EMBO/soda-nel huggingface dataset:
The CRHR1/CREB/REST signaling cascade regulates mammalian embryonic neural stem cell properties (2022)
Figure 5

Figure 5. REST is a direct transcriptional target of CREB in embryonic neural stem cells

A, B (A) qPCR (n = 5 biological replicates) and (B) Western blot analyses (n = 3 biological replicates) of E14.5 neural progenitor cells transduced with CREB-expressing lentiviral vectors, measuring REST mRNA and protein levels, respectively.

C, D Dose-dependent inhibition of endogenous REST expression by a CREB inhibitor, 666-15. E14.5 neural progenitor cells were treated with the indicated concentrations of 666-15 for 48 h and subjected to (C) qPCR (n = 3 biological replicates ) and (D) Western blot analyses (n = 4 biological replicates).

E, F Inhibition of CRHR1-induced REST expression by 666-15. E14.5 neural progenitor cells transduced with a lentiviral vector expressing CRHR1 were treated with 20 nM of 666-15, and after 48 h, cells were lysed and subjected to (E) qPCR (n = 3 biological replicates) and (F) Western blot analyses (n = 4 biological replicates).

G Identification of a CRE sequence within the mouse REST promoter region. Consensus CRE sequences, the heptamer GACGTCA, were identified within the mouse REST promoter at position -971, and a region from -1,500 to +172 of the REST genomic sequence was cloned into the luciferase plasmid pGL3-Basic. The function of this site was destroyed by mutating CG to AT.

H Luciferase activity was measured using E14.5 neural progenitor cells transfected with CREB expression vectors and pGL3-Basic luciferase plasmids harboring the wild-type or mutated REST promoter at 2 days post-transfection (n = 3 biological replicates).

I A representative gel image of ChIP analysis showing association of CREB proteins with regions of the REST promoter by PCR amplification of putative CRE sequences in the REST promoter (n = 3 biological replicates).

J Immunolabeling of E15.5 brain sections electroporated in utero with CREB-expressing plasmids with or without shREST vectors at E13.5 using anti-GFP antibody. GFP+ cell localization was quantified (right panel) (n ≥ 5 biological replicates). The upper and lower dashed lines indicate the boundaries between the CP and IZ, and IZ and VZ/SVZ, respectively. Scale bar, 20 µm.

Data information: Error bars represent SEM. Unpaired Student's t-test (A, B, I), ordinary one-way ANOVA (C, D, E, F), two-way ANOVA with Sidak's multiple comparisons test (H) and two-way ANOVA with Tukey's multiple comparisons test (J) were used to determine statistical significance. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns: not significant.

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Autophagic degradation of dBruce controls DNA fragmentation in nurse cells during late Drosophila melanogaster oogenesis. (2010)
Figure 1.

Autophagy is activated in dying nurse cells during late oogenesis in D. melanogaster. (A–C) Confocal micrographs of egg chambers of flies expressing the UASp-GFP-mCherry-DrAtg8a transgene exclusively in the germline (genotype: UASp-GFP-mCherry-DrAtg8a/+; nanos-VP-16 Gal4/+). Expression of UASp-GFP-mCherry-DrAtg8a exhibits punctate staining pattern (arrows). Note that during developmental stages early 12 (A) and late 12 (B), the punctate dots are yellow (merge). In late stage 13 (C), large red dots are evident, indicating the presence of acidic compartments (autolysosomes; arrows). The arrowheads in A point to the physical connection between the oocyte and the nurse cells during early stage 12 (not observed during late stage 12). (D and E) Transmission electron micrographs of nurse cells in stage 12 (D) and in stage 13 (E) egg chambers. Autophagosomes (inset in D) and autolysosomes (arrow in E) are evident in the remaining nurse cell cytoplasm (arrow in D indicates actin bundles). Note that the autolysosome shown in E contains condensed material resembling the material of the condensed and fragmented nurse cell nucleus (NCN), providing evidence for nuclear autophagy. (F) Nurse cell of a late stage 13 egg chamber expressing UASp-mCherry-DrAtg8a exclusively in the germline (genotype: UASp-mCherry-DrAtg8a/+; nanos-VP-16 Gal4/+). mCherry-DrAtg8a puncta are attached to or located adjacent to the fragmented nucleus (arrows and insets). Hoechst staining (blue) was performed to visualize the nuclei. Nurse cells are outlined with a white line. FC, follicle cell; FCN, follicle cell nucleus; OC, oocyte. Bars: (A–C) 10 µm; (D–F) 1 µm.
Autophagic degradation of dBruce controls DNA fragmentation in nurse cells during late Drosophila melanogaster oogenesis. (2010)
Figure 2.

