EMBO/soda-nel huggingface dataset:
Glutamine deprivation stimulates mTOR-JNK-dependent chemokine secretion (2014)
f3

(a) Colocalization of lysosomes (Lysotracker Green) with phagophores (mCherry-ULK1 and mCherry-ATG5 puncta) in glutamine-deprived U2OS cells. Scale bar, 25 μm. (b) Colocalization of LAMP1-positive lysosomes with GFP-LC3-labelled autophagosomes after glutamine deprivation in the absence or presence of 400 nM BafA1 in U2OS cells. Scale bar, 15 μm. (c) Immunofluorescence images of U2OS cells stained for p62 expression and LAMP1 following glutamine deprivation and BafA1 treatment. Nuclei were stained with DAPI. Scale bar, 15 μm. (d) Immunofluorescence images of cells stained with LAMP1 (lysosomes) and the Golgi marker, RCAS1, following glutamine deprivation. Nuclei were stained with DAPI. Scale bar, 15 μm. (e) U2OS cells were treated for 24 h with cell culture medium containing the indicated amino acids. Cell lysates were immunoblotted to detect changes in S6K phosphorylation and p62. (f) Immunofluorescence staining for LAMP1 (green) and mTOR (red) in U2OS cells subjected to glutamine deprivation (24 h), in the presence of 100 nM WYE-125132 (WYE-132). −AA denotes cells deprived of all amino acids. Scale bar, 15 μm. Error bars in all figures represent s.d. of three biological replicates. Ess., essential.
Glutamine deprivation stimulates mTOR-JNK-dependent chemokine secretion (2014)
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(a) Immunoblot of autophagy-related proteins in U2OS cells expressing a control TALEN (U2OS-TAL2-C1) or two independent subcloned TALEN cell lines targeting ATG7 (U2OS-TAL2-A7, U2OS-TAL2-A8). p62 long and short refer to either long or short exposure times during film development. (b) U2OS cells expressing an ATG7-TALEN (TAL2-ATG7) or control TALEN (TAL2-CTL) were subjected to glutamine starvation for 6 days followed by 6 days recovery in glutamine-replete medium. Cells were then stained with SRB and absorbance was read at 540 nM. Graph shows average results from five clonal sub-lines for each condition. The statistical significance (P value) was determined by a two-tailed, paired Student’s t-test. P0.05. (c) Representative images of SRB-stained clones in b. (d) IL-8 enzyme-linked immunosorbent assay (ELISA) of U2OS-TAL2-CTL-C1 (control) and U2OS-TAL2-ATG7-A8 (ATG7 knockout) following 24 h glutamine deprivation. (e) IL-8 ELISA of U2OS parental cells starved of glutamine for the indicated times with or without BafA1. Error bars in all figures represent s.d. of three biological replicates. OD, optical density.
Glutamine deprivation stimulates mTOR-JNK-dependent chemokine secretion (2014)
f5

