EMBO/soda-nel huggingface dataset:
Lipoic acid rejuvenates aged intestinal stem cells by preventing age-associated endosome reduction (2020)
Figure 6

Figure 6. ALA regulates autophagic activation

A-C. Immunofluorescence images of esg-GFP and Lysotracker staining with the midgut section from the R4 region in 14-day WT flies (A), 40-day WT flies (B), 40-day WT flies with ALA administration (C). esg-GFP (green; outlined by dotted lines), lysotracker (red). esg-GFP+ cells are outlined by red dotted lines in the images of lysotracker staining channel.

D. Quantification of the dot number of Lysotracker in esg positive cells from experiments (A-C). n is indicated. The numbers of quantified guts from left to right are 9, 11, and 8.

E. Quantification of the dot size of Lysotracker in esg positive cells from experiments (A-C). n is as indicated.

F-H. Expression of esg-GAL4-driven UAS-GFP-mCherry-Atg8a in 14-day Drosophila (F), 40-day Drosophila (G), 40-day Drosophila with ALA administration started at the middle age (26 days) (H). GFP (green) and mCherry (red). The red arrows indicate the autophagosomes.

I. Quantification of the dot number of mCherry in esg positive cells from experiments (F-H). n is indicated. The numbers of quantified guts from left to right are 11, 10, and 12.

J. Quantification of the dot size of mCherry in esg positive cells from experiments (F-H). n is as indicated.

K-L. Immunofluorescence images of esg-GFP and Lysotracker staining with the midgut section from the R4 region in 14-day flies carrying esgts-GAL4-driven UAS-lacZ (K), and 14-day flies carrying esgts-GAL4-driven Las RNAi (L). esg-GFP (green; outlined by dotted lines), lysotracker (red). esg-GFP+ cells are outlined by dotted lines.

M. Quantification of the dot number of Lysotracker in esg positive cells from experiments (K-L). n is indicated. The numbers of quantified guts from left to right are 10, and 9.

N. Quantification of the dot size of Lysotracker in esg positive cells from experiments (K-L). n is as indicated.

O-P. Expression of esg-GAL4-driven UAS-GFP-mCherry-Atg8a in 14-day Drosophila carrying esgts-GAL4-driven UAS-lacZ (flies were cultured at 18 °C and transferred to 29 °C after flies eclosion) (O), and 14-day Drosophila with Las depleted in ISCs and EBs (flies were cultured at 18 °C and transferred to 29 °C after flies eclosion) (P). GFP (green) and mCherry (red). The red arrows indicate the autophagosomes.

Q. Quantification of the dot number of mCherry in esg positive cells from experiments (O-P). n is indicated. The numbers of quantified guts from left to right are 11, and 10.

R. Quantification of the dot size of mCherry in esg positive cells from experiments (O-P). n is as indicated.

Data information: DAPI stained nuclei are shown in blue. Scale bars represent 5 μm (A-C and K-L) and 2 μm (F-H and O-P). Error bars represent SDs. Student's t-tests, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and non-significant (NS) represents p > 0.05. See also Figure EV4.
Lipoic acid rejuvenates aged intestinal stem cells by preventing age-associated endosome reduction (2020)
Figure 7

Figure 7. ALA administration rejuvenates aged ISCs via activation of autophagy process

A-C. Immunofluorescence images of midgut section from the R4 region in 40-day flies (A), 40-day flies with spermidine administration started at 26th day after fly eclosion (B), 40-day flies with spermidine and ALA administration started at 26th day after fly eclosion (C). esg-GFP (green) indicates ISCs and their differentiating cells. Dl (red) staining was used to visualize ISCs.

D. Quantification of the number of esg-GFP+ cells, Dl+ cells, pH3+ cells in experiments (A-C). n is indicated. The numbers of quantified guts from left to right are: 15, 18, 17, 15, 18, 17, 15, 18, and 17.

E. Quantification of luciferase activity of midguts indicated genotypes and manipulations. Error bars show the SD of six independent experiments.

F-I. Representative images of the midgut section from the R4 region of Drosophila carrying esgts-GAL4-driven expression of lacZ cDNA (F, control), Las RNAi (G), Las RNAi and Atg5 cDNA (H), or Las RNAi with spermidine administration (I). GFP (green) and Dl staining (red) was used to visualize ISCs.

