shown for each condition. For A and C, ** = p<.001, *** = p<.0001, unpaired student's t-test, n=3-5 independent observations. For B, **** = p<.00001, paired student's t-test, n=11 independent observations. For box and whisker plots, the horizontal lines represent the medians, the upper and lower boundaries of the boxes represent the upper and lower quartiles, and the whiskers represent the upper and lower extremes. Scale bars=10 µm.
Phosphoproteomics reveals conserved exercise-stimulated signaling and AMPK regulation of store-operated calcium entry (2019)
Figure 5

Figure 5. Phosphorylation of STIM1 by AMPK suppresses SOCE.

(A-C) L6 myoblasts were transfected with the genetic cytosolic Ca2+ sensor GCaMP6s. Cells were also transfected with non-targeted siRNA (Control) or siRNA directed at endogenous STIM1 (siSTIM1). Some cells with endogenous STIM1 knockdown were also transfected with full-length wild type (WT) or mutant (S257A, S257E or L251S) STIM1-mRuby3. (A) Representative microscopy images of GCaMP6s signal indicating SOCE in L6 myoblasts, (B) Ca2+ levels over time (fold of 0 mM Ca2+ + EGTA basal) and (C) quantification of SOCE in in L6 myoblasts incubated in buffer containing 1 or 0 mM Ca2+ and treated with 2 µM thapsigargin during the indicated times. For (C), "b" is significantly different from "a" and "c", and "c" is significantly different from "a" and "b", p < 0.05 by one-way ANOVA with Tukey's post-hoc test. For (B-C), data are represented as mean ± SEM, n=5 independent experiments (n=3 for L251S).
Phosphoproteomics reveals conserved exercise-stimulated signaling and AMPK regulation of store-operated calcium entry (2019)
Figure 6

Figure 6. STIM1 inhibition prevents exercise-induced fatigue.

(A, B) Negative geotaxis climbing assays in Drosophila (flies). Time required for 50% of flies to climb to the target line before or after 2.5 hr of continuous climbing exercise (A) in the yw strain with muscle-specific STIM knockdown using two independent RNAi sequences (RNAi26-1 or RNAi8-4) compared to GFP-expressing controls, or (B) in the w1118 strain with muscle-specific STIM knockdown using RNAiGD16187 compared to non-targeted RNAiGD12145 controls.

(C) Time to fatigue, when flies were no longer able to climb, during a 3-min period after negative geotaxis stimulation in STIM whole-body knockout flies overexpressing wild type (WT) or mutant (S257A or S257E) human STIM1-mRuby3.

Data information: * = p<0.05 by one-way ANOVA with Tukey's post-hoc test. Data are represented as mean ± SEM, n=3 independent experiments.
Role for intraflagellar transport in building a functional transition zone (2018)
Figure 1

Figure 1. Screen for retrograde IFT defects identifies a temperature-sensitive allele of the dynein heavy chain CHE-3, the orthologue of mammalian DYNC2H1.

(a) Genetic screen for retrograde IFT defects uncovers a temperature-sensitive (ts) mutation in C. elegans CHE-3 (Dynein-2 heavy chain). Paraquat-resistant strains were first screened for dye-filling defects (Dyf phenotype) typically indicative of cilia structure anomalies (examples of normal and defective DiI dye-filling are shown). Three different IFT reporters were then introduced into the dyf mutants (CHE-11::GFP shown as an example) to reveal candidate retrograde IFT mutants showing strong IFT protein accumulation at cilia tips. Whole-genome sequencing revealed the likely causative mutation in one of the strains, namely in the retrograde IFT dynein motor CHE-3 (DYNC2H1 human orthologue). A variable Dyf phenotype at 20ºC suggested the possibility of a ts mutation. Scale bar, 20 µm.

(b) The Dyf phenotype of the ts-che-3 mutant was confirmed, with animals grown at the permissive temperature (15ºC) showing the ability to uptake dye, while those raised at the restrictive temperature (25ºC) are Dyf. Scale bar, 20 µm.

(c) Using the homodimeric kinesin OSM-3 as a marker for IFT, proper localisation is seen in the ts-che-3 mutant at the permissive temperature, with the strongest signal at the basal body (bb). At the restrictive temperature, most of the OSM-3 signal is found within the axoneme of the short, bulbous cilia. Scale bar is 4 µm.

(d) The ts-che-3 mutant shows normal osmotic avoidance at permissive temperature but not at the restrictive temperature, indicating defective chemosensation. The osm-9 mutant is included as a positive control. n = 50 animals; error bars are standard error.
Role for intraflagellar transport in building a functional transition zone (2018)
Figure 2

Figure 2. Loss of IFT-dynein function at restrictive temperature results in TZ protein localisation defects that are reversed at permissive temperature.