(a) Immunofluorescence images of U2OS cells stained for LAMP1 and the Golgi marker RCAS1 after 24 h glutamine deprivation with or without treatment with 1 μM BrefA or 100 nM WYE-125132. Scale bar, 15 μm. (b) Cytokine array analysis of conditioned medium from U2OS cells subjected to glutamine deprivation in the presence of 1 μM BrefA or 100 nM WYE-125132. (c) IL-8 enzyme-linked immunosorbent assay in U2OS cells after 24 h glutamine deprivation with or without CCI-779 or WYE-125132. (d) RT–PCR analysis of IL-8 mRNA expression in U2OS cells after 24 h glutamine deprivation with or without CCI-779 or WYE-125132. Error bars in all figures represent s.d. of three biological replicates. OD, optical density.
Glutamine deprivation stimulates mTOR-JNK-dependent chemokine secretion (2014)
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(a) Immunoblot analysis of autophagy, ER stress- and mTOR-related proteins, and JNK in U2OS cells after 24 h glutamine deprivation with or without CCI-779. (b) RT–PCR analysis of IL-8 mRNA expression in U2OS cells transfected with two different siRNAs targeting JNK (JNK #1 and JNK #2) and subjected to 24 h glutamine deprivation. (c) IL-8 enzyme-linked immunosorbent assay (ELISA) of conditioned media from the experiment described in b. (d) Immunoblot analysis of JNK protein expression in JNK siRNA-treated cells. (e) RT–PCR analysis of IL-8 expression in U2OS cells after 24 h glutamine deprivation with or without the JNK inhibitor SP600125 (JNKi). (f) IL-8 ELISA of conditioned medium from the experiment described in e. (g) IL-8 ELISA of U2OS cells transfected with two different siRNAs targeting IRE1 (IRE-A and IRE-B) and subjected to glutamine deprivation for 24 h. (h) RT–PCR analysis of IL-8 mRNA expression from experiment described in g. (i) RT–PCR analysis of CHOP mRNA expression from experiment described in g. (j) RT–PCR analysis of IRE1 mRNA expression from experiment described in g. (k) Immunoblot analysis of experiment described in g showing phosphorylation of JNK. Error bars in all figures represent s.d. of three biological replicates. OD, optical density.
Glutamine deprivation stimulates mTOR-JNK-dependent chemokine secretion (2014)
f7

(a) IL-8 enzyme-linked immunosorbent assay (ELISA) on U2OS cells starved of glutamine or treated with BPTES (10 μM) for 24 h. (b) RT–PCR analysis of IL-8 and CHOP mRNA expression in U2OS cells starved of glutamine for 24 h and co-treated with DMαKG. (c) Metabolite analysis of U2OS cells starved of glutamine, treated with BPTES or glutamine-starved co-treated with DMαKG. (d) IL-8 ELISA of U2OS cells starved of glutamine or treated with BPTES for 24 h and co-treated with DMαKG. (e) Immunoblot analysis of pS6K and pJNK in U2OS cells starved of glutamine for 24 h and co-treated with DMαKG. Error bars in all figures represent s.d. of three biological replicates. OD, optical density.
Erythropoietin signaling in peripheral macrophages is required for systemic β-amyloid clearance (2022)
Figure 1

Figure 1 Aβ42 promotes its self-clearance through macrophage EPO signaling.

A, B Peritoneal macrophages (PMs) were treated with Aβ42 (0.5-5 μM) for 12 hr, phagocytosis of Aβ42 (A) and Aβ40 (B) was measured by flow cytometry (n = 4). Vehicle (cold) was macrophage treated with Aβ resolution incubated at 4℃. It was used as a negative control to exclude Aβ bound with macrophage from Aβ engulfment. Vehicle was macrophage treated with Aβ resolution incubated at 37℃ as control.

C, D WT mice (8w) were treated with Aβ42 (0.2 μmol/kg/d, i.v., twice per day) for 24 hr, splenic macrophages were purified for phagocytosis assay of Aβ42 (C) and Aβ40 (D) (n = 4).

E Splenic macrophages from young APP/PS1-21 mice (6-week-old) were isolated for phagocytosis assay of Aβ42 (n = 4).

F-H Macrophages from A-E were detected by immunoblotting with the indicated antibodies (n = 3).

I PMs from Lyz2-Cre+/+/Eporloxp/loxp mice (EPOR-MKO) or Lyz2-Cre+/+/Epor+/+ mice (EPOR-C) were treated with Aβ42 (5 μM) or vehicle for 12 hr. Phagocytosis of Aβ42 was measured by flow cytometry (n = 4).

J EPOR-MKO mice (8 w) were treated with Aβ42 (0.2 μmol/kg/d, i.v.) for 24 hr, splenic macrophages were used for phagocytosis assay of Aβ42 (n = 4).

Data information: For multiple groups, one-way ANOVA was used. Unpaired t-tests were used for the comparison of two groups. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance. Aβ, amyloid-β; EPO, erythropoietin. EPOR-KO macrophages were PMs isolated from EPOR-MKO mice.
Erythropoietin signaling in peripheral macrophages is required for systemic β-amyloid clearance (2022)
Figure 3

Figure 3 EPO promotes Aβ clearance via PPARγ in macrophages.