J. Quantification of the number of esg-GFP+ cells, Dl+ cells, and pH3+ cells in experiments (F-I). n is indicated. The numbers of quantified guts from left to right are 16, 17, 19, 20, 16, 17, 19, 20, 16, 17, 19, and 20.

K. Quantification of luciferase activity of midguts with indicated genotypes. Error bars show the SD of six independent experiments.

L. Quantification of three categories of midguts treated with the pH indicator Bromophenol Blue in Drosophila with indicated genotypes. The numbers of quantified guts from left to right are 90, 90, 90, 90, and 90. Error bars show the SD of three independent experiments.

M. Quantification of excretion numbers of Drosophila with indicated genotypes. Excretions are quantified in 30 fields for each group of 12 Drosophila. Tests were repeated as three independent experiments.

N. Survival (percentage) of female Drosophila with indicated genotypes. The numbers of quantified Drosophila: 100 (UAS-lacZ), 100 (Las RNAi), 100 (Las RNAi + UAS-Atg5), and 100 (Las RNAi + UAS-Atg8a). Three independent experiments were conducted.

O. Quantification of three categories of midguts treated with the pH indicator Bromophenol Blue in Drosophila with indicated genotypes. The numbers of quantified guts from left to right are 90, 90, 90, and 90. Error bars show the SD of three independent experiments.

P. Quantification of excretion numbers of Drosophila with indicated genotypes. Excretions are quantified in 30 fields for each group of 12 Drosophila. Tests were repeated as three independent experiments.

Data information: DAPI stained nuclei are shown in blue. Scale bars represent 10 μm (A-C and F-I). Error bars represent SDs. p-values for lifespan curves (N) were calculated by the log-rank test. The statistical tests used in other panels were student's t-tests. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and non-significant (NS) represents p > 0.05. See also Figure EV4 and Figure EV5.
Lipoic acid rejuvenates aged intestinal stem cells by preventing age-associated endosome reduction (2020)
Figure 8

Figure 8. ALA administration rejuvenates aged ISCs by modulating endocytosis

A-E. Expressions of Rab5-GFP reporter in ISCs of 14-day WT Drosophila (A), 40-day WT Drosophila (B), 40-day Drosophila with ALA administration (C), 14-day Drosophila carrying esgts-GAL4>UAS-lacZ (D), and 14-day Drosophila carrying esgts-GAL4>Las RNAi (E). GFP (green).

F. Quantification of fluorescence intensity of RAB5-GFP in experiments (A-E). Each dot corresponds to one cell. n is indicated. The numbers of quantified guts from left to right are 13, 11, 12, 10 and 13.

G-K. Expressions of Rab7-GFP reporter in ISCs of 14-day WT Drosophila (G), 40-day WT Drosophila (H), 40-day Drosophila with ALA administration (I), 14-day Drosophila carrying esgts-GAL4>UAS-lacZ (J), and 14-day Drosophila carrying esgts-GAL4>Las RNAi (K). GFP (green).

L. Quantification of fluorescence intensity of RAB7-GFP in experiments (G-K). Each dot corresponds to one cell. n is indicated. The numbers of quantified guts from left to right are 11, 10, 11, 13 and 12.

M-Q. Immunofluorescence images of midgut section from the R4 region of Drosophila carrying esgts-GAL4-driven expression of lacZ cDNA (M, control), constitutively active form of RAB5 (N), constitutively active form of RAB5 with ALA administration (O), constitutively active form of RAB7 (P), or constitutively active form of RAB7 with ALA administration (Q). GFP (green) and Dl staining (red) was used to visualize ISCs.

R. Quantification of the number of esg-GFP+ cells, Dl+ cells, and pH3+ cells in experiments (M-Q). n is indicated. The numbers of quantified guts from left to right are 17, 18, 18, 17, 19, 17, 18, 18, 17, 19, 17, 18, 18, 17, and 19.

S. Quantification of luciferase activity in Drosophila with indicated genotypes and applied manipulations. Error bars show the SD of six independent experiments.