(a) Three TZ markers (NPHP-4, CEP-290 and MKS-6) are shown at both the permissive (15ºC) and restrictive (25ºC) temperatures in the ts-che-3 mutant. At the permissive temperature, all three markers localise to the TZ, while at the restrictive temperature exhibit ectopic localisation (leakage) distally into the ciliary axoneme. While NPHP-4 and MKS-6 show significant accumulations, CEP-290 is grossly normal. At the permissive temperature, both IFT co-markers (CHE-13 and XBX-1) display strong localisation to the basal body (bb) and along the axoneme. At the restrictive temperature, the IFT markers show accumulation in the axoneme. Accumulations emphasized using asterisks.

(b) To determine if ciliary compartmentalisation could be restored, young ts-che-3 mutant adults were transferred to the permissive temperature after being raised at the restrictive temperature. The TZ proteins reassembled at the TZ, with loss of the ectopic accumulations, and IFT protein localisation was also restored-strongest at the basal body, similar to animals grown at the permissive temperature.

(c) After 24 hours at the restrictive temperature, TZ proteins appear to begin to accumulate distally within the axoneme in ts-che-3 mutants. This is seen, in small amounts, for NPHP-4 and CEP-290 but with stronger ectopic localisation for MKS-6. The IFT proteins CHE-13 and XBX-1 also show accumulations in the axoneme after 24 hours at the restrictive temperature.

Data information: For (A), (B), and (C), fluorescence quantification is shown for each marker at the indicated temperature in the heat maps on the right. Each point in the plot represents one pixel along the centre of the basal body (BB), transition zone (TZ) and middle segment (MS) regions. Dotted areas (three pixels) in the MS were used to quantify ectopic localisation (TZ markers) or accumulation (IFT markers) for statistical analyses. n = 10 cilia (4-7 animals), Kruskal-Wallis test, *P<0.05, **P<0.01. tz, transition zone; bb, basal body; scale bars are 4 µm.
Role for intraflagellar transport in building a functional transition zone (2018)
Figure 3

Figure 3. Abrogating CHE-3 function causes defects in transition zone gate function and ultrastructure.

(a) In the ts-che-3 mutant grown at the permissive temperature (15ºC), both RPI-2/RP2 and TRAM-1/TRAM1 are localised to the PCMC, just proximal to the ciliary axoneme. At the restrictive temperature, TRAM-1 shows leakage into the axoneme with its strongest localisation at the PCMC. Intriguingly, RPI-2 now displays strong localisation to the axoneme, indicating that in the absence of IFT, RPI-2 may be targeted into the cilium. When ts-che-3 mutant animals grown at the restrictive temperature (25ºC) are shifted at young adult to the permissive (15ºC) for 24 hours, both TRAM-1 and RPI-2 exhibit restoration of their PCMC localisation. Consistent with other TZ markers, both MKS-2 and MKSR-1 show ectopic localisation in the axoneme when grown at the restrictive temperature compared to the permissive temperature. Also consistent with what was observed for other markers, after 24 hours at the permissive temperature, animals grown at the restrictive temperature show loss of the ectopic localisation. Fluorescence quantification is shown for each marker at the indicated temperature in the heat maps on the right. Each point in the plot represents one pixel along the centre of the basal body (BB), transition zone (TZ) and middle segment (MS) regions. Dotted areas (three pixels) in the MS were used to quantify ectopic localisation (MKS-2 and MKSR-1) or accumulation (RPI-2 and TRAM-1) for statistical analyses. n = 10 cilia (4-6 animals), Kruskal-Wallis test, *P<0.05, **P<0.01; scale bars are 4 µm.

(b) Loss of CHE-3 function results in short, bulbous cilia containing electron dense accumulations and abnormal membrane-microtubule connections. Shown are transmission electron micrograph (TEM) cross sections of an amphid channel cilium in wild-type and che-3(e1124) null mutant animals. Representative images of wild-type cilia show intact middle segment (containing doublet microtubules) and transition zone (with Y-shaped links) (left top and bottom panels) regions. Representative images of che-3(e1124) cilia reveal apparently intact transition zones, with visible Y-link structures, but enlarged ciliary ends filled with electron-dense accumulations, which often appear vesicular. The bulbous structures reveal doublet microtubules associated with the membrane, and occasionally ectopic microtubule-to-membrane connections, which sometimes appear similar to transition zone Y-links in the region just distal to the seemingly 'normal' TZ structure. Schematics show longitudinal (left images) and cross section (right images) representations of wild-type and che-3(e1124) cilia. tz, transition zone; pcmc, periciliary membrane compartment; scale bars are as indicated in (nm) for TEM images.
Role for intraflagellar transport in building a functional transition zone (2018)
Figure 4

Figure 4. Ciliary maintenance and IFT function upon shift to the restrictive temperature (25ºC).