A-F Macrophages from Fig. 2A-F were detected by immunoblotting with indicated antibodies (n = 3).

G PMs from Lyz2-Cre+/+/Epor+/+ mice (EPOR-C) were pretreated with PPARγ antagonist GW9662 (10 μM, 12 hr), then with rhEPO (100 IU, 12 hr), and then applied for phagocytosis assay of Aβ42 (n = 3).

H PMs from Lyz2-Cre+/+/Eporloxp/loxp mice (EPOR-MKO) or from EPOR-C mice were pretreated with PPARγ agonist RSG (50 μM, 12 hr), and then applied for phagocytosis of Aβ42 by flow cytometry (n = 4).

I-L Splenic macrophages from EPOR-C mice treated with GW9662 (1 mg/kg/d, i.p.) or/and rhEPO (10000 IU/kg/d, i.p.) twice per day for 1 day (I), from EPOR-MKO mice treated with RSG (0.1 mg/kg/d, i.p.) for 24 hr (J), from APP/PS1-21 mice treated with GW9662 (1 mg/kg, i.p., every second day) and/or EPO (10000 IU/kg, i.p., every second day) for 5 days (K), or from APP/PS1-21+/+/Lyz2-Cre+/+/Eporloxp/loxp mice (APP/PS1-21+EPOR-MKO) treated with RSG (0.1 mg/kg, i.p., every second day) for 5 day (L), were purified for Aβ42 phagocytosis assay (n = 4).

M-O Macrophages from G-L were applied for detection of inflammatory factor expression by QPCR (n = 4).

Data information: All the vehicles of Fig 3 were PBS. For multiple groups, one-way ANOVA was used. Unpaired t-tests were used for the comparison of two groups. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance. GW, GW9662; RSG, Rosiglitazone. EPOR-KO macrophages were PMs isolated from EPOR-MKO mice.
Erythropoietin signaling in peripheral macrophages is required for systemic β-amyloid clearance (2022)
Figure 4

Figure 4 Peripheral macrophage EPO signaling regulates peripheral Aβ accumulation, brain Aβ efflux and central Aβ deposition.

A-F Flag-Aβ42 was injected into the lateral ventricles of Lyz2-Cre+/+/Eporloxp/loxp mice (EPOR-MKO) or Lyz2-Cre+/+/Epor+/+ mice (EPOR-C) (8 w) mice pretreated with rhEPO 10000 IU/kg/d, i.p.) or ARA290 (0.03 mg/kg/d, i.p.) for 2 days (twice per day) (n = 3). One day later, Flag-Aβ42 in spleens and brains was detected by immunoblotting (A, B, D and E). Aβ42 levels of spleen were measured by ELISA (C, F).

G-I Proteins were extracted from spleens and brains of APP/PS1-21 mice (5 m) treated with rhEPO (10000 IU/kg/d) or ARA290 (0.03 mg/kg/d) (i.p., once a day, for 14 days) (n=10), and soluble Aβ levels were measured by ELISA (n = 5) (G, H). Aβ plaques in cortex and hippocampus (n = 5) were detected by IHC (I).

J-M Proteins were extracted from spleens and brains of 5- or 11-month-old APP/PS-1-21+/+/Lyz2-Cre+/+/Eporloxp/loxp mice (APP/PS1-21+EPOR-MKO) or APP/PS-1-21+/+/Lyz2-Cre+/+/Epor+/+ mice (APP/PS1-21+EPOR-C), and soluble Aβ levels were measured by ELISA (n = 5) (J, K). Aβ plaques in the cortex and hippocampus of mice of 5- (L) or 11-month-old (M) (n = 5) were detected by IHC.

N Timeline of bone marrow transplantation experiment.

O Splenic macrophages from APP/PS1-21 mice transplanted with EPOR-C or EPOR-MKO bone marrow were purified for Aβ phagocytosis assay (n = 4).

