EMBO/soda-nel huggingface dataset:
The glucocorticoid receptor in brown adipocytes is dispensable for control of energy homeostasis (2019)
Figure 5

Fig 5. Deletion of GR from BAT does not affect glucose handling.

(A) Intraperitoneal GTT after 19 weeks of HFD-feeding, glucose injections at 1.5 mg per g of body weight (n = 11-13 animals per group).

(B) Area under the curve calculated from (A) (n = 11-13 animals per group).

(C) Plasma insulin levels during GTT (n = 11-13 animals per group).

(D). Intraperitoneal ITT after 20 weeks of high-fat diet intervention. Insulin was adjusted to 1 mU per g of body weight (n = 11-13 animals per group).

(E) Area under the curve determined from (D) (n = 11-13 animals per group).

(F) Plasma insulin levels after a 5-hour fast, before injecting insulin (n = 11-13 animals per group).

Data information: Error bars denote SEM.
The glucocorticoid receptor in brown adipocytes is dispensable for control of energy homeostasis (2019)
Figure 6

Fig 6. Weight gain in GRBATKO mice is comparable to Flox animals with prolonged high-fat feeding.

(A) Body weight in Flox and GRBATKO after 24 weeks of high-fat feeding (n = 10-13 animals per group).

(B) BAT and iWAT weight in Flox and GRBATKO mice fed HFD for 24 weeks (n = 10-13 animals per group).

(C) GR levels in BAT after 24 weeks high-fat feeding (n=10-13 mice per group).

(D) Representative H&E stained sections of BAT from Flox and GRBATKO animals. Scale bar 100 µm.

(E) Triglyceride content in BAT per gram of wet tissue (n = 10-13 animals per group).

(F) qRT-PCR analysis of thermogenic marker gene expression in BAT of Flox and GRBATKO mice after high-fat feeding (n = 9-13 animals per group).

(G) Representative immunohistochemistry for UCP1 in BAT. Scale bar 100 µm.

(H) Serum insulin levels determined by ELISA (n = 10-13 animals per group).

(I) Blood glucose in Flox and GRBATKO animals after 24 weeks HFD (n = 10-13 animals per group).

Data information: Data were analyzed with two-tailed Student's t-test (C, F). *, p < 0.05; **, p < 0.01. Error bars denote SEM.

Expanded View Figure Legends
CROP: A Retromer-PROPPIN complex mediating membrane fission in the endo-lysosomal system (2022)
Figure 1

Figure 1: Atg18 interacts with Vps26, Vps29 and Vps35

A Scatter plot of the log2 distribution of Atg18 partners identified by SILAC mass spectrometry. The abscissa shows log2 ratios of peptides found in standard media relative to the non-tagged negative control (medium/light; M/L). The ordinate shows log2 ratios of peptides found in salt shocked cells, relative to the non-tagged negative control (heavy/light; H/L).

B Interaction of Atg18 with retromer subunits. Genomically tagged yomCherry-fusions of each retromer subunit were expressed in SEY6210 atg18∆, atg21∆, hsv2∆ cells, together with a plasmid expressing Atg18HA3yEGFP. Cell lysates were subjected to immunoprecipitation using RFP-Trap magnetic beads and analyzed by SDS-PAGE and Western blotting against the indicated proteins.

C Salt-induced vacuole fragmentation. Live cell confocal imaging of Atg18HA3-yeGFP and Vps26yomCherry before and after a mild salt shock with 0.5 M NaCl for 15 minutes. Calcofluor white used to stain the cell walls is only shown in the merge. Arrows indicate sites of colocalization between Vps26yomCherry and Atg18HA3-yeGFP. Scale bar: 5 µm.

D Atg18-Vps35 interaction. Genomically tagged Vps35yomCherry was pulled down from lysates of SEY6210 WT or SEY6210 vps26∆ strains carrying genomically tagged Atg18HA3yEGFP. Adsorbed proteins were analyzed by SDS-PAGE and Western blotting using the antibodies indicated in brackets.

