EMBO/soda-nel huggingface dataset:
Oxidative stress-induced phosphorylation of JIP4 regulates lysosomal positioning in coordination with TRPML1 and ALG2 (2022)
Figure 3

Fig. 3. Spm/acrolein induces perinuclear lysosomal clustering through the TRPML1-ALG2-dynein pathway.

a, b. SH-SY5Y cells were transfected with the indicated siRNA for 48 h and then treated with 50 µM spm for 4 h. Cells were fixed and stained with an anti-LAMP2 antibody (green). Nuclei were stained with DAPI (blue). Scale bar, 20 µm.

c. SH-SY5Y cells were transfected with JIP4 siRNA or TMEM55B siRNA for 48 h, and then treated with 50 µM spm for 4 h. Cells were fixed and stained with an anti-LAMP2 antibody (red). Nuclei were stained with DAPI (blue). Scale bar, 20 µm.

d. SH-SY5Y cells were transfected with the indicated siRNAs for 48 h and then treated with 40 µM acrolein for 2 h. (upper) Cells were fixed and stained with an anti-LAMP2 antibody (green). Nuclei were stained with DAPI (blue). Scale bar, 10 µm. (lower) Clustered relative to whole lysosomes were quantified as lysosomal clustering value. *** p<0.0001; ** p<0.001; * p<0.01; N.S., not statistically different (Dunnett's test, vs Control si-acrolein) 4-6 images were quantified per each condition. Values are mean ± SD. At least three experiments were replicated.

e. Fura2-AM-loaded SH-SY5Y cells were treated with 40 μM acrolein and observed by time-lapse fluorescence microscopy at 5-second intervals. The fluorescence ratio (F340/F380) values in two representative cells are shown.

f. Cells transfected with control siRNA (left) and TRPML1 siRNA (right) for 48 h were loaded with Fura2-AM for 30 min. Cells were treated with 40 μM acrolein and observed by time-lapse fluorescence microscopy at 5-second intervals. The fluorescence ratio (F340/F380) values in two representative cells are shown.

g. Histogram of the spike frequency. Spikes with an amplitude of more than 0.05 for 40 min were counted. The value represents the percentage of cells with the indicated number of spikes to the total cells. Spike frequency "0" means no spikes in the cell.
Oxidative stress-induced phosphorylation of JIP4 regulates lysosomal positioning in coordination with TRPML1 and ALG2 (2022)
Figure 4

Fig. 4. Phospho-JIP4 mediates acrolein-induced lysosomal clustering.

a. Cells were transfected with the TMEM55B-myc-DDK plasmid for 24 h and then treated with 40 μM acrolein for 2 h. Cell lysates were immunoprecipitated with flag magnetic beads and immunoblotted with the indicated antibodies.

b. SH-SY5Y cells were treated with 40 μM acrolein for 2 h. Cells were fixed and stained with anti-JIP4 (green) and anti-LAMP2 (red) antibodies. Nuclei were stained with DAPI (blue). Scale bar, 20 μm.

c. SH-SY5Y cells treated with the indicated concentration of acrolein for 2 h were lysed, subjected to Phos-tag PAGE, and immunoblotted with an anti-JIP4 antibody.

d. SH-SY5Y cells were pre-treated with 10 µM Jak3 inhibitor VI (Jak3 i VI) for 1 h and then treated with 40 µM acrolein for an additional 2 h. Cells lysates were subjected to Phos-tag PAGE and immunoblotted with an anti-JIP4 antibody.

e. SH-SY5Y cells were pre-treated with 10 µM Jak3 inhibitor VI for 1 h and then treated with 40 µM acrolein for an additional 2 h. Cells were fixed and stained with anti-JIP4 (green) and anti-LAMP2 (red) antibodies. Nuclei were stained with DAPI (blue). Scale bar, 20 μm.