P, Q Soluble Aβ levels of spleens and brains were measured by ELISA (n = 4).

R Aβ plaques in the cortex and hippocampus (n = 4) were detected by IHC.

Data information: For multiple groups, one-way ANOVA was used. Unpaired t-tests were used for the comparison of two groups. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance. ARA, ARA290; ELISA, enzyme linked immunosorbent assay; IHC, Immunohistochemical staining.
Erythropoietin signaling in peripheral macrophages is required for systemic β-amyloid clearance (2022)
Figure 6

Figure 6 EPO and ARA290 restore EPO signaling and dysregulated functions of peripheral macrophages in old APP/PS1-21 mice.

A-E Splenic macrophages from APP/PS1-21 (5- or 11-month-old) or matched WT mice were detected by immunoblotting with the indicated antibodies (n = 3) (A, B). Their phagocytosis of Aβ42 (C, D) were measured by flow cytometry (n = 4). The mRNA levels of IDE and NEP were measured by QPCR (n=4) (E).

F Splenic macrophages from APP/PS1-21 mice at different ages were purified for detection of inflammatory factor expression (n = 6).

G-J Old APP/PS1-21 mice (11 m) and WT mice at the same age were treated with rhEPO (10000 IU/kg/d), ARA290 (0.03 mg/kg/d) or vehicle (i.p., once a day, for 14 days). Splenic macrophages were isolated for detection by immunoblotting with the indicated antibodies (n = 3) (G). Phagocytosis of Aβ42 by the splenic macrophages was measured by flow cytometry (n = 4) (H). Protein levels of IDE and NEP were detected by immunoblotting with indicated antibodies (n = 3) (I). The mRNA levels of inflammatory factors were measured by QPCR (n = 5) (J).

K-M PMs were treated with Aβ42 (0, 5, 7.5 or 10 μM) for 12 hr, and cell lysates were detected by immunoblotting with the indicated antibodies (n = 3) (K). Phagocytosis of Aβ42 and Aβ40 was measured by flow cytometry (n = 4) (L). Protein levels of IDE and NEP were detected by immunoblotting with indicated antibodies (n = 3) (M).

Data information: All the vehicles of Fig 6 were PBS. For multiple groups, one-way ANOVA was used. Unpaired t-tests were used for the comparison of two groups. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance. IDE, insulin degrading enzyme; NEP, neprilysin.
Erythropoietin signaling in peripheral macrophages is required for systemic β-amyloid clearance (2022)
Figure 7

Figure 7 EPO and ARA290 ameliorate peripheral and central Aβ accumulation, brain AD-type pathologies and behavioral deficits in old APP/PS1-21 mice.

A Timeline of rhEPO (10000 IU/kg/d) or ARA290 (0.03 mg/kg/d) treatments of old APP/PS1-21 mice (11 m) (n = 5).

B Plasma Aβ levels were measured by ELISA (n = 5).

C, D Soluble Aβ levels in spleens (C) and brains (D) were measured by ELISA (n = 5).

E Aβ plaques in the cortex and hippocampus (n = 5) were detected by IHC.

F Cortex and hippocampus were taken, and the mRNA levels of inflammatory factors were measured by QPCR (n = 5).

G Expression of MAP-2 or the apoptotic neurons of hippocampus were detected by IF staining and then statistically quantified (n = 5).

H-I Both non-cognitive and cognitive behavioral impairments were analyzed by nest construction assay, social interaction assay, NORT, FCT and Morris water maze test (n = 10).

