EMBO/soda-nel huggingface dataset:
Mutations in TIMM50 compromise cell survival in OxPhos-dependent metabolic conditions (2018)
Figure 7

Figure 7 - Characterization of RNASEH1 and ISCU mutant fibroblasts

A Western-blot of whole cell extracts for TIMM50 and GAPDH in control and TIMM50, RNASEH1 and ISCU mutant fibroblasts grown in either glucose or galactose containing medium. Other components of the protein import machinery are shown in Appendix Fig S8A and their quantification in Appendix Fig S8B.

B Mitochondrial membrane potential in control and TIMM50, RNASEH1 and ISCU mutant fibroblasts grown in glucose or galactose using JC-1 staining in untreated or FCCP treated conditions. Data are shown as mean ± SD, n = 4 biological replicates; ∗p < 0.05, ∗∗p < 0.01, ∗*∗p < 0.001. Exact P-values are reported in Table EV1.

C Western blot for representatives of all five respiratory complexes in control and TIMM50, RNASEH1 and ISCU mutant fibroblasts grown in glucose or galactose. GAPDH is from the same blot as panel A. Quantification based on two biological replicates for RNASEH1 and ISCU mutant fibroblasts are shown in Appendix Fig S9. Significant changes in protein levels in RNASEH1 and ISCU mutant fibroblasts compared to controls have been boxed in red.

D Oxygen consumption measurements in control and TIMM50, RNASEH1 and ISCU mutant fibroblasts grown in glucose or galactose. Values of basal and maximal respiration along with ATP-production dependent, proton leak respiration and spare capacity are shown. Data are shown as mean ± SD, n = 4 biological replicates; ∗p < 0.05, ∗∗p < 0.01, ∗*∗p < 0.001. Exact P-values are reported in Table EV1.

E Steady state levels of ROS related proteins SOD2 and ACO2 and mitophagy related proteins VDAC1, p62 and LC3 in control and TIMM50, RNASEH1 and ISCU mutant fibroblasts growing in either glucose or galactose were assessed by Western blot using GAPDH as internal control. GAPDH is from the same blot as panel A. Quantification are shown in Appendix Fig S10.

F Quantification of the number of late apoptotic cells in control and TIMM50, RNASEH1 and ISCU mutant fibroblasts grown in glucose or galactose. Data are shown as mean ± SD, n = 4 or more biological replicates; ∗*∗p < 0.001. Exact P-values are reported in Table EV1.
Autophagy modulates miRNA‐mediated gene silencing and selectively degrades AIN‐1/GW182 in C. elegans (2013)
Figure 1

Loss of autophagy activity suppresses the retarded heterochronic defects in miRISC mutants. (A) Schematic illustration of seam cells in a wild‐type L1 larva. (B) Division pattern of seam cells at the postembryonic stage in hermaphrodites. Seam cells V1–V4 and V6 divide symmetrically at the L2 stage (highlighted in red), thus doubling in number. (C–F) At the young adult stage, wild‐type and atg‐2 mutant animals have 16 seam cells on each side, visualized by the seam cell‐specific marker scm::gfp. Cuticular alae (arrow) run continuously from anterior to posterior. The number of seam cells is shown in brackets. (G,H) dcr‐1 young adults have an increased number of seam cells (shown in brackets) and gaps in the alae (between arrows). (I,J) atg‐2 mutations suppress the retarded heterochronic defect in dcr‐1 mutants. Complete alae (arrow) are formed in dcr‐1; atg‐2 double mutants. (K–M) Confocal images showing expression of hbl‐1::gfp::hbl‐1 reporter in VNC in wild type (K), atg‐2 (L) and epg‐6 (M) L3 larvae. (N) Distribution of relative fluorescence intensity of HBL‐1::GFP in 50 unit areas in VNC in the indicated strains. (O–Q) Confocal images showing the expression of the col‐10::gfp::lin‐41(3′UTR) reporter in wild type (O), atg‐2 (P) and atg‐3 (Q) late L4 larvae. (R) Distribution of relative fluorescence intensity of col‐10::gfp::lin‐41(3′UTR) in 50 unit areas of hypodermal cells in the indicated strains. (S,T) Distribution of relative fluorescence intensity of hbl‐1::gfp in VNC and col‐10::gfp::lin‐41(3′UTR) in hypodermal cells in 50 unit areas in wild type, let‐363(RNAi) or starvation‐treated animals. Black lines in (N,R–T) represent the average fluorescence intensity of the reporter in each strain. P values are determined by Student's two‐tailed unpaired t‐test in (N,R–T). (C,E,G,I) Scale bar, 20 μm; (D,F,H,J–M,O–Q) scale bar, 10  μm. GFP, green fluorescent protein; miRISC, miRNA‐induced silencing complex; RNAi, RNA‐mediated interference; UTR, untranslated region; VNC, ventral nerve cord.
Autophagy modulates miRNA‐mediated gene silencing and selectively degrades AIN‐1/GW182 in C. elegans (2013)
Figure 2