f, g. SH-SY5Y cells transfected with indicated siRNAs for 48 h were treated with 40 μM acrolein for 2 h. Cell lysates were subjected to Phos-tag PAGE and immunoblotted with anti-JIP4 antibody (f, left). CaMK2G knockdown efficiency was confirmed by qRT-PCR.; ***p <0.001, n=3 technical replicates. Values are mean ± SD. At least three experiments were replicated (f, right). Cells were fixed and stained with anti-LAMP2 and anti-JIP4 antibodies. Scale bar, 20 μm (g).

h. Peptide sequence detected by mass spectrometry. The underlined amino acid was modified with phosphoric acid.

i. The amount of each phosphorylated peptide when cells were treated with 40 μM acrolein or co-treated with acrolein and JAK3 inhibitor VI compared with the control was determined by SWATH-MS analysis. ***p <0.001, **p <0.01; N.S., not statistically different. (Tukey-Kramer's test); n=3 technical replicates. Values are mean ± SD.

j. His6-tagged recombinant truncated JIP4 (100-300 aa) was purified from E. coli and incubated with recombinant CaMK2G with/without Ca2+ and calmodulin at 30°C　for 1 h. The reactant was subjected to Phos-tag PAGE and immunoblotted with anti-His antibody. Jak3 inhibitor VI was pre-reacted with CaMK2G for 30 min.

k. JIP4 KO cells were transfected with flag-tagged JIP4 (WT and T217A) for 24 h and treated with 40 μM acrolein for 2 h. Cells were fixed and stained with the indicated antibodies. Scale bar, 20 μM.
Oxidative stress-induced phosphorylation of JIP4 regulates lysosomal positioning in coordination with TRPML1 and ALG2 (2022)
Figure 5

Fig. 5. JIP4-dependent lysosomal retrograde transport requires TRPML1-ALG2 signalling.

a. SH-SY5Y cells were transfected with the indicated siRNAs for 48 h and then treated with 40 µM acrolein for 2 h. Cells were fixed and stained with anti-JIP4 (green) and anti-LAMP2 (red) antibodies. Nuclei were stained with DAPI (blue). Scale bar, 20 μm.

b. TRPML1-mCherry-stably expressing SH-SY5Y cells were transfected with GFP-ALG2 for 48 h and then treated with 40 µM acrolein for 2 h. Cells were fixed, and then green and red fluorescence was detected by confocal microscopy.

c. GFP or GFP-ALG2 transfected TRPML1-mCherry stably expressing cells were treated with acrolein for 2 h. Cell lysates were immunoprecipitated with anti-RFP magnetic beads and immunoblotted with the indicated antibodies.

d. TRPML1-mCherry-stably expressing SH-SY5Y cells were treated with 40 µM acrolein for 2 h. Cells were fixed and stained with an anti-JIP4 (green) antibody.

e. SH-SY5Y cells were transfected with GFP-ALG2 for 48 h and then treated with 40 µM acrolein for 2 h. Cells were fixed and stained with an anti-JIP4 (red) antibody.

f. SH-SY5Y cells transfected with GFP or GFP-ALG2 for 24 h were treated with acrolein for 2 h and then spatial proximity of JIP4 and GFP was detected by a PLA using JIP4 and GFP antibodies. Scale bar, 10 µm. ***p <0.001; N.S., not statistically different (Tukey-Kramer's test). 3-5 images were quantified per each condition. Values are mean ± SD. At least three experiments were replicated.
Oxidative stress-induced phosphorylation of JIP4 regulates lysosomal positioning in coordination with TRPML1 and ALG2 (2022)
Figure 6

Fig. 6. Phosphorylation state of JIP4 acts as the switch that controls lysosomal positioning.

a, b. Parental and JIP4 KO SH-SY5Y cells were treated with 40 µM acrolein with or without 100 nM bafilomycin A1 for 2 h (a). SH-SY5Y cells were treated with 40 µM acrolein with or without 100 nM bafilomycin A1 in the presence of 10 µM Jak3 inhibitor VI (Jak3 i VI) for 2 h (b). Cell lysates were immunoblotted with the indicated antibodies. Autophagic flux was determined as the LC3-II production rate calculated as the level of LC3-II with bafilomycin A1 treatment.