Genetic inhibition of autophagy in the germline prevents DNA fragmentation. Confocal micrographs of stage 13/14 egg chambers after TUNEL staining. (A) Wild-type (wt) stage 13 egg chamber contains nurse cell nuclei exhibiting fragmented DNA (positive TUNEL staining; arrows). (B) Wild-type stage 14 egg chambers do not contain nurse cells. Arrows point to the respiratory appendages (RA). (C–E) Stage 14 atg1−/− (C), atg13−/− (D), and vps34−/− (E) germline mutant chambers have persisting nurse cell nuclei that do not contain fragmented DNA (negative TUNEL staining; arrows). Nurse cells are outlined with a white line. Draq5/Hoechst staining (blue) was performed to visualize the nuclei. PhC, phase contrast. Bars, 50 µm.
Autophagic degradation of dBruce controls DNA fragmentation in nurse cells during late Drosophila melanogaster oogenesis. (2010)
Figure 3.

Germline autophagy mutant egg chambers exhibit reduced expression of cleaved caspase-3. Confocal micrographs of stage 13/14 egg chambers stained for cleaved caspase-3 (casp-3). (A) Wild-type (wt) late stage 13 egg chamber showing cleaved caspase-3 staining in the degenerating nurse cell cluster. (B) In wild-type stage 14 egg chamber, nurse cells are completely degraded (arrows). (C–E) atg1−/− (C), atg13−/− (D), and vps34−/− (E) germline mutant stage 14 egg chambers exhibit significantly reduced staining for cleaved caspase-3 and contain persisting nurse cell nuclei. Nurse cells are outlined with a white line. Draq5/Hoechst staining (blue) was performed to visualize the nuclei. PhC, phase contrast. (F) Quantification of mean intensity of cleaved caspase-3 staining observed in the nurse cells of stage 12–14 egg chambers in wild-type and germline autophagy mutant egg chambers. Wild type (WT): three independent experiments, n = 30 egg chambers; atg1−/− GLCs: four independent experiments, n = 30 egg chambers; atg13−/− GLCs: four independent experiments, n = 30 egg chambers; vps34−/− GLCs: three independent experiments, n = 30 egg chambers. Data are presented as mean ± SD. Difference was significant with P < 0.001 for all values versus wild type. Bars, 20 µm.
Autophagic degradation of dBruce controls DNA fragmentation in nurse cells during late Drosophila melanogaster oogenesis. (2010)
Figure 4.

dBruce colocalizes with the autophagic marker Atg8a-GFP in the nurse cells during late oogenesis and is accumulated in autophagy germline mutants. Confocal micrographs of late stage egg chambers expressing Atg8a-GFP and stained for dBruce. (A–C) Stage 10 (A), early stage 12 (B), and late stage 12 (C). dBruce colocalizes with Atg8a-GFP in punctuate structures during stages 12 and 13 (insets in B and insets and arrows in C) and not during stage 10 (insets in A). (D–G) Confocal micrographs of stage 14 wild-type (D) and autophagy mutant egg chambers (E–G) stained for dBruce. dBruce is accumulated in atg1, atg13, and vps34 mutant nurse cells (arrows). Draq5/Hoechst staining (blue) was performed to visualize the nuclei. Nurse cells are outlined with a white line. OC, oocyte; PhC, phase contrast. Bars, 10 µm.
Autophagic degradation of dBruce controls DNA fragmentation in nurse cells during late Drosophila melanogaster oogenesis. (2010)
Figure 5.