E Signals from the blots in d were quantified on an infrared fluorescence scanner and normalized to the amount of pulled-down Vps35yomCherry. n=3 independent experiments were analyzed using an unpaired Student's t-test. Bars represent the mean and errors bars the SEM. *** P < 0.001.

F Atg18-Vps26 interaction. Genomically tagged Vps26yomCherry was pulled down from lysates of SEY6210 WT, SEY6210 vps35∆ or SEY6210 vps29∆ strains carrying genomically tagged Atg18HA3yEGFP. Adsorbed proteins were analyzed as in D.

G Proteins from F were quantified as in E. n=3 independent experiments were analyzed using an unpaired Student's t-test. Bars represent the mean and errors bars the SEM. *** p < 0.001).
CROP: A Retromer-PROPPIN complex mediating membrane fission in the endo-lysosomal system (2022)
Figure 3

Fig. 3. Interaction of Atg18 and CSC

A Vps17 labilizes the Atg18-Vps26 interaction. Wildtype or vps17∆ cells expressing ATG18HA3-yEGFP from a centromeric plasmid were logarithmically grown in YPD. Genomically tagged Vps26yomCherry was pulled down from whole-cell extracts and analyzed for associated Atg18HA3-yEGFP by SDS-PAGE and Western blotting. Glucose-6-phosphate dehydrogenase (Zwf1) serves as a loading control. The intensity of the interacting Atg18HA3-yEGFP was quantified on a LICOR fluorescence imager and normalized to the amount of Vps26yomCherry. Values of the wildtype interaction were used as the reference and set to 1. n = 4 independent experiments were analyzed using an unpaired Student's t-test. Bars represent the mean and errors bars the SEM, ** p < 0.01)

B Epistasis of ATG18 and retromer genes concerning vacuolar morphology. The indicated cells were logarithmically grown in YPD medium, stained with FM4-64 and calcofluor white as in Fig. 2A and analyzed by confocal microscopy. Scale bar: 5 µm.

C Quantification of vacuole morphology. The number of vacuoles per cell was measured in the cells from B. The graph shows the fractions of cells displaying the indicated numbers of vacuolar vesicles (n = 3 biological experiments with at least 100 cells per condition and experiment were analyzed using an unpaired Student's t-test. Bars represent the mean and errors bars the SEM. ** p < 0.01
CROP: A Retromer-PROPPIN complex mediating membrane fission in the endo-lysosomal system (2022)
Figure 4

Figure 4: Formation of CROP from pure components and interference by SNX.

A Pure recombinant Atg18 was titrated from 0 to 75 µM using the shift of fluorescence of Vps29GFP (2.5 nM), which is induced by Atg18 binding. n = 3 independent experiments were analyzed using non-linear curve fitting (least squares regression method) with GraphPad Prism 9. Bars represent 95% confidence intervals using an asymmetrical likelihood function.

B Purified Atg18 and CSC-GFP were mixed in a 1:1 ratio, incubated together in PBS and analyzed by native PAGE and Western blotting using the antibodies indicated in brackets.
CROP: A Retromer-PROPPIN complex mediating membrane fission in the endo-lysosomal system (2022)
Figure 5

Figure 5: Substitutions labilizing the Atg18-Vps26 interaction

A Sequence alignment of various Atg18 and WIPI1/2 orthologs showing a conserved stretch (in red) of residues in blade 2.

B The stretch containing T56 is mapped on the structure of Atg18 from S. cerevisiae (pdb #6KYB) (Lei et al, 2020), the LFSTSL motif in from S. cerevisiae is shown in orange. Arrows show the Thr56 residue.