c. Parental and JIP4 KO SH-SY5Y cells were treated with 40 µM acrolein and 500 µM H2O2 or cultured in starvation medium for 2 h and then stained with an anti-LAMP2 antibody (red). Nuclei were stained with DAPI (blue). Scale bar, 20 μm

d. (upper) SH-SY5Y cells were pretreated with 10 µM BAPTA-AM or 10 µM Jak3 inhibitor VI for 1 h and then treated with 500 µM H2O2 for an additional 2 h or cultured in starvation medium for an additional 2 h. Cells were fixed and stained with anti-JIP4 (green) and anti-LAMP2 (red) antibodies. Nuclei were stained with DAPI (blue). Scale bar, 20 μm. (lower) Clustered relative to whole lysosomes were quantified as lysosomal clustering value. ****p<0.0001, ***p <0.001; N.S., not statistically different (Tukey-Kramer's test). 4-10 images were quantified per each condition. Values are mean ± SD. At least three experiments were replicated.

e. SH-SY5Y cells were treated with 40 µM acrolein and 500 µM H2O2 or cultured in starvation medium for 2 h. Cells were lysed, subjected to Phos-tag PAGE, and immunoblotted with anti-JIP4 antibody.

f. (left) SH-SY5Y cells were transfected with TMEM55B siRNAs for 48 h and then treated with 40 µM acrolein and 500 µM H2O2 or cultured in starvation medium for 2 h. Cells were fixed and stained with anti-γ-tubulin (green) and anti-LAMP2 (red) antibodies. Nuclei were stained with DAPI (blue). Scale bar, 20 μm. (right) Clustered relative to whole lysosomes were quantified as lysosomal clustering value. ***p<0.001,**<0.01; N.S., not statistically different (Tukey-Kramer's test). 8-12 images were quantified per each condition. Values are mean ± SD. At least three experiments were replicated.
Oxidative stress-induced phosphorylation of JIP4 regulates lysosomal positioning in coordination with TRPML1 and ALG2 (2022)
Figure 7

Fig.7 Schematic illustration of the lysosomal retrograde transport in response to oxidative stress.

a. Proposed lysosomal retrograde transport signalling in response to oxidative stress. Oxidative stress induces Ca2+ release from TRPML1, resulting in JIP4 T217 phosphorylation by CaMK2G. Phosphorylated JIP4 complexes with ALG2 and TRPML1, thereby promoting lysosomal retrograde transport.

b. Parental and JIP4 KO SH-SY5Y cells were treated with various concentrations of acrolein for 24 h. Cytotoxicity was measured by an LDH assay. ***p<0.0001 (Tukey-Kramer's test); n=3 from independent cultures. Values are mean ± SD. At least three experiments were replicated.

c. SH-SY5Y cells were transfected with TRPML1 siRNAs for 48 h and then treated with various concentrations of acrolein for 24 h. Cytotoxicity was measured by an LDH assay. ***p<0.0001 (Tukey-Kramer's test); n=3 from independent cultures. Values are mean ± SD. At least three experiments were replicated.
Learning causal networks using inducible transcription factors and transcriptome-wide time series (2020)
Figure 2

Figure 2. Characterizing the downstream responses of Aft1 induction. A. Heatmap summary of Aft1 induction experiment showing all genes that change with |log2(fold-change)| > 1.5. B. Parametric summaries of representative sigmoidal activation and inhibition timecourses and impulse (double sigmoid) modeling of transitory inhibition. Sigmoids are summarized by half-max time (trise) and the asymptote (vinter), while impulses include a second half-max (tfall) time and final asymptote (vfinal). The strongest supported model for each timecourse is shown as a filled in line, while the alternative model is shown with a dashed line. C. K-mers enriched in the promoters of regulated genes are overlaid on summary of each gene's trise and vinter. Presence of the Aft1 motif is associated with early activation, while early inhibition is associated with the PAC motif (Tod6/Dot6). D. Response kinetics are overlaid on gene coordinates based on synthetic lethality as a surrogate for functional similarity. Up-regulated genes are enriched for vesicle trafficking/glycosylation processes and down-regulated genes are enriched for mitochondrial/mitotic/rRNA processes.
Regulation of the RNA-binding protein Smaug by the GPCR Smoothened via the kinase Fused (2020)
Figure 2

Figure 2: SMO/HH regulates Smaug levels and activity.