Data information: DAPI stained nuclei are shown in blue. Scale bars represent 2 μm (A-E, G-K), and 10 μm (M-Q). Error bars represent SDs. Student's t-tests, **p < 0.01, ***p < 0.001, ****p < 0.0001, and non-significant (NS) represents p > 0.05. See also Figure EV5.
Lipoic acid rejuvenates aged intestinal stem cells by preventing age-associated endosome reduction (2020)
Figure 9

Figure 9. ALA functions upstream of endocytosis to prevent the functional decrease of ISCs

A-D. Representative images of midgut sections from the R4 region of Drosophila carrying esgts-GAL4-driven expression of lacZ cDNA (A, control), Las RNAi (B), Las RNAi and RAB5-CA (C), or Las RNAi and RAB7-CA (D). GFP (green), Dl staining (red) was used to visualize ISCs.

E. Quantification of the number of esg-GFP+ cells, Dl+ cells, and pH3+ cells in experiments (A-D). n is indicated. The numbers of quantified guts from left to right are 19, 19, 20, 21, 19, 19, 20, 21, 19, 19, 20, and 21.

F. Quantification of the luciferase activity of midguts of Drosophila with indicated genotypes and manipulations. Error bars show the SD of six independent experiments.

G. Quantification of esg-GFP+ cells, Dl+ cells, and pH3+ cells in midguts of Drosophila with indicated genotypes and manipulations. n is indicated. The numbers of quantified guts from left to right are 18, 19, 18, 18, 19, 18, 19, 18, 18, 19, 18, 19, 18, 18, and 19.

H. Quantification of luciferase activity of midguts of Drosophila with indicated genotypes and manipulations. Error bars show the SD of six independent experiments.

Data information: DAPI stained nuclei are shown in blue. Scale bars represent 10 μm (A-D). Error bars represent SDs. Student's t-tests, **p < 0.01, ***p < 0.001, ****p < 0.0001, and non-significant (NS) represents p > 0.05. See also Figure EV5.
Dynamic re-configuration of pro-apoptotic BAK on membranes (2021)
Figure 3

Fig 3. BAK α1 and α7 dissociate upon activation on membranes.

A HDX-MS reveals a significant increase in deuteration at positions E24 (α1) and R169 (α7) following activation with cBID. These positions are conserved suggesting a potentially important hydrogen bonding interaction.

B BAK, or BAK with cysteine introduced at the indicated positions (on a BAK∆Cys background) were stably expressed in Bax-/-Bak-/- MEFs and assess for BAK expression by immunoblotting (inset panel; * indicates non-specific band.) and apoptotic activity in response to etoposide treatment. Data is mean +/-SD of 3 independent experiments.

C E24 and R169 are proximal in inactive BAK on mitochondria. Mitochondria-enriched membrane fractions from cells in (B) were incubated with oxidant (CuPhe) and induced intramolecular disulphide linkage of BAK was assessed on non-reducing SDS-PAGE.

D E24 and R169 dissociate during BAK activation. Mitochondria-enriched membrane fractions from cells in (B) were incubated with cBID (100 nM) prior to oxidant (CuPhe) and the induced intramolecular and intermolecular disulphide linkage of BAK was assessed on non-reducing SDS-PAGE.
Dynamic re-configuration of pro-apoptotic BAK on membranes (2021)
Figure 4

Fig 4. Mutation of the BAK BH4 promotes constitutive activation.

A HDX-MS reveals a constrained BH4 domain in inactive BAK that is rapidly deuterated after activation. BAK BH4 residues in the α1 (V34, F35, S37 and Y38) corresponding to the BH4 motif Φ1Φ2XXΦ3Φ4 ((Kvansakul et al., 2008) are indicated as sticks.

B BAK wild-type, or BAK engineered with the indicated mutations (on a BAK∆Cys background) were stably expressed in Bax-/-Bak-/- MEFs and expression was assessed by immunoblotting following 3 h doxycycline treatment.

C Bax-/-Bak-/- MEFs expressing the indicated BAK variants were treated with doxycycline (Dox, 3 h), incubated for a further 24 h in the presence or absence of BH3-mimetic compounds, and cell death was assessed by PI uptake. Data is expressed as mean +/-SD of 3 independent experiments, **p<0.01, unpaired Student's T-test.

