EMBO/soda-nel huggingface dataset:
CSF progranulin increases in the course of Alzheimer's disease and is associated with sTREM2 (2018)
Figure 4

Figure 4. CSF PGRN as a function of cognitive function

Scatter plots representing the association of CSF PGRN with different cognitive tests. Only the subjects of the Alzheimer's continuum group (n = 474) were included. In all tests studied, higher levels of CSF PGRN were associated with worse cognitive performance (namely, lower scores in ADNI-Mem, ADNI-EF and MMSE and higher scores in ADAS-Cog11, ADAS-Cog13 and CDR-SB). The analysis and the graphs are excluding PGRN outliers; including them rendered similar results (Appendix Table S6).

Each point depicts the value of CSF PGRN and the corresponding cognitive test score of a participant. The solid lines indicate the regression line and the 95% confidence interval (CI) calculated by a linear model (Model 1, unadjusted). Table 5 shows the standardized regression coefficients (β) and the P-values calculated by different models.

Abbreviations: ADAS-Cog, Alzheimer's disease Assessment Scale - cognitive subscale; ADNI-Mem: ADNI memory composite score; ADNI-EF: ADNI executive function composite score; CDR-SB: clinical dementia rating Sum of Boxes; MMSE, Mini-Mental State Examination.
CSF progranulin increases in the course of Alzheimer's disease and is associated with sTREM2 (2018)
Figure 5

Figure 5. CSF PGRN as a function of neuroimaging biomarkers

Scatter plots representing the association of CSF PGRN with temporo-parietal FDG-PET uptake (A) and total hippocampal volume (B) within the subjects of the Alzheimer's continuum group (n = 474).

Each point depicts the value of CSF PGRN and the corresponding neuroimaging biomarker of a participant. The solid lines indicate the regression line and the 95% confidence interval (CI). The regression coefficients (β) and the P-values the calculated by a linear model adjusted by age, gender, APOE ε4 and education.

Abbreviations: FDG-PET: Fludeoxyglucose positron emission tomography; SUVR: standardized uptake value ratio.
CSF progranulin increases in the course of Alzheimer's disease and is associated with sTREM2 (2018)
Figure 6

Figure 6. Association of CSF PGRN with AD core CSF biomarkers in ADAD (DIAN)

Scatter plots representing the associations of CSF PGRN with CSF sTREM2 and each of the AD CSF core biomarkers (T-tau, P-tau181P and Aβ1−42) in non-carriers (NC, blue; A, C, E and G) and in mutation carriers (MC, red; B, D, F and H). Each point depicts the value of CSF PGRN and the corresponding biomarker of a subject and the solid lines indicate the regression line and the 95% confidence interval (CI) for each of the groups. The standardized regression coefficients (β) and the P-values are shown and were computed using a linear model adjusting for age, gender and APOE ε4. The sample contained some outliers (defined as 3 SDs below or above the group mean) of the CSF core markers of AD. The results shown in the Table are excluding these outliers. We also performed the analysis including these outliers which yielded similar results (Appendix Table S8).

Abbreviations: Aβ1-42: amyloid-β 42; T-tau: total tau; P-tau: tau phosphorylated at Threonine 181.
4-aminopyridine promotes functional recovery and remyelination in acute peripheral nerve injury (2016)
Figure 4

Figure 4. Long-term/local 4-AP treatment promoted remyelination and increased the number of myelinated axons.

(A, B) Sustained local 4-AP administration was associated with increased myelin area and close-to-normal Garea-ratio compared to untreated group, as determined by analysis of 40 randomly chosen myelinated axons from sections of 4 nerves for each group. (**: p<0.0001; ANOVA, with post-hoc comparisons using two-tailed unpaired t-test). 4-AP treated mice had a greater proportion of axons for which the associated myelin area was greater than the mean value for uninjured mice (shown in the green line), with inset figure displaying all axons with values above this mean. (C, D) 4-AP treated nerves also showed increases in the levels of P0 protein as detected by western blot analysis (**:p<0.01; ANOVA, with post-hoc comparisons using two-tailed unpaired t-test). (E) Increases in P0 protein over time also were observed by immunofluorescence analysis at different time points, with P0 protein expression increasing to a greater extent in nerves of mice treated with 4-AP. Scale bar = 200 m. (F, G) 4-AP treatment increased the number of myelinated axons (**: p=0.002, n=4; ANOVA, with post-hoc comparisons using two-tailed unpaired t-test). Even though the 4-AP treated group also exhibited a greater number of total axons, this difference was not statistically significant. All myelinated and total axons were counted in 5 randomly chosen grids from each of 4 nerves for each group. This experiment represents a single group of mice of one of three replicates on NCV recovery.
Lipin1 deficiency causes sarcoplasmic reticulum stress and chaperone-responsive myopathy (2018)
Figure 1