(A) Smaug repression assay. The assay is based on the dual expression of a construct encoding a λN-HA-SNAP-Smaug chimeric protein (abbreviated λN-SNAP-Smaug) and a second that is transcribed into a reporter mRNA (called SNAP-GUS-5BoxB) that carries a translational fusion between SNAP and GUS coding regions followed by five Box B hairpins (5BoxB) inserted in the 3'UTR. A plasmid encoding a GFP-SNAP fusion (not shown here) was also used as a transfection and loading control.

(B, C) Smaug downregulates reporter expression. Mean values of the relative levels of SNAP-GUS reporter (SNAP-GUS/GFP-SNAP ratio, in arbitrary units) in extracts of transfected Cl8 cells in the absence (black) or presence of λN-SNAP-Smaug (red), λN-SNAP (pale grey) or SNAP-Smaug (dark grey). See Fig EV3A for the representative gel and Fig EV3B which shows that λN-SNAP-Smaug has no effect on a SNAP-GUS construct that lacks the 5BoxB. N=3 (biological replicates). See also Fig EV3A-C.

Here and in D-F: SNAP-GUS/GFP-SNAP ratios were set to 1 in cells expressing λN‐SNAP-Smaug. The error bars correspond to the standard deviation of the mean (SD). Statistical analysis was done by a Kruskal-Wallis test by ranks followed by a Dunn test and p values are: black bracket <0.05, red bracket <0.01, green bracket <0.0001. Here and in D-E: 15 ng of pAct.λN-SNAP-smaug plasmid were used per transfection.

(D, E) HH/SMO reduces Smaug levels and its overall negative effect on reporter expression. Mean values of the relative levels of reporter expression (determined as above) (D) or of λN-SNAP-Smaug (ratio λN-SNAP-Smaug/GFP-SNAP) (E) in the absence of SMO constructs (red) or in the presence of SMOWT-HA (green), SMOPKA-SD-HA (blue) or SMO∆958-HA (yellow), without HH (plain boxes) or in presence of HH (striped boxes). N=6 (independent biological replicates), except for "no SMO, no HH" and "SMOWT with HH" conditions where n=10 (independent biological replicates). A representative gel is shown in Fig EV3D. Note that SMOWT-HA or HH alone had reproducible weak effects that were observed in two independent biological triplicates but were not statistically significant. See also Fig EV3E-F.

(F) HH/SMO reduces Smaug's intrinsic repressive ability. A similar experiment was conducted with different amounts of λN-SNAP-smaug expressing plasmid (ranging from 0 to 50 ng) as indicated). The mean values of the relative levels of reporter (y-axis) are plotted against the relative levels of λN-SNAP-Smaug (x-axis). SNAP-GUS/GFP-SNAP ratios were set to 1 in cells expressing λN‐SNAP-Smaug in absence of SMOPKA-SD/HH. The colored area represent the extent of the values observed for the relative amounts of reporter expression obtained with (blue) and without (red) SMOPKA-SD/HH, respectively. N=3 (biological triplicates). See Fig EV3H and also a representative gel in Fig EV3G.
Regulation of the RNA-binding protein Smaug by the GPCR Smoothened via the kinase Fused (2020)
Figure 3

Figure 3: SMO/HH activation promotes the phosphorylation of Smaug.

(A) HH/SMO promotes slow-migrating forms of Smaug. Western blot analysis of Cl8 cells that transiently express HA-SmaugWT alone, or together with either SMOWT-GFP or SMOPKA-SD-GFP, in the presence or in absence of HH. GMAP serves as a loading control. Here and in the other panels, the antibodies used are indicated on the left. Here and in (B,C), the black arrows indicate the unphosphorylated form of HA-SmaugWT and the brackets indicate the slower migrating phosphorylated forms of HA-SmaugWT. NT: not transfected. See a phosphatase assay in Fig EV4A.