D Mutation of BH4 promotes BAK activating conformation change. Cells in (B) were treated with doxycycline (3 h) to induce BAK expression followed by incubation with BH3-mimetics where indicated (for 2 h) and conformation change of BAK was assessed by intracellular flow cytometry with an antibody that recognises activated BAK (G3172). FACS profiles from one experiment representative of the 3 independent experiments shown in Fig EV3. Collated data from 3 independent experiments is expressed as percent G3172 positive cells (mean +/- SD), **p<0.01 unpaired Student's T-test.
Dynamic re-configuration of pro-apoptotic BAK on membranes (2021)
Figure 5

Fig 5. Residues preceding the BAK BH3 domain do not promote, and are not required for, pore formation.

A BAK-6H on liposomes was activated (+cBID, bottom) or not (Control, top) with cBID for 60 mins prior to deuteration for 10 s, 60 s, 600 s, or 3600 s. Incorporation of deuterium was assessed by mass spectrometry and is expressed relative to a non-deuterated sample and a theoretical maximum. HDX-MS reveals that following activation with cBID, maximal deuteration of the BAK N-terminus (up to amino acid 58) occurred within 10 secs.

B BAK mutant with an engineered thrombin cleavage site in α1-α2 loop of hBAK∆N22/∆C25/C166S with a 6xHis C-terminal tag for recombinant protein expression or as full-length protein for ectopic expression in MEFs (BAKthrombin).

C BAK cleaved within its α1-2 loop by thrombin is able to mediate liposome permeabilisation. Recombinant BAK-6Hthrombin pre-cleaved or not with thrombin (see Fig EV4) was incubated with liposomes at either 20 or 60 nM and liposome permeabilisation induced by cBID was monitored over time. Data is presented as percentage liposome permeabilisation relative to detergent-lysed liposomes, mean +/-SEM of 3 independent experiments.

D BAK wild-type, or BAK with an engineered α1-α2 loop thrombin cleavage site were stably expressed in Bax-/-Bak-/- MEFs. Membrane fractions were incubated with thrombin prior to incubation with copper phenanthroline (CuPhe) to induce disulphide-linkage. Samples were run on SDS-PAGE under non-reducing or reducing conditions and immunoblotted for BAK with an antibody recognising the BAK N-terminus upstream of the thrombin cleavage site (aa23-38) or an antibody recognising the BH3 domain (4B5) downstream of the cleavage site. Schematic indicates the nature of the BAK protein detected by each antibody. Disulphide-linkage indicated by a hashed line. Note that the N-terminal portion of thrombin cleaved BAK could not be detected with the N-terminal antibody unless it was intramolecularly disulphide-linked. *cBID (100 nM) was added to activate BAK during thrombin cleavage. Data representative of two independent experiments.

E Cleavage of the BAK α1-α2 loop potentiates MOMP. Membrane fractions from Bax-/-Bak-/- MEFs expressing BAK or BAKthrombin were incubated or not with thrombin in the presence or absence of cBID (10 nM) for the indicated times. Membrane (P) and supernatant (S) fractions were separated and immunoblotted for cytochrome c or BAK 4B5. Data representative of three independent experiments.

Expanded View Figure Legends
Effector caspase Dcp-1 and IAP protein Bruce regulate starvation-induced autophagy during Drosophila melanogaster oogenesis. (2008)
Figure 1.