C Pull-down. Cells (SEY6210 atg18∆, atg21∆) expressing genomically tagged Vps26yomCherry and the indicated Atg18HA3-yEGFP variants were logarithmically grown in SC-URA media. Vps26yomCherry was pulled down from whole-cell extracts with RFP-trap magnetic beads and analyzed by SDS-PAGE and Western blotting against the indicated proteins. Bands were quantified on a LICOR fluorescence imager. Signals of Atg18HA3yEGFP were normalized relative to those of Vps26yomCherry. Vps1 served as a loading control. n = 3 independent replicates. Data were subjected to an unpaired t-test. Bars represent the mean and errors bars the SEM, *** P < 0.001).

D Influence of substitutions on Atg18 localization. The cells from c were stained with calcofluor white to mark the cell walls and analyzed by confocal microscopy. The calcofluor signal (blue) is only shown in the merge. Scale bar: 5 µm.
CROP: A Retromer-PROPPIN complex mediating membrane fission in the endo-lysosomal system (2022)
Figure 6

Figure 6: The Atg18-CSC interaction in CROP is essential for vacuole fission.

A, Effect of Atg18T56E on salt-induced vacuole fission. Cells expressing Atg18WTHA3-yEGFP or Atg18T56E-HA3-yEGFP from centromeric plasmids in a SEY6210 atg18∆, atg21∆ strain were logarithmically grown in SD-URA medium. They were stained and imaged before and after the induction of vacuole fission by a short salt shock as in Fig. 1D. Calcofluor white-stained cell walls are only represented in the merge (blue). Scale bar: 5 µm.

B, Quantification of the number of vacuoles per cell from A, n = 3 biological experiments with at least 100 cells per condition were scored; bars represent the mean and error bars the SEM.

C, Epistasis of atg18T56E over a vps17∆ mutation. The indicated variants of Atg18-HA3-yEGFP were expressed from plasmids in a SEY6210 atg18∆, atg21∆, vps17∆ strain. Cells were logarithmically grown in SD-URA and imaged as in Fig. 1D. Scale bar: 5 µm.

D, Quantification of the experiments from C, n = 3 independent experiments with at least 100 cells per condition scored; bars represent the mean and error bars the SEM .
CROP: A Retromer-PROPPIN complex mediating membrane fission in the endo-lysosomal system (2022)
Figure 7

Figure 7: Impact of phosphoinositides on CROP binding to membranes

A. Atg18-dependent recruitment of CSC-GFP to small liposomes. SUVs were incubated (10 min, 25°C) with purified CSC (1.5 µM) alone or in combination with the indicated recombinant Atg18 variants (1.5 µM). The vesicles were sedimented by centrifugation and supernatants (Sup.) and pellets were analyzed by SDS-PAGE and Coomassie staining.

B. Quantification of the Coomassie signals for Vps35 by densitometry on a LICOR Odyssey scanner. n = 3 independent experiments. Bars represent the mean and error bars the SEM, ** p < 0.01; * p < 0.05.

C. Quantification as in B, but for the Atg18 variants.

D. CROP recruitment to GUVs. GUVs containing the 5% of the indicated phosphoinositides and the fluorophore CY5.5-PE were left to sediment (30 min, 25°C) in wells that were supplemented with recombinant Atg18 (2.5 nM) covalently linked to dylight550 and CSCGFP (100 nM) as indicated. Samples were incubated for 30 min before acquisition on a confocal microscope. Scale bar: 20 µm.
CROP: A Retromer-PROPPIN complex mediating membrane fission in the endo-lysosomal system (2022)
Figure 9

Figure 9: Effect of retromer and WIPI1 on human endosomes

A Colocalization of WIPI1 with Vps26A. The indicated WIPI1mCherry variants and VPS26EGFP were expressed for 18 h in HK2 cells, from which endogenous WIPI1 had been deleted (WIPI1-KO). The cells were analyzed by confocal microscopy. Scale bars: 10 μm. Insets show enlargements of the outlined areas.

B Colocalization between WIPI1 variants and Vps26 was quantified in the cells from A using the Manders' colocalization coefficient M2. The red line indicates the mean; n = 120 cells per condition, pooled from three independent experiments. P-values were calculated by t-test (analysis performed with 99% confidence ***p < 0.0001).