Fig.1 HSA-Cre mice develop myopathy

(A) Quantitative RT-PCR shows that Lpin1 expression is depleted in the skeletal muscles Gastrocnemius (GC) and Tibialis Anterior (TA), but not in White Adipose Tissue (WAT) from 3-month-old HSACre/+/Lpin1fEx2-3/fEx2-3 mice (HSA-Cre) compared to control mice. Expression levels are corrected for expression of the control gene Pinin and presented in the graph as fold change relative to control mice. Data are mean ± SEM. (n = 5; **P<0.01).

(B) PAP1 and PAP2 activities were respectively substantially decreased and increased in TA, of HSA-Cre mice as compared to control mice, respectively (n = 6; *P<0.05).

(C) Measurements of muscle/body weight ratio of GC and TA of 3-month-old HSA-Cre and control mice. Data are mean ± SEM. (n=6; *P<0.05).

(D) Hematoxylin and Eosin (HE) staining of representative transverse sections of GC muscles from 3-month-old HSA-Cre and control mice. The black arrows indicate fibers with centrally located nuclei and yellow arrows indicate mononuclear cell infiltration. Scale bars: 40 μm

(E) Relative force measurements after normalization of absolute tetanic force to GC muscle weight from 3-month-old HSA-Cre and control male mice. Data are mean ± SEM. (n=3; **P<0.01).
Lipin1 deficiency causes sarcoplasmic reticulum stress and chaperone-responsive myopathy (2018)
Figure 3

Fig.3 Dysregulation of lipid metabolism in HSA-Cre mice muscles

(A) HE, Oil Red O (ORO), Cytochrome c Oxidase (COX) and Succinate Deshydrogenase (SDH) staining of representative frozen sections of TA muscle from 3-month-old HSA-Cre and control mice. Scale bars: 40 μm.

(B) Quantification of triacylglycerol (TAG) levels in GC muscles from 3-month-old HSA-Cre and and control mice. Data are mean ± SEM (n = 5; *P<0.05).

(C) Content of TAG, (D) Cholesterol, (E) Cholesteryl ester (CE), (F) DAG was assessed by MS in HSA-Cre and control GC muscles. Data are mean ± SEM (n=3; *P<0.05, **P<0.01).
Lipin1 deficiency causes sarcoplasmic reticulum stress and chaperone-responsive myopathy (2018)
Figure 4

Fig.4 Lipogenic gene expression program in HSA-Cre mice muscles

(A) Quantitative RT-PCR detection of the level of expression of Fatty acid synthase (Fasn), ATP citrate lyase (Acly), Thyroid hormone-inducible hepatic protein 1 (Thrsp1), Angiopoietin-like protein 4/ fast-induced adipose factor (ANGPTL4/Fiaf), Insulin-induced gene 1 (Insig1) and Peroxisome proliferator-activated receptor gamma (PPARγ) in TA from 3-month-old HSA-Cre and control mice. Expression levels are corrected for expression of the control gene Pinin and presented in the graph as fold change relative to control mice. Data are mean ± SEM (n = 5; *P<0.05, **P<0.01).

(B) Immunoblot analysis of Fasn in TA muscle of 3-month-old HSA-Cre and control mice.

(C) Quantitative RT-PCR measurement of Carnitine palmitoyltransferase 1B (Cpt1b) and Medium-chain acyl-CoA dehydrogenase (Mcad) in TA from 3-month-old HSA-Cre and control mice. Data are mean ± SEM (n = 5; ***P<0.001).

(D) Immunoblot analysis of full length and N-terminal SREBP1c and SREBP2 protein levels in GC muscle of 4-month-old HSA-Cre and control mice (n=7-4). The ratio of the densitometric assay of the N-term and Full length forms is presented. Data are mean SEM (****P<0.0001).
Lipin1 deficiency causes sarcoplasmic reticulum stress and chaperone-responsive myopathy (2018)
Figure 6

Fig.6 Lipin1 deficiency triggers an ER stress in muscles and leads to Unfolded Protein Response activation

(A) UPR activation was analysed by immunoblotting with indicated antibodies in GC muscles of 4-month-old HSA-Cre and control mice (n=7-4). Quantification by densitometric analyses of Fgf21, Bip, sXbp1 Atf6 cleaved form, p-Eif2a and Chop protein levels is presented as a graph. Data are normalized to Gapdh and expressed as a fold change relative to the control mice. Data are mean ± SEM (n=7-4; *P<0.05, **P<0.01, ****P<0.0001).