(a) Using the IFT marker BBS-7, cilia lengths were measured for wild-type and the ts-che-3 mutant animals raised at the permissive (15ºC) and shifted to the restrictive temperature (25ºC) for three time points. After 12 hours some accumulations (indicated by asterisks in the fluorescence images) are observed within the cilia but there is not a significant reduction in cilium length. After 18 and 24 hours, significant reduction in ciliary length is observed and large accumulations of BBS-7 is seen within short bulbous cilia, similar to what is observed in ts-che-3 animals raised at the restrictive temperature. n = 30-34 cilia, one per animal; Kruskal-Wallis test, **P<0.01; error bars are mean absolute deviation; scale bars are 4 µm.

(b) IFT velocities were measured for wild-type or ts-che-3 mutant animals (grown at permissive temperature) and imaged at the permissive temperature, restrictive temperature or after 3 hours at the restrictive temperature. Compared to wild-type, anterograde velocities in the ts-che-3 dynein mutant are similar, while IFT retrograde rates are highly reduced for both the middle and distal ciliary segments. Interestingly, the ts-che-3 IFT dynein mutant appears to not only have slow retrograde IFT trains, but the trains also appear much larger and less frequent. Initially upon the temperature shift (<15min) IFT rates are much faster for both wild-type and ts-che-3 dynein mutant, although retrograde remains much slower in the mutant. After 3 hours at the restrictive temperature, IFT velocities are intermediate between the rates seen at 15ºC and those observed at the initial shift to 25ºC. Error bars are 95% C.I. Wild-type (10-15 animals): anterograde n = 141-326, retrograde n = 100-271; for che-3(nx159ts) (18-25 animals): anterograde n = 234-312, retrograde n = 64-103; Y-axis scale bar is 2 seconds; X-axis scale bar is 1 µm.
Role for intraflagellar transport in building a functional transition zone (2018)
Figure 5

Figure 5. Transition zone repair potential decreases with age while older adults show reduced TZ protein localisation defects.

A, B To assess the effects of IFT restoration on TZ assembly, ts-che-3 mutant animals were shifted from the restrictive to the permissive temperature at different time points during their development (days 1, 4 and 8 of adulthood). The TZ markers MKS-6 (a) and NPHP-4 (b) show strong ectopic ciliary accumulations when raised at the restrictive temperature until day 1 of adulthood. After a shift to the permissive temperature for 24 hours, day 2 adults show 'repaired' TZ protein localisation resembling that of wild-type animals. For both markers, Day 4 adults (maintained at the restrictive temperature) show less ectopic TZ protein localisation than Day 1 adults. The correction of ectopic localisation following shift to the permissive temperature for 24 hours was reduced in day 4 (to day 5) of adulthood compared to that observed in day 1 (to day 2) adults. For MKS-6, but not NPHP-4, day 8 adults (maintained at the restrictive temperature) also display less ectopic accumulation than day 1 adults. The correction of ectopic TZ protein localisation in animals shifted to the permissive temperature after 8 days of adulthood is also much smaller in magnitude for both markers, indicating that this repair is most effective in day 1 adults. In summary, as the adults age, ectopic TZ protein localisation is reduced, while the ability to correct this phenotype by restoring IFT function also reduces. tz, transition zone; scale bars are 4 µm. Heat map graphs corresponding to the bar graphs are located in Fig. EV5. n = 10 cilia (4-7 animals), Kruskal-Walls test, *P<0.05, **P<0.01, error bars are mean absolute deviation.
Non-canonical Wnt signaling promotes colon tumor growth, chemoresistance and tumor fibroblast activation (2023)
Figure 1

Figure 1. Inactivation of non-canonical Wnt signaling decreases the expression of Snail1 and other mesenchymal markers in colon tumor cell lines.

A, SW620 and HCT116 cells were treated with the indicated concentrations of SB505124 (SB) or LGK974 (LGK) for 16 h. Total cell extracts were analyzed by WB. Protein quantification was analyzed by densitometry of three different experiments and represented (mean ± SD) (right panel).

B, SW620 and HCT116 cells expressing a shCtl or shRor2 vectors were analyzed by WB. Quantification of the WBs from three biological replicates are presented in the right panel. Values correspond to fold variation in shRor2 versus shCtl cells (mean ± SD).

C, RNA was isolated from shCtl or shRor2 SW620 and HCT116 cells and the expression of the indicated genes was assessed by qRT-PCR. Values correspond to fold variation in shRor2 versus shCtl cells. Results are the mean ± SD of at least three biological replicates performed in triplicate.

D, SW620 and HCT116 cells expressing a shCtl or shRor2 vectors were analyzed by WB. Quantification of the WBs are presented in Appendix Fig S3D as in B.