C Expression levels of WIPI1mCherry variants. Lysates (50 μg of protein per sample) from the cells in A were analyzed by SDS-PAGE and Western blot against WIPI1 and tubulin.

D Depletion of VPS35. HK2 cells expressing the indicated WIPI1eGFP variants were transfected with siRNA against VPS35 (VPS35 KD) or a control siRNA pool. Lysates (50 μg per sample) from the cells were analyzed by SDS-PAGE and Western blot against Vps35 and tubulin.

E Blots from D were quantified on a LICOR Odyssey fluorescence imager. n=3. Red lines indicate the means, n = 3 independent experiments, using a Welch's t-test statistical analysis. Bars represent the mean and error bars the SD.

F Cells from D were analyzed by confocal microscopy 18 h after transfection. Scale bar: 10 μm.

G Quantification of tubule length in the cells from F. Data are means ± SD. of n=210 tubules per condition, from three independent experiments. p values were calculated by unpaired Student's t-test. 99% confidence: *** = p < 0.0001.
CROP: A Retromer-PROPPIN complex mediating membrane fission in the endo-lysosomal system (2022)
Figure 10

Figure 10: Effect of CROP on protein exit from mammalian endosomes

A, Tf recycling. WIPI1-KO cells were transfected with WIPI1WT-eGFP, WIPI1S69E-eGFP or WIPI1S69A-eGFP for 18h. Then, they were serum-starved for 1 h, loaded with Alexa Fluor 568-conjugated Tf, chased at 37°C for 1 h without labeled Tf, and analyzed by confocal microscopy. Scale bar: 10 μm. White dashed lines delineate the circumference of the cells.

B, Quantification of Tf-fluorescence in the cells from a that expressed WIPI1 variants. Total cell fluorescence was integrated and corrected for background fluorescence. Mean values ± SD are shown. n = 3 independent experiments with a total of 150 cells analyzed per condition. P values were calculated by unpaired Student's t-test. 99% confidence: *** = p < 0.0001.

Extended View Figures
Ash1 counteracts Polycomb repression independent of histone H3 Lysine 36 methylation (2019)
Figure 1

Figure 1. Ash1 is the only H3K36-specific methyltransferase critical to counteract Polycomb repression of the Drosophila HOX genes. (A) The schematic of the Drosophila Ash1 protein organization. Ash1 is 2226 amino acid long and contains eight domains (indicated by colored rectangles). The SET domain together with the AWS (Associated With SET) and the post-SET domains are necessary and sufficient for Ash1 histone methyltransferase (HMTase) activity. The functions of the BAH (Bromo Adjacent Homology), PHD (Plant homeodomain) and AT-hook domains are unknown. The positions of ash122 and ash121 point mutations are indicated by arrows. (B) Segmental expression of the Drosophila bithorax complex genes. The three genes of the complex Ubx, abd-A and Abd-B are shown as colored rectangles. The expression of Ubx gives identity to the third thoracic (T3) and the first abdominal (A1) segments, the expression of abd-A defines the second, third and fourth abdominal segments (A2-A4) and the expression of Abd-B gives identity to the rest of the abdominal segments (illustrated with corresponding color code). (C) Adult phenotypes of the ash1 and NSD mutants. In ash122/ash121 mutants (designated as ash1-) the loss of Abd-B expression results in partial transformation of abdominal segments 6 and 5 toward segments 5 and 4, which is visible from the partial loss of pigmentation on tergites 5 and 6 (t5 and t6) and appearance of bristles on sternite 6 (s6, marked with black arrows). The loss of Ubx expression causes transformation of the third thoracic to the second thoracic segment visible as partial haltere (H) to wing and third leg (3L) to second leg (2L) transformations. The former is evident from the change in the haltere shape and the appearance of multiple bristles (black arrowheads). The latter is indicated by the apical and pre-apical bristles (red arrows) on the tibia of the third leg of ash1 mutants. These are normally present on 2L but absent on 3L (compare to wild-type). Also note the appearance of additional hypopleural bristles on the third thoracic segment of the ash1- flies (red arrowheads), which indicate its transformation towards the second thoracic segment. Phenotype of the NSDds46/NSDds46 (NSD-) flies is indistinguishable from wild-type and the phenotype of the double ash122,NSDds46/ ash122,NSDds46 (ash1-,NSD-) flies is no more severe than that of the single ash122/ash121 (ash1-) mutants. (D) Ubx expression in the haltere imaginal discs. The expression was assayed by immunostaining with antibodies against Ubx (red) and acetylated H3K18 (green, positive control). While ash122/ash121 (ash1-) larvae show stochastic clonal loss of the Ubx immunostaining in haltere discs (yellow dashed lines), Set2- larvae have uniform expression of Ubx throughout the haltere disc, resembling that in the wild-type larvae. Scale bars indicate 100μm.
Ash1 counteracts Polycomb repression independent of histone H3 Lysine 36 methylation (2019)
Figure 2