(B) Quantitative RT-PCR measurement of Fgf21, Gdf15, Bip/Grp78, hsp90b1, and Edem1 in GC muscles from 4-month-old HSA-Cre and control mice. Data are mean ± SEM (n = 7-4; *P<0.05, **P<0.01).

(C) EM analysis of EDL fibers of HSA-Cre mice revealed the presence of areas in which the non-junctional SR is more vesiculated and fragmented compared to control (black arrows). Remodeling of the SR is also accompanied by modification in the positioning of mitochondria (white arrows). A representative Calcium Release Unit (CRU) is indicated (empty arrow). Scale bars: 0.50 μm.
Lipin1 deficiency causes sarcoplasmic reticulum stress and chaperone-responsive myopathy (2018)
Figure 7

Fig.7 mtDNA levels and mitochondrial morphology are impaired in HSA-Cre muscles

(A) The relative mtDNA copy number was determined by the ratio of mitochondrial DNA-encoded Cytochrome B (CytB) and nuclear-encoded NADH dehydrogenase (ubiquinone) flavoprotein 1 (Ndufv1) genes in GC and TA from HSA-Cre and control mice. Data are mean ± SEM (n=4; *P<0.05).

(B) Mitochondria-encoded gene and Nuclear-encoded gene expression levels by quantitative RT-PCR in GC muscles from 4-month-old HSA-Cre and control mice. Data are mean ± SEM (n = 7-4; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001).

(C) Electron micrographs of HSA-Cre EDL muscles of 3-month-old mice showed that mitochondria are almost exclusively positioned at the two sides of Z lines in EDL fibers from control mice (black arrows). On the other hand, in Lipin1 deficient EDL fibers mitochondria are more often clustered and display electron-dense contact sites (white arrows). Scale bars: 0.20 μm.

(D) Ultrastructural analysis of HSA-Cre EDL muscles revealed the presence of numerous autophagic vacuoles of different morphologies (autophagosomes and lysosomes, black arrows; multilamellar bodies, white arrow) containing amorphous material and possibly degraded mitochondria (M). Scale bars: 0.50 μm
Lipin1 deficiency causes sarcoplasmic reticulum stress and chaperone-responsive myopathy (2018)
Figure 8

Fig.8 TUDCA treatment rescues Lipin1 deficient myopathy

(A) Representative immunoblots from protein extract of GC muscles of 6-month-old control, HSA-Cre untreated and HSA-Cre TUDCA-treated mice, with the indicated antibodies.

(B) Quantitative RT-PCR measurement of Fgf21 and GDF15 expression in GC muscle of 6-month-old control, HSA-Cre untreated and HSA-Cre TUDCA-treated mice. Data are mean ± SEM (n=4, *P<0.05, ** P<0.01).

(C) Histological analysis of TA muscles of 6-month-old control, HSA-Cre untreated and HSA-Cre TUDCA-treated mice by Oil Red O staining to detect the neutral lipids. Scale bars: 40 μm.

(D) TAG level measurement in GC muscles of 6-month-old control, HSA-Cre untreated and HSA-Cre TUDCA-treated mice. Data are mean ± SEM (n=4; *P<0.05, ** P<0.01).

(E) In vivo force measurements performed on GC muscles of 6-month-old control, HSA-Cre untreated and HSA-Cre TUDCA-treated mice. Data are mean ± SEM (n= 4; *P<0.05).

(F) Representative staining of IgG positive fibers on frozen sections of TA of 6-month-old control, HSA-Cre untreated and HSA-Cre TUDCA-treated mice. The sections were stained with a secondary fluorescent anti-mouse IgG antibody. Quantification of the number of necrotic fibers over the total number of fibers. Data are mean ± SEM (n=4; ** P<0.01). Scale bars: 40 μm.
Lipin1 deficiency causes sarcoplasmic reticulum stress and chaperone-responsive myopathy (2018)
Figure 9

Fig.9 Bezafibrate treatment rescues Lipin1 deficient myopathy

(A) Representative immunoblots from protein extract of GC muscles of 4-month-old control, HSA-Cre untreated and HSA-Cre Bezafibrate-treated mice, with the indicated antibodies.