dBruce is required for nurse cell survival by controlling DNA fragmentation during oogenesis. (A) Confocal micrographs of dBrucee00984 mutant ovarioles stained for TUNEL and DNA. Arrows point to degenerating stage 8/9 egg chambers that are TUNEL positive. (B and C) Confocal micrographs of vps34/dBruceE81 and atg, dBruceE81 double mutant egg chambers stained for TUNEL (green and red, respectively) and DNA. (B) vps34−/−GLCs; dBruceE81/dBruceE81 stage 14 mutant egg chamber contain persisting TUNEL-positive nurse cell nuclei (outlined; arrow). (C) atg1−/−GLCs, dBruceE81/dBruceE81 stage 14 mutant egg chamber contain persisting TUNEL-positive nurse cell nuclei. atg1 mutant nurse cells are identified by the lack of GFP staining (outlined; arrow). Draq5/Hoechst staining (blue) was performed to visualize the nuclei. PhC, phase contrast. Bars, 50 µm.
TBK1 suppresses mutant HTT induced toxicity and pathology in models of Huntington's disease (2020)
Figure 1

Figure 1: Identification of TBK1 as a kinase that efficiently phosphorylates HTT at S13 in cellular models of HD:

(A) Potential Phosphorylation sites in the HTT N17 domain (T3, S13 and S16).

(B) List of all the different substrates used for the in vitro kinase validation: Nt17, HTTex1 and HTT longer fragments.

(C) Mass spectra of recombinant HTTex1 23Q after 16 hour of co-incubation with by TBK1 showing complete phosphorylation of S13 and S16. The lower panel is a representative western blot of the same phosphorylation reaction (upper panel) using anti-pT3 (CHDI-90001528-1), pS13 HTT (CHDI-90001039-1) and pS16 (ZCH11020 -generated in house), antibodies (ab) after the indicated phosphorylation reaction times.

(D) Representative western blot of HTT (ab-MAB5492), HTT pS13 (ab-CHDI-90001039-1) and HTT pT3 (ab-CHDI-90001528-1) upon coexpression of HTTex1 16Q and 72Q eGFP with the indicated kinases for 48 hour in HEK293T cells.

(E) Quantification of the fold changes in the HTT pS13 and pT3 ratios to total HTT compared to empty vector (EV) control upon coexpression of the indicated kinase from the experiments like in D.

Data information: Graph report mean ± SEM, n=3. Significance was assessed using 2-tailed Student's t-test, < 0.001 - ***, 0.001 to 0.01 **, 0.01 to 0.05 *.
TBK1 suppresses mutant HTT induced toxicity and pathology in models of Huntington's disease (2020)
Figure 2

Figure 2: TBK1 phosphorylates HTT in cellular models:

(A) Representative western blot of HTT (ab-MAB5492), HTT pS13 (ab-CHDI-90001039-1) and HTT pT3 (ab-CHDI-90001528-1) upon coexpression of HTTex1 16Q eGFP, mutants with S to A substitutions at T3 or S13/S16 or HTTex1 72Q eGFP with TBK1 or TBK1 kinase-dead (KD) for 48 hour in HEK293T cells (the additional high molecular weight HTTex1 and HTT pS13 band is possibly due to the co-occurrence of phosphorylation at both S13 and T3).

(B) Quantification of the fold changes in the HTT pS13 (upper panel) and pT3 (lower panel) ratio to total HTT compared to those of the kinase-dead mutant from the experiments like in A (n=3).

(C) Representative western blot of HTT (ab-MAB2166) and HTT pS13 (ab-CHDI-90001039-1) upon coexpression of HTT N543 55Q with the indicated kinase for 24 hour in HEK293T cells.

(D) Quantification of HTT pS13 using the Singulex assay (using ab-MW1 as capture and CHDI-90001039 or 2B7 as detection antibodies for pS13 or total HTT) upon coexpression of HTT N543 55Q with the indicated kinase for 24 hour in HEK293T cells from the experiment in C (representative of 3 repeats).

(E) Representative western blot of HTT (ab-MAB2166) and HTT pS13 (ab-CHDI-90001039-1) upon coexpression of HTT-FL 48Q or HTT-FL 48Q S13A/S16A with the indicated kinase for 24 hour in HEK293T cells.

(F) Quantification of HTT pS13 using the Singulex assay (using ab-MW1 as capture and CHDI-90001039 or 2B7 as detection antibodies for pS13 or total HTT) upon coexpression of HTT-FL 48Q or HTT-FL 48Q S13A/S16A with the indicated kinase for 24 hour in HEK293T cells from the experiments likein E (n=3).