Figure 2. Ash1 SET domain is required to counteract Polycomb repression. (A) Adult phenotypes of the ash122/ash19011 flies supplemented with transgenic constructs expressing either the full-length Ash1 (Ash1FL) or the truncated variants lacking the PHD (Ash1∆PHD) or the SET (Ash1∆SET) domains. Note the third leg (L3) to second leg (L2), haltere (H) to wing (black arrows), t5 to t4 and t6 to t5 transformations in the Ash1∆PHD and the Ash1∆SET but not in the Ash1FL flies. The latter are evident from the partial loss of pigmentation in t6 and t5, or the appearance of small bristles (trichomas) on t6 of the Ash1∆PHD and the Ash1∆SET flies in the area that is normally naked (Ash1FL, yellow dashed line). (B) Two-fold dilutions of total nuclear protein extracts from the third instar larvae of the ash122/ash19011 mutants supplemented with various transgenic constructs and wild type flies were analyzed by western blot with anti-Ash1 antibodies. Arrowhead indicates the position of Ash1 protein. Note that transgenic proteins are expressed at comparable level. (C) Coomassie staining of SDS-PAGE separated protein extracts from (B) was used to control the loading.
Ash1 counteracts Polycomb repression independent of histone H3 Lysine 36 methylation (2019)
Figure 3

Figure 3. Western blot analysis of the bulk H3K36 methylation in larval tissues of various mutants. Two-fold serial dilutions of the total protein extracts from the wild-type, ash122/ash19011 (ash1-), ash122,NSDds46/ash19011,NSDds46 (ash1-, NSD-) and Set21 (Set2-) larval brains, imaginal discs and salivary glands were analyzed by western blot with antibodies against H3K36me1 (A), H3K36me2 (B) and H3K36me3 (C). Note the strong (>10-fold) reduction of H3K36me3 signal in the Set2- extract and the slight (~2-fold) reduction of H3K36me1 signal in the ash1- and ash1-, NSD- extracts. The protein extracts from the wild-type, double ash1-, NSD- and single NSD- and Set2- mutants (right panels) were analyzed together on the same membrane, however the images of the H3K36me1 and H3K36me3 western blots were modified to splice out the marker lane between the ash1-,NSD- and the Set2- extracts. Western blots with constitutively expressed BEAF-32 protein were used as loading controls.
Ash1 counteracts Polycomb repression independent of histone H3 Lysine 36 methylation (2019)
Figure 4

Figure 4. ChIP and quantitative PCR (ChIP-qPCR) analysis of H3K36 methylation and Ash1 binding. Chromatin from the wild-type (dark blue bars), ash122/ash19011 (ash1-, red bars) and transgenic ash122/ash19011 (Ash1FL, light blue bars; Ash1∆PHD, green bars; Ash1∆SET, orange bars) larvae was subjected to immunoprecipitation with the antibodies against H3K36me1 (A), H3K36me2 (B), and Ash1 (C). Histograms show the mean of the two independent experiments (n=2) with dots indicating individual experimental results. An intergenic region on chromosome 3R (intergenic) and the constitutively active TBP-associated factor 4 (Taf4) gene serve as controls. The loss of Ash1 ChIP signal in the ash1- larvae indicates that the selected genes are the genuine Ash1 binding sites.
Ash1 counteracts Polycomb repression independent of histone H3 Lysine 36 methylation (2019)
Figure 5