(B) Quantitative RT-PCR measurement of Fgf21 and GDF15 expression in GC muscle of 4-month-old control, HSA-Cre untreated and HSA-Cre Bezafibrate-treated mice. Data are mean ± SEM (n=4, *P<0.05, ** P<0.01, *** P<0.001).

(C) Histological analysis of TA muscles of 4-month-old control, HSA-Cre untreated and HSA-Cre Bezafibrate-treated mice by Oil Red O staining to detect the neutral lipids. Scale bars: 40 μm.

(D) TAG level measurement in GC muscles of 4-month-old control, HSA-Cre untreated and HSA-Cre Bezafibrate-treated mice. Data are mean ± SEM (n=4; *P<0.05).

(E) In vivo force measurements performed on GC muscles of 4-month-old control, HSA-Cre untreated and HSA-Cre Bezafibrate-treated mice. Data are mean ± SEM (n= 4; *P<0.05).

(F) Representative staining of IgG positive fibers on frozen sections of TA of 4-month-old control, HSA-Cre untreated and HSA-Cre Bezafibrate-treated mice. The sections were stained with a secondary fluorescent anti-mouse IgG antibody. Quantification of the number of necrotic fibers over the total number of fibers. Data are mean ± SEM (n=4; *** P<0.001). Scale bars: 40 μm.

Extended Views

Fig. EV1- Fiber damage of Lipin1 deficient muscles
Golgi membrane-associated degradation pathway in yeast and mammals (2016)
Figure 1

Figure 1. Induction of proteolysis in atg5Δ yeast cells by AmphoB

(A–E) GFP–pho8Δ60-expressing wild-type (WT), atg5Δ, atg7Δ, and atg5Δpep4Δ cells were starved (A) or treated with AmphoB (B–E) and subjected to western blotting. Cells were treated with 2.5 µg/ml AmphoB for the indicated time (B) or with the indicated doses for 24 hr (C-E). Generation of free GFP was a marker of proteolysis. (F) GFP–pho8Δ60- and Pep4–mRFP-expressing atg5Δ cells were cultured with (AmphoB) or without (NT) AmphoB (2.5 µg/ml, 24 hr), and localization of GFP–pho8Δ60 and Pep4–mRFP was observed by confocal microscopy. Arrowheads indicate the cells in which GFP–pho8Δ60 was delivered to the vacuoles. Scale bars = 2 µm. (G, H) Accumulation of autophagic body (AB)-like structures by AmphoB in atg5Δpep4Δ cells. pep4Δ and atg5Δpep4Δ cells were starved for 3 hr or treated with AmphoB (2.5 µg/ml, 24 hr). Scale bars = 2 µm. Cells containing AB-like structures were counted under phase-contrast microscopy. Representative images (G) and the percentage of cells with AB-like structures (H) are shown. *p < 0.01 vs the value of pep4Δ cells. (I–K) Induction of AB-like structures in atg5Δpep4Δ cells treated with AmphoB (2.5 µg/ml, 24 hr). Representative images of quick-frozen replicas (I) and thin section of the frozen-substituted material (J) are shown. (I) AB-like structures accumulated in the vacuole. Scale bar = 0.5 µm. (J) AB-like structures containing lipid particles and mitochondria accumulated in the cross-section of the vacuole. An autophagosome (AP)-like double-membrane compartment containing a ribosome and Golgi granule is present in the cytosol. Scale bar = 0.5 µm. A magnified photo is available in Appendix Fig. S4B. (K) The cells containing AB-like structures were counted under electron microscopy [no treatment (NT): n = 20, AmphoB: n = 192]. * p < 0.01 vs the value of NT.
Golgi membrane-associated degradation pathway in yeast and mammals (2016)
Figure 2