E, SNAI1 promoter activity was analyzed in HCT116 and SW620 cells and represented with respect to the activity of the -869/+59 promoter for each cell line (mean ± SD from three biological replicates performed in triplicate).

F, HCT116 cells were treated with the indicated concentrations of Stat3 inhibitor S3I-201 or JNK inhibitor II for 16 h. Cells were lysed and Snail1 levels determined by WB. Quantification of three biological replicates is presented Appendix Fig S3E.

G, H, In G, shCtl or shRor2 SW620 cells were transiently transfected with a plasmid encoding Snail1-HA. Cells were treated with cycloheximide (CHX) (50 μg/ml) for the indicated times and ectopic Snail1 was analyzed by WB. A quantification of four biological replicates (mean ± SD) is presented in H.

Data information: *, p < 0.05; **, p < 0.01; ***, p < 0.001 (unpaired t-test). The dashed line in A, B and C corresponds to the protein or RNA value in untreated or shCtl-transfected cells.

Source data are available online for this figure.
Non-canonical Wnt signaling promotes colon tumor growth, chemoresistance and tumor fibroblast activation (2023)
Figure 2

Figure 2. Wnt5a, Ror2 and Snail1 form a self-stimulatory signaling pathway.

A, RNA was isolated from shCtl or shRor2 HCT116 and SW620 cells, treated with LGK (10 μM) for 16 h when indicated and expression of WNT5A assessed by qRT-PCR. Results are presented relative to control (mean ± SD of three biological replicates performed in triplicate)

B, Wnt5a protein levels were determined by WB in the conditioned medium of shCtl or shRor2 SW620 cells.

C, MSC were treated with the specified conditioned media for the indicated times and pStat3 was analyzed by WB. Results from two different experiments are represented in Appendix Fig S5B.

D, E, HCT116 or SW620 cells were treated with conditioned medium from control or Wnt5a-producing cells for 16 h and expression of the indicated genes assessed by WB (D) or qRT-PCR (E) (mean ± SD of three biological replicates performed in triplicate in E). Results from three different experiments corresponding to D were densitometered and represented in Appendix Fig S5E.

F, SW480 cells were stably transfected with Ror2-HA or the empty vector phrGFP. Two independent clones of control and Ror2 were lysed and analyzed by WB. Quantification of the results is presented in Appendix Fig S5G.

G, WB analysis of HT29 M6 and SW480 overexpressing Snail1. Quantification is presented in Appendix Fig S5I.

H, I, SW620 and HCT116 cells were transfected with control or Snail1 siRNA and analyzed by WB (H) or qRT-PCR (I, mean ± SD of three biological replicates performed in triplicate). Quantification of the results from H is presented in Appendix Fig S5K.

Data information: *, p < 0.05; **, p < 0.01; ***, p < 0.001 (unpaired t-test). The dashed line in A, E and I corresponds to the 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 3

Figure 3. Non-canonical Wnt stimulates TGFβ production by tumor cells and fibroblast activation.

A, Smad2 phosphorylation was determined in extracts from shCtl or shRor2 SW620 and HCT116 cells. The quantification is presented in the right panel (mean ± SD or three biological replicates).

B, RNA was isolated from HCT116 and SW620 control, stably-depleted of Ror2 or treated with LGK for 16 h. Expression of TGFB1 was assessed by qRT-PCR (mean ± SD of three biological replicates performed in triplicate).

C, D, MSC were treated with the conditioned media from shCtl or shRor2 SW620 (C, D) for the indicated times. TGFβ receptor inhibitor SB505124 (SB, 5 μM) was added when indicated in D. Cells were lysed and proteins analyzed by WB. Quantification of the protein levels from D is represented at the lower panel (mean ± SD of three biological replicates); from C, in Appendix Figs S6B.

E, GFP-labelled MSC were seeded in Matrigel-coated transwells with SW620 and HCT116 cells, and supplemented with SB when indicated. After 48 h non-invading cells were removed from the upper surface of the membrane and cells present at the lower surface were fixed. GFP-labelled cells were counted in five different fields per filter by ImageJ software and represented (bottom). A representative picture of SW620 cells in each condition is shown (top). The bar corresponds to 500 μm. The figure shows the mean ± SD of three experiments.

F, MSC were treated with the conditioned media from Ctl or Snail over-expressing HT29 M6 cells for the indicated times. Cells were lysed and proteins analyzed by WB. Quantification of protein levels is represented in Appendix Figs S6C.

Data information: ns, not significant, *, p < 0.05; **, p < 0.01; ***, p < 0.001 (unpaired t-test). The dashed line in B corresponds to the RNA value in control cells. Source data are available online for this figure.