(B) Smaug phosphorylation requires its interaction with SMO. Western blot analysis of Cl8 cells expressing HA-SmaugWT, in presence of HH, in combination with SMO∆958-GFP, SMOPKA-SD-GFP or SMOPKA-SD, ∆978-GFP, as indicated.

(C) FU controls Smaug phosphorylation. Extracts of Cl8 cells expressing SNAP-Smaug with SMOPKA-SD FU-SD-GFP (with HH) in combination with GFP-FUWT, GAP-GFP-FUWT, Myc-FUEE or GAP-GFP-FUDANA (as indicated) were labeled for the SNAP tag before electrophoresis. After quantification of the gels, the percentage (%) of phosphorylated Smaug (% of phosphorylated forms of SNAP-Smaug total amounts of SNAP-Smaug) was estimated. In C to E, the mean values and (SD) for independent biological triplicates (N=3) are shown in the graph at the bottom, and the statistical analysis was done by one tailed bivariate Wilcoxon rank test. See also fig EV4C, D.

(D) Phosphorylation of Smaug by activated FU requires the co-expression of SMO. Mean values of the percentage of phosphorylated Smaug in extracts of Cl8 cells that express λN-SNAP-Smaug with GAP-GFP-FUWT and SMOWT alone or together (as indicated) N=3 (billogical triplicates). Note that the effect of GAP-GFP-FUWT and SMOWT together is lower than the one observed with GAP-GFP-FUWT and SMOPKA-SD FU-SD-GFP together (in panel C). This could have multiple nonexclusive causes, including the fact that SMOWT is present at much lower levels than SMOPKA-SD FU-SD [38] and/or the involvement of another kinase. See also Fig EV4B. Note that Smaug does not coIP FU unless SMO is present see Appendix Figure S2.

(E) Forcing the interaction of Smaug with activated FU leads to a SMO-independent phosphorylation of Smaug. Mean values of the percentage of phosphorylated Smaug in Cl8 cells coexpressing λN-SNAP-Smaug with GFP-FU, FU-SBR, FUEE-SBR or FUDANA-SBR, with or without HH, as indicated. N=3 (biological triplicates). See also Fig EV4C and D. Note that the levels of Smaug phosphorylation observed in presence of FUDANA-SBR are not lower than those seen with GFP-FU. This suggests an incomplete inhibition of endogenous FU, which may be due to the trapping of FUDANA-SBR by Smaug. See a representative blot in Fig EV3C and a phosphatase assay in Fig EV4D.

The mapping of the phosphosites is shown Fig EV4E-G.
Regulation of the RNA-binding protein Smaug by the GPCR Smoothened via the kinase Fused (2020)
Figure 4

Figure 4: FU downregulates Smaug levels and repressive effect.

(A and C) Effect of FUEE-SBR on Smaug phosphorylation and levels. Extracts of Cl8 cells transfected with λN-SNAP-smaug (25 ng) and SNAP-GUS-5BoxB (300 ng) plasmids in absence or with different amounts of FUEE expressing plasmid (ranging from 15 to 60 μg, as indicated) were analyzed by electrophoresis before quantification as above. See a reprenstive gel in Fig EV5A. The % of phosphorylated SNAP-Smaug protein (determined as in Fig 3) (A) and relative levels of of λN-SNAP-Smaug protein (C) (determined as in Fig 2) are represented as a function of the levels of FUEE-SBR. The mean values and (SD) for independent biological triplicates (N=3) are shown in the graph at the bottom. The statistical analysis was done by one tailed bivariate Wilcoxon rank test with a p value= 0.05 (*). Here CLIP-GUS was used as an internal control.

(B) Effect of FUEE-SBR on Smaug activity. The relative levels of reporter expression obtained in the same experiment as in (A-C) are represented as a function of the % of phosphorylated Smaug. Color of data points correspond to ng of transfected FUEE-SBR in A-C.

See also Fig EV5B-D.