Figure 2. Golgi involvement in AmphoB-induced proteolysis

(A–D) Generation of Golgi stacks by AmphoB. Stacked Golgi cisternae are observed in quick-frozen replicas (A–C) and substituted thin section (D) of atg5Δpep4Δ cells treated with AmphoB (2.5 µg/ml) for the indicated time. (A) Scale bar = 1 µm. (B) The magnified image shows the area indicated by the square in (A). Scale bar = 0.5 µm. (C, D) A representative Golgi stack with four cisternae is shown. Scale bar = 0.5 µm. (E-G) Generation of AP-like double-membrane compartment from the stacked Golgi. A quick-frozen replica (E) and a substituted thin section (F, G) are shown. Scale bars = 0.5 µm. The distal stacks are extended, curved, and form AP-like structure (E, F). Mitochondria are encircled by the Golgi membrane (F). (G) The AP-like structure was separated away from Golgi stacks. (H, I) The scene that AP-like structure was fused with vacuole. A magnified image of (H) is shown in (I). In atg5Δpep4Δ cells treated with AmphoB (2.5 µg/ml, 24 hr), outer membrane of AP-like structure (red arrows) was fused with vacuolar membrane (blue arrows). Inner membranes of AP-like structure (yellow arrowheads) were entered into the vacuoles to become AB-like structure. Green arrows indicate the membrane fusion site. Scale bars = 0.5 µm (H) and 0.2 µm (I). (J) The number of elongated single cisternae (green bars), Golgi stacks (red bars), and AB-like structures (blue bars) was counted over time using electron microscopy images. Error bars indicate s.e.m. (3 hr: n = 21, 6 hr: n = 20, 12 hr: n = 193, 24 hr: n = 192). (K) Translocation of the trans-Golgi protein to the vacuolar membrane induced by AmphoB treatment. GFP–Sft2-expressing atg5Δ cells (trans-Golgi marker protein) were incubated with or without AmphoB (2.5 µg/ml, 24 hr), and localization of GFP–Sft2 was assessed by confocal microscopy. Arrowhead indicates the GFP signal on the vacuolar membrane. Scale bars = 2 µm. (L–O) Grh1 and Gvp36 involvement in AmphoB-induced autophagy-like proteolysis. (L, M) Representative electron microscopy images of grh1Δatg5Δpep4Δ (L) and gvp36Δatg5Δpep4Δ cells (M) treated with AmphoB (2.5 µg/ml, 24 hr). AP-like and AB-like structures were absent. (L) A unilamellar Golgi cisterna was observed in the cytoplasm (dotted square). Scale bar = 1 µm. (M) Formation of a Golgi stack, but not Golgi curvature, was observed (dotted square). Bar = 0.5 µm. (N) Indicated cells were treated with AmphoB for 24 hr and the number of cells containing AB-like structures was counted by phase-contrast microscopy. *p < 0.01 vs the value of atg5Δpep4Δ cells. (O) GFP–pho8Δ60-expressing atg5Δ, grh1Δatg5Δ and gvp36Δatg5Δ cells were cultured with or without AmphoB (2.5 µg/ml, 24 hr) and subjected to western blotting for GFP. An unidentified band at 32kD (asterisk) is non-specific band because it was present in Pep4-lacking grh1Δatg5Δ cells.
Golgi membrane-associated degradation pathway in yeast and mammals (2016)
Figure 3