Autophagy regulates developmental processes specified by the let‐7 miRNA family and lsy‐6 miRNA. (A–C) Wild‐type and atg‐2 hermaphrodites have a single vulva (arrow). (B) let‐60(n1046gf) mutants show a Muv phenotype. Black arrows indicate pseudovulvae. (D) Loss of autophagy activity suppresses the Muv phenotype in let‐60(n1046) mutants. The number of adult animals examined was: wild type (n=450), let‐60 (n=406), epg‐6; let‐60 (n=444), let‐60; atg‐2 (n=803), epg‐5; let‐60 (n=672), epg‐5; let‐60; ain‐1(ku322) (n=635). P values are determined by a two‐tailed χ2 test. (E,F) lsy‐6 mutants show defects in ASEL fate specification and do not express the ASEL‐specific reporter lim‐6pro::GFP. (G) Expression of lim‐6pro::GFP in various strains. aCompared with lsy‐6(ot150).bCompared with lsy‐6(ot150); ain‐1(ku322). P values are determined by a two‐tailed χ2 test. (A–C) Scale bar, 20 μm; (E,F) scale bar, 10 μm. ASEL, ASE left; ASER, ASE right; GFP, green fluorescent protein; miRNA, microRNA; Muv, multivulva.
Autophagy modulates miRNA‐mediated gene silencing and selectively degrades AIN‐1/GW182 in C. elegans (2013)
Figure 3

AIN‐1 is selectively degraded by autophagy. (A,B) AIN‐1::GFP is weakly and diffusely expressed in the cytoplasm during embryogenesis in wild‐type animals. (A) Nomarski image of the embryo shown in (B). Scale bar, 10 μm for whole embryo. C. elegans embryos remain the same size during embryogenesis and loss of autophagy activity has no effect on the embryo size. Thus, scale bars for embryos are only shown once in each figure. (C) AIN‐1::GFP forms a large number of aggregates in epg‐6 mutant embryos. (A–C) are confocal images. (D) Western blotting shows that endogenous AIN‐1 protein levels are dramatically elevated in autophagy mutant embryos compared with wild type. (E) No AIN‐1::GFP aggregates are formed in sqst‐1 mutant embryos. (F) AIN‐1::GFP accumulates into numerous aggregates in epg‐7 mutant embryos. (G,H) Number of AIN‐1::GFP (G) and ALG‐2::GFP (H) aggregates per focal plane in the indicated strains. Error bars indicate the s.d. of four examined embryos. (I,J) ALG‐2::GFP is diffusely localized in the cytoplasm in wild‐type embryos. (I) Nomarski image of the embryo shown in (J). (K) ALG‐2::GFP forms a few aggregates in epg‐6 mutant embryos. (L) ALG‐2::GFP forms a large number of aggregates in epg‐6 mutant embryos under stress conditions. GFP, green fluorescent protein; s.d., standard deviation.
Autophagy modulates miRNA‐mediated gene silencing and selectively degrades AIN‐1/GW182 in C. elegans (2013)
Figure 4

AIN‐1 colocalizes with SQST‐1 aggregates in autophagy mutants and directly interacts with LGG‐1. (A–D) Both AIN‐1::GFP and SQST‐1, detected by anti‐SQST‐1 antibody, are very weakly expressed and diffusely localized in wild‐type embryos. (E–H) AIN‐1::GFP aggregates colocalize with SQST‐1 aggregates in atg‐3 mutant embryos. Scale bars, 10 μm for insets that show a magnified view. (I–L) AIN‐1::GFP aggregates are separable from PGL granules, detected by anti‐SEPA‐1, in atg‐3 mutant embryos. (M–P) Colocalization of AIN‐1::GFP aggregates with LGG‐1 puncta in epg‐6 mutant embryos. (Q) % of AIN‐1::GFP aggregates colocalized with SQST‐1 aggregates, SEPA‐1 aggregates and LGG‐1 puncta in autophagy mutants. Error bars indicate the s.d. of four examined embryos. (R) A co‐IP assay shows that AIN‐1 associates with LGG‐1 in vivo. (S) Direct interaction between AIN‐1 and LGG‐1 in an in vitro pull‐down assay. 5% of the His fusion proteins used for pull‐down are shown as input. Corresponding Ponceau‐stained blots show the amount of proteins used for pull‐down. (T) A fragment of AIN‐1 directly binds to LGG‐1 in an in vitro pull‐down assay. (U) Model for the role of autophagy in modulating the miRNA pathway. co‐IP, coimmunoprecipitation; GFP, green fluorescent protein; MBP, maltose‐binding protein; miRNA, microRNA; IP, immunoprecipitant; RISC, RNA‐induced silencing complex; s.d., standard deviation.
Beclin‐1 is required for chromosome congression and proper outer kinetochore assembly (2013)
Figure 1