(D) Effect of FUEE-SBR on Smaug subcellular localization.Representative fluorescent images of Cl8 cells expressing GFP-SmaugWT (1), FUEE-SBR-mCherry (2), FUDANA-SBR-mCh (3) or expressing GFP-SmaugWT together with FUEE-SBR-mCherry (4-4'') or GFP-SmaugWT together with FUDANA-SBR-mCh (5-5''). The merge images in 4" and 5" show GFP-SmaugWT in green and FUEE-SBR-mCh or FUDANA-SBR-mCh in red. (6) In presence of FUEE-SBR, 274 of a total of 294 cotransfected cells show a diffuse localization of Smaug and FU and no foci. In presence of FUDANA-SBR, 100% of the cotransfected cells (n=241) show Smaug and FU distributed in bodies and colocalized. 250 ng of plasmid were used for each construct. Scale bar (shown in D1, identical for all panels): 10μm.
Regulation of the RNA-binding protein Smaug by the GPCR Smoothened via the kinase Fused (2020)
Figure 5

Figure 5: HH signaling and Smaug control each other in vivo

(A) Effect of FUEE-SBR and HH/SMO on endogeneous Smaug target mRNAs in Cl8 cells.

Relative mRNA levels estimated using semi-quantitative RT-PCR for various Smaug's targets in Cl8 cells expressing FUEE-SBR (black) , FUEE (dark grey) , or FUDANA-SBR (light grey) were reported with respect to levels of a GFP control. mRNA levels were set to 1 for the GFP control. The list of genes and the sequences of the primers used are shown in Appendix Table S2 and S3. See also Fig EV5E.

Here and in Fig EV5D: RNA encoding mitochondrial proteins are written in blue. N=3 (biological triplicates) and the quantifications were repeated 3 times on each sample.The results are presented as the means ± standard error. The differences between groups were assessed using Student's t-tests with Prism V8.2.1 software. A p value <0.05 was considered to indicate a statistically significant difference*.

(B) Effect of HH on endogeneous targets of Smaug in the wing imaginal disc. Relative levels of Smaug's targets mRNAs in MS1096; UAS-hh or hhts and their respective controls (as in Fig EV5X). N=3, with xx disc for each phenotype.

(C) Effect of smaug mutation on [fu1] wing class distribution. Percent distribution of phenotypic classes (defined in Appendix Figure S4) in fu1 males in presence of zero (fu1), one (fu1;;smaug47/+) or two copies (fu1;; smaug47/smaug47) of the smaug47 allele. The wings were double-blindly classified in "weak" and "strong" according to the LV3-V4 defects (see Fig EV6B). *Chi2 statistical analysis gave a p of 0,015 for fu1 and fu1;;smaug47/+ distributions and of 0,0024 when comparing fu1;;smaug47/+ to fu1;; smaug47/smaug47.
Linear ubiquitination by LUBEL has a role in Drosophila heat stress response (2016)
Figure 1

Figure 1. Conservation of functional domains of HOIP in Drosophila CG11321.

A Schematic diagram of HOIP orthologues in Drosophila melanogaster (D. melanogaster) and Homo sapiens (H. sapiens). D. melanogaster CG11321 is predicted to contain B-box (blue), Npl4 zinc finger (NZF) (navy), ubiquitin (Ub)-associated (UBA1 and UBA2) (pink), and a C-terminal catalytic region, RING between RING (RBR)-C. The RBR-C region consists of RING1 (R1; red), In Between RING (IBR; yellow), RING2 (R2; light green) and Linear Ub chain Determining Domain (LDD; green). PNGase/Ub-associated (PUB) (sky blue) and Zinc finger (ZF) (lavender) domains were found in H. sapiens but not in D. melanogaster.

B Ub chain interaction of the LUBEL-NZF and UBA2 domains. Immobilized GST-NZF or GST-UBA2 was incubated with Lys (K) 48-, K 63-linked, or linear di-Ub (Ub2) chains and bound Ub chains were subjected to immunoblotting using anti-Ub antibody or a linear linkage-specific Ub antibody (anti-linear Ub antibody). Loading of GST-fusion proteins was visualized by Ponceau S staining.