Quantification of starvation-induced autophagy in D. melanogaster l(2)mbn cells. (A) Flow cytometry analysis of l(2)mbn cells starved for 2 h (2h S) or 4 h (4h S) showed an increase in LTG fluorescence levels (x axis) compared with control cells in full-nutrient medium (C). The gate shown on the histogram represents the LTGhigh population. (B) Representative images of GFP-LC3 puncta in control and 2-h starved l(2)mbn cells. An increase in GFP-LC3 puncta was observed in the starved cells (left). Bar, 10 μm. (C) Flow cytometry analysis of 4-h starved cells were incubated with 3MA (4h S + 3MA) and Baf (4h S + Baf). Both autophagy inhibitors reduced the LTG fluorescence levels compared with starved cells (4h S). Control cells in nutrient-full medium (C) are represented by the brown line. (D) Both autophagy inhibitors, 3MA and Baf, reduced the LTGhigh population significantly. (3MA, P = 0.00001; and Baf, P = 0.00006). (E) RNAi of representative Atg genes decreased the LTR fluorescence levels compared with control. (Atg1, P = 0.01; Atg5, P = 0.01; Atg7, P = 0.002; Atg8a, P = 0.008; Atg8b, P = 0.006; and Atg12, P = 0.02). (F) RNAi of all tested genes in the TOR–PI3K pathways had a statistically significant effect on LTG fluorescence levels. Known negative regulators of autophagy are shown with gray bars; positive regulators are shown with white bars. (Pten, P = 0.007; Tsc1, P = 0.027; Tsc2, P = 0.025; RheB, P = 0.005; Tor, P = 0.016; and S6k, P = 0.012). dsRNA corresponding to a human gene (Hs) was used as a negative control in E and F. Results represent the mean value ± SD from at least three independent experiments.
Effector caspase Dcp-1 and IAP protein Bruce regulate starvation-induced autophagy during Drosophila melanogaster oogenesis. (2008)
Figure 2.

Identification of known cell death–related genes in autophagy regulation in l(2)mbn cells using RNAi. (A) The percentage of LTGhigh cells was reduced by hid-RNAi (P = 0.006) but not by rpr, grim, and skl-RNAi. (B) Knockdown of Ras, phl, and rl expression by RNAi resulted in an increase in the percentage of LTGhigh cells. (Ras, P = 0.003; phl, P = 0.001; and rl, P = 0.028). (C) th-RNAi treatment (24 h) had no significant effect on LTG levels; in contrast, RNAi of Bruce resulted in an increase in LTG fluorescence levels (P = 0.01). (D) Reduction of debcl, Buffy, or p53 expression by RNAi resulted in a decrease in LTG fluorescence levels. (debcl, P = 0.018; Buffy, P = 0.006; and p53, P = 0.004). (E) RNAi of effector caspase Dcp-1 resulted in a significant decrease in the LTGhigh population (P = 0.001). (F) Representative images of GFP-LC3 puncta in cells treated with the indicated RNAi after a 2-h starvation treatment. Bar, 10 μm. (G) Quantification of cells with GFP-LC3 puncta after RNAi treatment. Cells with more than three GFP-LC3 punctate dots were considered to be GFP-LC3–positive cells. Cells treated with the RNAi indicated here all showed a significant difference (P < 0.05) in the percentage of GFP-LC3–positive cells compared with the human (Hs) RNAi control. (Pten, P = 0.006; Tor, P = 0.034; Buffy, P = 0.005; debcl, P = 0.003; Bruce, P = 0.003; Dcp-1, P = 0.007; hid, P = 0.002; Ras, P = 0.006; and phl, P = 0.050). Results represent the mean value ± SD from three independent experiments.
Effector caspase Dcp-1 and IAP protein Bruce regulate starvation-induced autophagy during Drosophila melanogaster oogenesis. (2008)
Figure 3.

Nutrient deprivation induces autophagy at region 2 within the germarium and in dying midstage egg chambers. (A) GFP-LC3 proteins were expressed in nurse cells (NC) but not in follicle cells (FC) by using the UASp/nanos Gal4 system. DAPI staining of nuclei is shown in blue. (B) UASp-GFP-LC3; nanos GAL4 flies were conditioned on yeast paste and had a diffuse GFP-LC3 pattern. Numerous GFP-LC3 puncta (green) at region 2 within germarium were observed in nutrient-deprived flies. Ovaries were stained with LTR in w1118 flies. Germarium of nutrient-deprived w1118 flies had an increase in punctate LTR staining (red) compared with well-fed germarium. (C) Degenerating stage 8 egg chambers (arrows) had numerous GFP-LC3 puncta (green) and an increase in LTR-positive dots (red) compared with healthy egg chambers (arrowheads). DAPI (blue) staining of nuclei is shown in the two panels on the right. (D) Degenerating stage 8 egg chambers (arrows) of nutrient-deprived Atg7 mutants (Atg7d77/Atg7d14) showed a dramatic decrease in LTR staining. DAPI staining of nuclei is shown in blue. Bars: (A and B) 20 μm; (C and D) 50 μm.
Effector caspase Dcp-1 and IAP protein Bruce regulate starvation-induced autophagy during Drosophila melanogaster oogenesis. (2008)
Figure 4.