Beclin‐1 is required for proper mitotic progression. (A) Western blot of Beclin‐1 and FACS profiles following siRNA treatment in HeLa cells and cells stably expressing a HA‐Beclin‐1 construct resistant to the siRNA targeting endogenous Beclin‐1. (B) FACS profiles of cells treated with indicated siRNA following nocodazole blockage and release. (C,D) Quantification of cells in mitosis (C) and quantification of each step of mitosis (D) by H3Ser10P staining for Control and Beclin‐1 siRNA‐treated cells. Error bars represent standard deviations (s.d.) (n=number of counted cells) calculated on three independent experiments. Two and three asterisks indicate significant results (P=0.0028 and P=0.0006, respectively). (E) Knockdown of Beclin‐1 increases the number of mitotic cells with misaligned chromosomes. siRNA‐treated HeLa cells were fixed in PFA, immunolabelled for α‐tubulin (green) and stained with DAPI (blue). White arrows indicate misaligned chromosomes. Quantification of cells with misaligned chromosomes is shown at the right (n=number of counted cells). Three asterisks indicate significant results (P=0.0001). Scale bar, 10 μm. DAPI, 4′,6‐diamidino‐2‐phenylindole; HA, haemagglutinin; PFA, paraformaldehyde; siRNA, small interfering RNA.
Beclin‐1 is required for chromosome congression and proper outer kinetochore assembly (2013)
Figure 2

Beclin‐1 depletion prolongs prometaphase by delaying chromosome congression. (A–H) Time‐lapse films of HeLa cells stably expressing YFP‐α‐tubulin and H2B‐Cherry and treated with the indicated siRNA. The pictures are acquired every 10 min. Time lapse starts at nuclear envelope breakdown. (A) Mitosis in control cells. (B) Western blot of whole‐cell lysates from Control, Beclin‐1 and Beclin‐1+Mad2 siRNA‐treated cells. (C–E) Three different classes of mitotic progression were observed upon Beclin‐1 depletion. Higher magnification of lagging chromatids in the anaphase of Beclin‐1‐depleted cells is shown on the right of (D). (F) The prometaphase delay is dependent on MAD2 and the spindle checkpoint. Cells treated simultaneously with siRNA targeting MAD2 and Beclin‐1 rapidly exit mitosis. (G) Average duration of mitosis in different classes of Beclin‐1‐depleted cells, compared to control and MAD2 depletions. n=number of counted cells in three independent experiments. Error bars on graphs represent s.d. (H) The frequency of the different Beclin‐1 phenotypes depended on the efficiency of Beclin‐1 knockdown. Mitotic progression in cells treated with increasing concentrations of Beclin‐1 siRNA was followed by video‐microscopy. Left panel: western blot of Beclin‐1 upon RNAi treatment; right panel: quantification of the appearance of the three different mitotic progression patterns, that is, prolonged mitosis with normal anaphase (as C), with lagging chromatids (as D) and mitotic exit without clear anaphase (as E) upon increased Beclin‐1 knockdown experiments. NEB, nuclear envelop breakdown; RNAi, RNA interference; siRNA, small interfering RNA; YFP, yellow fluorescent protein.
Beclin‐1 is required for chromosome congression and proper outer kinetochore assembly (2013)
Figure 3

Beclin‐1 knockdown affects kinetochore–microtubule attachment. (A) Inter‐kinetochore distance is reduced in unaligned chromosomes of Beclin‐1‐depleted cells. The distance between each kinetochore pair was quantified in siRNA‐treated and control cells treated with nocodazole for 3 h before fixation. Cells were stained with anti‐CREST antibody and DAPI. Quantification of the distance between kinetochore pairs (indicated by coloured bars) is reported at the bottom and done on sister kinetochores present in the same focal plane. One asterisk indicates significant result (P=0.0365). Graphs represent three independent experiments (error bars=s.d.). (B) Depletion of Beclin‐1 leads to defects in K‐MT attachment. siRNA‐treated cells were cooled on ice before fixation to reveal stable k‐fibres and then stained with anti‐α‐tubulin (red) and anti‐CREST (green) antibodies. Numbers point to magnified areas and indicate the mode of attachment of k‐fibres to kinetochores (1, bi‐oriented; 2, unattached; 3, mono‐oriented kinetochores). Scale bar, 10 μm. DAPI, 4′,6‐diamidino‐2‐phenylindole; NS, not significant; siRNA, small interfering RNA.
Beclin‐1 is required for chromosome congression and proper outer kinetochore assembly (2013)
Figure 4