Data information: All the vehicles of Fig 7 were PBS. For multiple groups, one-way ANOVA was used. Unpaired t-tests were used for the comparison of two groups. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance. IF, Immunofluorescent; MAP-2, microtubule-associated protein 2; NeuN, neuronal nuclei; TUNEL, Terminal dUTP Nick End Labeling.
TMEM59 defines a novel ATG16L1‐binding motif that promotes local activation of LC3 (2013)
Figure 1

TMEM59 induces LC3 activation. (A) Clone P15 induces HALC3 lipidation. 293T cells were transfected with P15 plus plasmids expressing HALC3A and/or the apoptotic inhibitor p35 (as shown), and lysed for western blotting against the indicated molecules (anti-HA for HALC3A). The figure shows that expression of P15 induces HALC3A conversion to a lower molecular weight form indicative of protein lipidation. This activity remains unchanged by p35. (B) P15 induces GFPLC3 translocation to a vesiculated pattern. 293T cells were transfected with the indicated plasmids mixed with vectors expressing GFPLC3A and p35. The known autophagic inducer bNIP3L constituted a positive control. Representative confocal pictures are shown. (C) TMEM59 induces HALC3 lipidation in different cell lines lacking the SV40 large T-antigen plasmid amplification system. Cells were transfected with the shown plasmids, vectors expressing HALC3A (left) or HALC3B (right) and GST (as transfection control), and lysed for western blotting against the indicated molecules. (D) TMEM59 induces GFPLC3 activation. 293 cells were transfected with the indicated plasmids and GFPLC3A (top) or GFPLC3B (bottom). Representative confocal pictures are shown. (E) Quantification of the phenotype in D. The number of GFPLC3-positive vesicles per transfected cell was scored for at least fifty cells. Data are expressed as means ±s.d. of one representative experiment of three repetitions.Source data for this figure is available on the online supplementary information page.
TMEM59 defines a novel ATG16L1‐binding motif that promotes local activation of LC3 (2013)
Figure 2

The ID of TMEM59 is necessary and sufficient for LC3 activation. (A) TMEM59 ID is required for HALC3 lipidation. 293 cells were transfected with full-length TMEM59 (FL) or a deleted version lacking the ID (?ID, ?263323), HALC3A (left) or HALC3B (right) and GST. Cells were lysed for western blotting against the shown molecules. (B) The ID is necessary for GFPLC3 activation. 293 cells were transfected with the indicated TMEM59 constructs and GFPLC3A or GFPLC3B (as indicated). Representative confocal pictures are shown. (C) Quantification of the phenotype in B. Scoring and data expression were as in Figure 1E. (D) TMEM59 ID (amino acids 263323) suffices for HALC3A lipidation. 293 cells were transfected with the indicated CD16:7 chimera (Control: empty chimera) and vectors encoding HALC3A and GST, subjected to aggregation with the shown amounts of anti-CD16 antibody and lysed for western blotting. The right panel shows control WBs demonstrating equal loading (ACTIN), transfection (GST) and chimera expression (CD16) in unaggregated samples, as all experimental points per chimera derive from a single transfection. (E) Comparable surface levels of CD16:7 constructs. Transfected 293 cells were processed for anti-CD16 flow cytometry. The graph displays percentages of positive cells (left axis) and means of fluorescence of positive cells (MF, right axis) obtained from triplicates. Data are expressed as means ±s.d. of the triplicates. (F) The ID suffices for HALC3B lipidation. (G) The ID suffices for GFPLC3 activation. 293 cells were transfected with the indicated chimeras and GFPLC3A or GFPLC3B, aggregated and mounted. Representative confocal pictures are shown. Activated GFPLC3 appears as a collapsed mass of indiscernible vacuoles. (H) Quantification of the phenotype in G. As individual vesicles could not be counted, quantification was done as the percentage of transfected cells showing redistributed GFPLC3. At least 10 different fields were counted (about 400 cells). The experiment was repeated three times. Data are expressed as means ±s.d. of the triplicates.Source data for this figure is available on the online supplementary information page.
TMEM59 defines a novel ATG16L1‐binding motif that promotes local activation of LC3 (2013)
Figure 3