The effector caspase Dcp-1 is not only required for nutrient starvation–induced autophagy but is also sufficient for the induction of autophagy during D. melanogaster oogenesis. (A) Germaria of the nutrient-deprived Dcp-1Prev flies showed a dramatic decrease in LTR staining compared with nutrient-deprived wild-type flies shown in Fig. 3 B. DAPI staining of nuclei is shown in blue. (B) Degenerating stage 8 egg chambers (arrows) of nutrient-deprived Dcp-1Prev flies showed a dramatic decrease in LTR staining compared with nutrient-deprived wild-type flies shown in Fig. 3 C. (C) Lack of Dcp-1 function (UASp-GFP-LC3 Dcp-1Prev/Dcp-1Prev; nanos-GAL4/+) resulted in uniform diffuse staining of GFP-LC3 rather than the punctate pattern observed in wild-type degenerating stage 8 egg chambers shown in Fig 3 C. (D) Dying egg chambers (arrows) of NGT/+; nanos-GAL4/UASp-fl-Dcp-1 flies that were conditioned on yeast paste showed a significant increase in punctate LTR staining (red) compared with healthy egg chambers (arrowheads). (E) Expression of activated Dcp-1 (a truncated form) and GFP-LC3 in the germ line (UASp-GFP-LC3/+; nanos-GAL4/nanos-GAL4 UASp-tDcp-1) resulted in abundant degenerating stage 8 egg chambers (arrows) with numerous GFP-LC3 puncta (green). DAPI staining of nuclei is shown in blue. Bars: (A) 20 μm; (B–E) 50 μm.
Effector caspase Dcp-1 and IAP protein Bruce regulate starvation-induced autophagy during Drosophila melanogaster oogenesis. (2008)
Figure 6.

Dcp-1 is required for nutrient starvation–induced germarium cell death, and the IAP protein Bruce inhibits germarium and mid-oogenesis cell death. (A) Ovaries were stained with TUNEL (green) to detect DNA fragmentation. Clusters of cysts with TUNEL staining were observed in region 2 in nutrient-deprived w1118 files. In Dcp-1Prev flies, fewer TUNEL-positive cysts in region 2 were observed. Under well-fed conditions, numerous TUNEL-positive cysts were observed in BruceE81 flies. DAPI staining of nuclei is shown in white. (B) Numerous degenerating stage 8 egg chambers (arrows) with TUNEL-positive staining (green) were observed in well-fed BruceE81 flies. DAPI staining of nuclei (white) is shown on the right. Bars: (A) 20 μm; (B) 50 μm.
Effector caspase Dcp-1 and IAP protein Bruce regulate starvation-induced autophagy during Drosophila melanogaster oogenesis. (2008)
Figure 7.

Lack of Atg7 or Atg1 function reduces DNA fragmentation during mid-oogenesis cell death. (A) TUNEL-positive staining was observed in dying stage 8 egg chambers (arrows) of starved control flies (CG5335d30/Atg7d14). DAPI staining of nuclei (white) is shown on the right. (B) In nutrient-deprived Atg7 mutants (Atg7d77/Atg7d14), degenerating stage 8 egg chambers (arrows) showed no or low levels of TUNEL staining. Nuclear DNA condensation, detected by DAPI, was still observed. (C) Dying stage 8 egg chambers (arrows) from nutrient-deprived control siblings (Atg1Δ3D/TM3) generated from the same cross in D had abundant TUNEL-positive staining. (D) In nutrient-deprived Atg1 GLCs, degenerating stage 8 egg chambers (arrows) showed no or low levels of TUNEL staining. Nuclear DNA condensation (DAPI, right) in degenerating egg chambers appeared to occur as in the controls. Bars, 50 μm.