Figure 5. Zygotic replacement of replication coupled H3.2K36 with H3.2R36 does not cause ectopic repression of HOX genes. (A) Chromosomal positions of various histone H3 genes, ash1 and marker transgenes. 23 histone gene repeat units, each containing single His1, His2B, His2A, His4, and His3.2 gene, are clustered near the centromere (black circle) of chromosome 2. These histone genes are removed by the ∆HisC deletion. To select the animals homozygous for the deletion, the ∆HisC chromosomes are marked with insertions of either Elav-Gal4 or UAS-2xYFP transgenes (black triangles on chromosome 2L). The His3.3A and His3.3B genes encode the same protein, but reside on chromosomes 2L and X. The transgenes carrying twelve copies of either the wild-type histone repeat unit (12xWT) or the unit in which H3 gene is altered to have K36 replaced with arginine (12xH3K36R) are inserted in the same attP site (black triangle on chromosome 3L) [47]. The ash1 gene is located on the same chromosome arm. (B) The ∆HisC; 12xH3K36R and control ∆HisC; 12xWT flies show no homeotic transformations and are indistinguishable from the wild-type.
Ash1 counteracts Polycomb repression independent of histone H3 Lysine 36 methylation (2019)
Figure 6

Figure 6. Zygotic replacement of H3.3K36 and H3.2K36 with H3.2R36 does not cause ectopic repression of Abd-B in the central nervous systems. (A) The Abd-B expression in the Central Nervous System (CNS) of stage 16 embryos was assayed by immunostaining with antibodies against Abd-B (red). In the ash122/TM3, Sb, e Kr::GFP or ash19011/TM3, Sb, e Kr::GFP embryos (heterozygous control), Abd-B is expressed in parasegments 14 to 10 in a gradient that recedes from the posterior to anterior parasegment. In the ash122/ash19011 mutants the Abd-B gradient is much steeper, with reduced staining of parasegments 13 and 12 and at the edge of the detection in parasegments 11-10. Heterozygous control and ash122/ash19011 mutant embryos were stained together and separated by strong GFP expression (green) in the Bolwig's organs (white arrows). Here and in B the embryos are oriented with anterior pole facing the top and scaling bars correspond to 50μm. (B) Unlike ash1 mutants, embryos homozygous for His3.3A, His3.3B, ∆HisC deletions and supplemented with 12xH3K36R transgene (His3.2-,His3.3-,H3K36R) display the wild-type Abd-B immunostaining pattern (red), the same as the control embryos heterozygous for His3.3A, and ∆HisC deletions. In this experiment, immunostaining with antibodies against acetylated H3K18 (green) served as positive control. (C) The schematic of the Abd-B expression in the Drosophila embryo. Although homeotic transformations are often described according to the adult segments affected, the expression of the bithorax complex genes is regulated at the level of embryonic parasegments (PS), which are directly related to adult segments but slightly shifted relative to one another [5,64]. (D) Reverse transcription and quantitative PCR (RT-qPCR) measurement of Abd-B expression in His3.2-,His3.3-,H3K36R embryos. Expression of Abd-B in stage 16 embryos, which are homozygous for His3.3A, His3.3B, and ∆HisC deletions and carry 12xH3K36R transgene (mutant), is not reduced compared to their wild-type counterparts (control 1) or embryos heterozygous for His3.3A, and ∆HisC deletions (control 2). Histograms show the mean of the two independent experiments (n=2) with dots indicating individual experimental results.

Expanded View Figure legends