A minimal 19-amino-acid subdomain between amino acids 263281 holds the autophagic activity of TMEM59. (A, B) Ability of serial C-terminal deletions of TMEM59 to activate LC3. 293 cells were transfected with the indicated TMEM59 C-terminal deletions, HALC3A (A) or HALC3B (B) and GST, and lysed for western blotting against the indicated molecules. (C) Amino acids 263281 retain the full potential of TMEM59 ID to promote HALC3 conversion. 293 cells were transfected with the shown CD16:7 chimeras, HALC3A or HALC3B (as indicated) and GST, and subjected to anti-CD16 aggregation before lysing them for western blotting. The lower panels show control WBs (unaggregated samples). (D) Amino acids 263281 suffice for GFPLC3 activation and retain the full potential of TMEM59 ID to promote GFPLC3 activation. 293 cells were transfected with the indicated CD16:7 chimeras and GFPLC3A or GFPLC3B (as indicated), and subjected to anti-CD16 aggregation before fixing them for microscopy. The graph shows percentages of transfected cells exhibiting redistributed GFPLC3. Quantification and data expression were done as in Figure 2H. (E) Amino acids 282323 of TMEM59 lack LC3 activation potential. 293 cells were transfected with the shown chimeras, HALC3A or HALC3B and GST, and processed for western blotting against the indicated molecules. Control WBs of unaggregated samples are shown in the lower panels. (F) Surface expression levels of CD16:7 chimeras. Procedures and data expression were as in Figure 2E. The figure shows that the functional differences observed between CD16:7 chimeras (CE) are not caused by differential surface expression. (G) Scheme of TMEM59 showing amino-acid positions and the minimal active subdomain (amino acids 263281). ED, extracellular domain; TM, transmembrane domain.Source data for this figure is available on the online supplementary information page.
TMEM59 defines a novel ATG16L1‐binding motif that promotes local activation of LC3 (2013)
Figure 4

The minimal active subdomain of TMEM59 promotes LC3 labelling and lysosomal degradation of its own vesicular compartment. (A) Endocytic vesicles containing aggregated CD16:7263281 become labelled with GFPLC3. JAR cells were transfected with vectors expressing the indicated CD16:7 chimeras and GFPLC3A, and subjected to anti-CD16 aggregation for 8 h before staining for the endocytosed chimera (red). Representative confocal pictures are shown. The inset highlights a vesicle where the peripheral GFPLC3 staining is particularly distinguishable. The right panel displays control WBs showing that chimera aggregation for 8 h activates HALC3A conversion in JAR cells. (B) Colocalization of endocytosed CD16:7263281 with EEA1 and CD63. JAR cells were transfected with CD16:7263281 and subjected to aggregation before staining them for the endocytosed chimera (red) and EEA1 or CD63 (green; FITC-coupled primary antibodies), as indicated. Representative confocal pictures are shown. (C) Immunoelectron microscopy assay showing that endocytosed CD16:7263281 localizes to LC3-labelled, single-membrane vesicles. JAR cells were transfected with the CD16:7263281 chimera and a construct expressing human IgG1 fused to LC3A. Cells were subjected to anti-CD16 aggregation for 8 h and processed for immunoelectron microscopy. Thick gold signal (18 nm): aggregated, endocytosed chimera; thin gold signal (12 nm): IgG1LC3. Arrows indicate single membrane (black) or IgG1LC3A (white). Scale bar: 400 nm. (D) Lysosomal inhibition increases HALC3II levels promoted by CD16:7263281. 293 cells were transfected with the indicated chimera and HALC3A, and subjected to anti-CD16 aggregation in the absence or presence of bafilomycin (200 nM, added 4 h post aggregation) before lysing them for western blotting against the indicated molecules. (E) Aggregation of CD16:7263281 promotes its own degradation. 293 cells were transfected with the indicated constructs, aggregated and lysed for western blotting. Shown are overexposed anti-CD16 WBs. (F) Degradation of CD16:7263281 is inhibited by ATG5 depletion. 293 cells were transfected with the indicated siRNAs and subsequently with the shown CD16:7 constructs, aggregated and lysed for anti-CD16 western blotting (left panel). The right panel displays control WBs showing ATG5 depletion.Source data for this figure is available on the online supplementary information page.