(G) Representative western blot of immunoprecipitated (IP) HTT (ab-MAB2166) and HTT pS13 (ab-CHDI-90001039-1) after lentivirus (LV)-mediated overexpression of TBK1 or TBK1 KD for 72 hour in rat primary striatal neuronal cells.

(H) Quantification of the fold changes in the HTT pS13 ratio to total HTT compared to those of the kinase-dead mutant from the experiments like in G (n=3).

Data information: Graphs report mean ± SEM. Significance was assessed using 2-tailed Student's t-test, < 0.001 - ***.
TBK1 suppresses mutant HTT induced toxicity and pathology in models of Huntington's disease (2020)
Figure 3

Figure 3: TBK1 overexpression affects subcellular localization and reduces aggregation and cytotoxicity of HTT:

(A) Representative western blot of soluble HTT (ab-MAB5492), HTT pS13 (ab-CHDI-90001039-1) and HTT pT3 (ab-CHDI-90001528-1) upon coexpression of HTTex1 16Q eGFP or 72Q eGFP or its phosphorylation-incompetent mutants with the indicated kinases for 48 hour in HEK293T cells.

(B) The graph indicates the fold change in HTT in samples like illustrated in A, relative to empty vector controls normalized to GAPDH.

(C) Representative immunoblot of the HTT (ab-MAB5492) after filter retardation assay from the insoluble cellular fraction upon expression of HTTex1 16Q eGFP or 72Q eGFP or its phosphorylation-incompetent mutants with the indicated kinases for 48 hour in HEK293T cells.

(D) Quantification of the fold change in HTT aggregates compared to EV expressing HTTex1 16Q from the experiments like in C.

(E) Representative immunofluorescence images of HTT aggregates (eGFP) and TBK1 (ab-anti-MYC) upon coexpression of HTTex1 72Q eGFP or its phosphorylation-incompetent mutants with the indicated kinases for 48 hour in HEK293T cells (scale bar 20μm).

(F) Quantification of the percentage of co-transfected cells presenting aggregates upon expression of the indicated kinase, from the experiments like in E.

(G) Quantification of the percentage of cells presenting nuclear aggregates from the experiments like in E.

(H) Representative western blots of HTT (ab-MAB5492), HTT pS13 (ab-CHDI-90001039-1) in soluble fraction and aggregates from HEK293T cells transfected first with HTTex1 72Q eGFP and then after 48 hour (to enable the formation of aggregates before the kinase expression); with TBK1; cells were lysed at the indicated time after kinase transfection.

(I) Quantification of the percentage of cells presenting aggregates and kinase expression from the experiments in H.

Data information: Graphs report mean ± SEM, n=3. Significance was assessed using 2-tailed Student's t-test, < 0.001 - ***, 0.001 to 0.01 **, 0.01 to 0.05 *.
TBK1 suppresses mutant HTT induced toxicity and pathology in models of Huntington's disease (2020)
Figure 5

Figure 5: TBK1 overexpression modulates HD pathology in C. elegans:

(A) Representative immunofluorescence image of C. elegans overexpressing HTT N513 Q15 and HTT N513 Q128 (scale bar 20μm). Top panel complete worm, lower panel head region.

(B) Western blot showing HTT (ab-MAB5492), HTT pS13 (ab-CHDI-90001039-1) and HTT pT3 (ab-CHDI-90001528-1) levels in C. elegans overexpressing HTT N513 Q15 and HTT N513 Q128.

(C) Quantification of the fold changes in ratios of HTT pS13 and pT3 to total HTT in C. elegans overexpressing HTT N513 Q15 and HTT N513 Q128 from the experiments like in B (n=3).

(D) Representative western blots showing the HTT (ab-MAB5492) and HTT pS13 (ab-CHDI-90001039-1) levels from the detergent soluble fraction and HTT aggregates (ab-MAB5492) from the detergent insoluble fraction in three different transgenic C. elegans lines overexpressing TBK1 or the TBK1 KD.

(E) The bottom graph indicates the fold change in the HTT pS13 (ab-CHDI-90001039-1) ratios to total HTT (ab-MAB5492) compared to those in the kinase-dead mutant line 1 from the experiments like in D. The top graph indicates the fold change in HTT aggregates (ab-MAB5492) compared to that in the kinase-dead mutant the experiments like in D (n=3).