Figure 3. Reduction in Golgi PI(4)P levels is required for GOMED

(A) Localization of cellular PI(4)P. GFP–2xPHOsh2-expressing atg5Δ, sac1-23/atg5Δ, and pik1-83/atg5Δ cells were incubated with or without AmphoB (2.5 µg/ml) for 6 hr at 37 °C, and their localization was observed by confocal microscopy. Scale bars = 2 µm. (B) The same experiments were performed using GFP–GOLPH3 (a Golgi PI(4)P-monitoring protein) instead of GFP–2xPHOsh2. Scale bars = 2 µm. (C, D) Reduction of AmphoB-induced GOMED by the deletion of Golgi PI(4) phosphatases. (C) Indicated cells expressing GFP–pho8Δ60 were cultured with or without AmphoB (2.5 µg/ml, 24 hr) and subjected to western blotting for GFP. (D) The number of AP-like and AB-like structures was counted using EM images (Fig. EV2B, C). (E−H) Induction of GOMED by the deletion of Golgi PI(4) kinase. (E) Indicated cells were incubated at 37 °C (temperature shift) for 6 hr, and cells containing AB-like structures were counted by phase-contrast microscopy. *p < 0.01 vs the value of atg5Δpep4Δ cells. (F) Representative electron microscopy image of pik1-83/atg5Δpep4Δ cells at 3 hr after temperature shift. AP-like structure (arrow) and AB-like structures were generated. Scale bar = 0.2 µm. (G) The number of AP-like and AB-like structures was counted using EM images. Error bars indicate s.e.m. (atg5Δpep4Δ cells at 37 °C: n = 54 and pik1-83/atg5Δpep4Δ cells at 37 °C: n = 63). *p < 0.01 vs the value of atg5Δpep4Δ cells. (H) Sec7–HA-expressing pik1-83/atg5Δ cells were incubated at 37 °C (temperature shift) for 3 hr, and freeze replica immunolabelling was performed as described in Methods. Sec7–HA-positive signals (labelled with 15 nm gold particle) were observed on the surface membrane of AP-like structure and Golgi membrane. Scale bar = 0.1 µm. (I−L) Genetic (I, J) and pharmacological (K, L) alterations in anterograde trafficking from the Golgi are required for GOMED. (I) Indicated cells were incubated at 37 °C (temperature switch) for 3 hr, and the cells containing AB-like structures were counted by phase-contrast microscopy. *p < 0.01 vs the value of atg5Δpep4Δ cells. (J) Indicated cells were also observed using EM. Arrow indicates AP-like structure. Scale bar = 0.5 µm. (K, L) atg5Δpep4Δ cells were treated with (CBM) or without (NT) CBM (1 mM, 3 hr), and cells containing AB-like structures were counted by phase-contrast microscopy (K). * p < 0.01 vs the value of NT. Cells were also observed using EM (L). AB-like structures were observed. Scale bar = 0.5 µm. (M−O) Disturbance of anterograde trafficking from the Golgi to the PM in the cells with GOMED. Indicated cells expressing Hsp150–HA(3×) were treated as follows: NT, no treatment; starve, starvation for 6 hr; AmphoB, 2.5 µg/ml for 24 hr (M); CBM, 1 mM for 24 hr (N); temperature shift, from 25 °C to 37 °C for 3 hr (O). The cells were then lysed and examined for Hsp150–HA(3×) expression by western blotting. Accumulation of O-glycosylated Hsp150–HA(3×) indicated the trafficking failure from the Golgi to the PM.
Golgi membrane-associated degradation pathway in yeast and mammals (2016)
Figure 5

Figure 5. Induction of GOMED in Atg5-deficient MIN6 cells

(A) Suppression of insulin secretion in Atg5 knockout (KO) MIN6 cells after glucose deprivation. Atg5 KO MIN6 cells were incubated with normal culture medium (NT) or glucose-depleted medium for 1 hr. The amount of secreted insulin is expressed as the percentage of that of the untreated control (NT) (mean ± s.e.m, n = 3). *p < 0.01 vs the value of NT. (B, C) EM analysis revealed the induction of GOMED structures. Atg5 KO MIN6 cells were subjected to glucose deprivation for 1 hr. Representative images are shown. The white arrow and black arrows indicate the AP-like structure and insulin vesicles, respectively. Scale bars = 0.2 µm. (D) The number of AP-like and AL-like structures was calculated from the EM images of cells left untreated (NT) or subjected to glucose deprivation. Error bars indicate s.e.m. (NT: n = 17, glucose deprivation: n = 84). *p < 0.01 and #p < 0.05 vs the value of NT. (E) Involvement of Golgi membrane in the generation of GOMED. Lamp1–GFP/mCherry–Syntaxin 6-expressing Atg5 KO MIN6 cells were subjected to glucose deprivation, and analysed by TEM and confocal microscopy. CLEM analysis revealed that AL-like structures (arrows) merged with Lamp1–GFP fluorescence and Syntaxin 6 fluorescence. Scale bars = 5 µm. A magnified image of the dashed square is shown in the inset. Scale bars = 2 µm. (F) GOMED-mediated degradation of (pro)insulin by glucose deprivation of Atg5 KO MIN6 cells. Atg5 KO MIN6 cells were incubated in glucose-depleted medium in the absence or presence of bafilomycin A1 (10 nM) for the indicated times. The cell lysates were then subjected to immunoblot analysis using an anti-(pro)insulin antibody. (G) Immunofluorescence analysis of (pro)insulin and Lamp2. Atg5 KO MIN6 cells were left untreated (NT) or subjected to glucose deprivation for 1 hr in the presence of E64d (25 µM) or bafilomycin A1 (10 nM), and were stained with anti-(pro)insulin and anti-Lamp2 (lysosomal marker) antibodies and observed by fluorescence microscopy. Scale bar = 1 µm. Arrowheads indicate the colocalization of (pro)insulin with GOMED structures.