(F) Mobility of the Q15 and Q128 C. elegans lines upon overexpression of TBK1 or TBK1 KD (n= 26-29).

(G) Survival graph of the Q15 and Q128 C. elegans lines upon overexpression of TBK1 or TBK1 KD (n=30).

(H) Representative western blots showing HTT (ab-MAB5492) and HTT pS13 (ab-CHDI-90001039-1) from the detergent soluble fraction and HTT aggregates (ab-MAB5492) from the detergent insoluble fraction in TBK1 orthologue (ikke-1 or unc-51) RNAi-treated C. elegans lines expressing HTT N513 Q15 and HTT N513 Q128.

(I) Quantification of the fold change in the ratio of pS13 HTT to total HTT compared to non-targeting RNAi from the experiments like in H (n=4).

Data information: Graphs report mean ± SEM. Significance was assessed using 2-tailed Student's t-test, < 0.001 - accept for (F) where 2-way ANOVA with Sidak post-hoc test was used. ***, 0.001 to 0.01 **, 0.01 to 0.05 *.
TBK1 suppresses mutant HTT induced toxicity and pathology in models of Huntington's disease (2020)
Figure 6

Figure 6: Autophagy inhibition blocks the HTTex1 72Q aggregate-reducing effect of TBK1: (A) Representative immunoblot of a filter retardation of HTT (ab-MAB5492) from the insoluble cellular fraction; HEK293T cells co-expressed HTTex1 72Q eGFP and TBK1 or TBK1 KD for 48 hour, and for the last 16 hour, they were treated with the indicated proteasome inhibitor (MG132, 5 µM) or autophagy inhibitor (Baf A1, 200 nM, NH4Cl, 10 mM, 3-MA,5 mM).

(B) Quantification of the fold change in HTT aggregates compared to TBK1 KD treated with DMSO from the blots like in A (n=3).

(C) Immunoblot of LC3 (ab48394) from soluble HEK293 cellular fractions overexpressing TBK1 or TBK1 KD for 24 hour; for the last hour, Baf A1 (500 nM) was added as indicated.

(D) Fold change in LC3-II levels (lower band) relative to the respective untreated kinase dead mutant normalized to actin from the blots like in C (n=3).

(E) Immunoblot of LC3 (ab48394) from soluble rat primary neuronal cells overexpressing lentivirus-mediated TBK1 and TBK1 KD for 96 hour. For the last 1 or 4 hour, Baf A1 (500 nM) was added as indicated.

(F) Quantification of the fold change in LC3-II levels (lower band) compared to the kinase dead mutant, which was untreated, normalized to GAPDH from the blots like in E (n=3).

Data information: Graphs report mean ± SEM. Significance was assessed using 2-tailed Student's t-test, 0.01 to 0.05 *.
TBK1 suppresses mutant HTT induced toxicity and pathology in models of Huntington's disease (2020)
Figure 7

Figure 7: TBK1 deficient in the autophagy adaptor binding domain does not reduce HTTex1 72Q eGFP S13A/S16A aggregates:

(A) Western blot of soluble HTT (ab-MAB5492), HTT pS13 (ab-CHDI-90001039-1) and HTT aggregates (ab-MAB5492), upon coexpression of the HTTex1 72Q eGFP or HTTex1 72Q eGFP S13A/S16A variant with TBK1 KD, TBK1 or TBK1 Δ690-713 for 48 hour in HEK293 cells.

(B) The graph indicates the calculated fold changes in HTT, HTT pS13 ratio to total HTT and HTT aggregates all compared to the levels in TBK1 KD from the experiments like in A (bottom panel normalized to Tubulin, n=3).

(C) Representative immunofluorescence images of eGFP HTTex1 72Q or its phosphorylation-deficient variant S13A/S16A upon coexpression with TBK1 KD or TBK1 or TBK1 Δ690-713 (ab-anti-Myc) with for 48 hour in HEK293 cells (scale bar 20μm).

(D) The graph indicates the percentage of co-transfected cells presenting aggregates upon expression of the indicated kinase, from the experiments like in C (n=3).

Data information: Graphs report mean ± SEM. Significance was assessed using 2-tailed Student's t-test, < 0.001 - ***, 0.001 to 0.01 **.
Natural variants suppress mutations in hundreds of essential genes (2021)
Figure 3

Figure 3. Mapping suppressor loci by sequencing segregant pools.

A Example of the mapping results. Wild allele frequency in progeny of a cross of

the gab1-1 temperature sensitive allele to the YJM975 strain (y-axis) along the yeast genome (x-axis) at the permissive 26°C (blue) and restrictive 34°C (TS allele loss-of-function, cyan). The allele frequency change between the two temperatures is used in mapping. Labels: selected loci in the cross. Blue regions: called suppressor loci.

B Suppressors are plentiful. The average number of suppressor loci per cross

(y-axis) at a given allele frequency change cut-off (x-axis) with either the wild allele beneficial (blue) or the reference allele beneficial (yellow). Vertical line at an allele frequency change of 0.2: the cut-off used for calling suppressor loci.

C Suppressors are reproducible across TS alleles. Allele frequency change of

suppressor loci in crosses using different TS alleles of the same gene (x- and y-axis) crossed with the same wild strain. Colours: gene and wild strain combinations.

D Suppressors are reproducible across wild strains. Allele frequency change of

suppressor loci at 34°C in crosses using the same TS allele and a different wild strain (x- and y-axis). Colours: TS alleles.
Natural variants suppress mutations in hundreds of essential genes (2021)
Figure 4

Figure 4. Identifying and validating suppressor candidates.

A Mapping results for segregant pools involving the indicated TS alleles and wild

strains. The change in S288C allele frequency between the meiotic progeny isolated at 26°C and 34°C is plotted by genomic coordinate. Causal suppressor genes are indicated for regions that show selection for the wild strain sequence. Genes in brackets have not been validated experimentally.

B Comparison of the change in reference allele frequency, either for suppressor

loci for which a causal suppressor gene was validated (N=17), or for loci for which we were unable to validate a suppressor gene (N=10). Loci for which all experiments failed due to technical reasons were excluded from the analysis. Statistical significance was determined using a two-tailed Mann-Whitney's U test (** = p < 0.005). Boxes: first, second, and third quartiles; whiskers: 1.5 interquartile range away from the first and third quartiles.

C A cartoon of the TORC2 signalling pathway, highlighting suppression of a

TORC2 mutant (tsc11) by mutations in ORM2.

D Suppressor prediction within the chromosome XV QTL of rpn11 TS mutants.

The functional information prioritisation score (y-axis) for genes in the suppressor region (x-axis) identified RPT4 and RPN8 as the two highest-scoring suppressor candidates.

E Experimental validation of RPN8 as the causal suppressor of the rpn11-8 TS

mutant. Cultures of the indicated strains were diluted to an optical density at 600 nm of 0.1 and a series of ten-fold dilutions was spotted on agar plates and incubated for 2-3 days at 34°C. UWOPS = UWOPS87-2421. See also Figure EV4.
Natural variants suppress mutations in hundreds of essential genes (2021)
Figure 5

Figure 5. The NSE1 allele of UWOPS87-2421 can suppress NSE3 and NSE4 TS mutants.

A,B Suppression of nse3-ts4 and nse4-ts4 temperature sensitivity (A) and DNA

damage sensitivity (B) by the NSE1 allele of UWOPS87-2421. Cultures of the indicated strains were diluted to an optical density at 600 nm of 0.1 and a series of ten-fold dilutions was spotted on agar plates and incubated for 2-3 days. UW = UWOPS87-2421. The plates shown in (B) were incubated at 30°C.

C A cartoon of the SMC5/6 complex.

D An illustration of the various types of genetic interactions that have been

observed between different alleles of NSE1 and NSE3.

E Recruitment of Smc6-FLAG by ChIP-qPCR at two known SMC5/6 binding sites

(pericentromere of chromosome XIV and CEN3) and one negative control locus (arm of chromosome III) in G2/M arrested strains. Relative enrichment corresponds to the ratio of the signal after immunoprecipitation (FLAG) over beads alone, normalised to the ratio in wild-type cells at CEN3. Error bars: standard error of the mean of three independent experiments. Statistical significance was determined using an unpaired, two-tailed t-test (* = p < 0.05, ** = p < 0.005).

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