Beclin‐1 depletion reduces targeting of several outer kinetochore proteins. Control and Beclin‐1‐depleted cells were treated with nocodazole before fixation, and labelled with the indicated antibody. (A) CENP‐E and CREST. Two asterisks indicate significant results, P=0.0053. (B) CENP‐F and CREST. Three asterisks indicate significant results, P=0.0006. (C) ZW10 and CREST. Three asterisks indicate significant results, P=0.0007. (D) MAD2 and CREST. Two asterisks indicate significant results, P=0.0032. (E) HEC1 and CREST. (F) KNL‐1 and CREST. On the right, magnified areas and quantification of the fluorescence intensity of the protein of interest normalized to the fluorescence intensity of CREST staining are shown. Graphs represent three independent experiments (error bars=s.d.). Scale bar, 10 μm. (G) Western blot of whole‐cell lysates from control and Beclin‐1 siRNA‐treated cells. siRNA, small interfering RNA.
Beclin‐1 is required for chromosome congression and proper outer kinetochore assembly (2013)
Figure 5

Beclin‐1 interacts directly with Zwint‐1 and localizes to the mitotic apparatus. (A) Immunoprecipitation of endogenous Zwint‐1 in HeLa cell extracts expressing HA‐Beclin‐1 or HA‐GFP. Input is 3.5% of the immunoprecipitated material. (B) GST‐Zwint‐1 or GST‐beads were used to pull down recombinant purified His6‐Beclin‐1. (Left) Coomassie‐blue‐stained gel of the purified recombinant proteins (1 μg). (Right) A western blot of GST proteins and His6‐Beclin‐1 in the input and bound fractions. His6‐Beclin‐1 in the input represents 4% of the amount used for the pull‐down. (C) Zwint‐1 levels do not change in Beclin‐1‐depleted cells. Cells were treated with nocodazole before fixation. On the right, magnified individual kinetochore pairs stained for Zwint‐1 (red) and CREST (green), and quantification of Zwint‐1 levels normalized to CREST signal from three independent experiments (error bars=s.d.). (D) Beclin‐1 is associated with both the centrosome and the mitotic spindle during mitosis. HeLa cells were permeabilized before fixation and immunostained for Beclin‐1 (green), α‐tubulin (red) and DNA (DAPI, blue). (E) Beclin‐1 is retained on the k‐fibres. HeLa cells were cooled on ice before permeabilization and fixation, and stained with anti‐α‐tubulin (red) and anti‐Beclin‐1 (green) antibodies. (F) Beclin‐1 forms discrete foci in the vicinity of the outer kinetochore in a microtubule‐dependent manner. Untreated cells (left) and nocodazole‐treated cells (right) were pre‐extracted and fixed before labelling with Beclin‐1 (green) and Zwint‐1 (red) antibodies. (G) The fluorescent‐tagged Cherry‐Beclin‐1 forms foci in the vicinity of the outer kinetochore. HeLa cells transfected with indicated constructs were pre‐extracted and fixed before labelling with Zwint‐1 (green) antibody. Magnified areas are shown on the right. (H) Beclin‐1 localization close to the kinetochore does not depend on the ZW10 protein. Cells treated with Control, Beclin‐1 and ZW10 siRNA were pre‐extracted and fixed before labelling with Beclin‐1 (green), CREST (blue) and ZW10 (red) antibodies. DNA was visualized with DAPI. (C–H) Scale bar, 10 μm. DAPI, 4′,6‐diamidino‐2‐phenylindole; DNA, deoxyribonucleic acid; GFP, green fluorescent protein; GST, glutathione S‐transferase; HA, haemagglutinin.
UV irradiation resistance‐associated gene suppresses apoptosis by interfering with BAX activation (2011)
Figure 1

Genotoxic or metabolic stress‐induced UVRAG expression in tumour cells. (A,B) Expression of UVRAG under cell stress. HL60 and HCT116 cells were treated with doxorubicin (1 μg/ml), cisplatin (10 μm) and UV irradiation (5 min after 50 mJ/cm2) for the indicated time, and then apoptosis, autophagy and fluorescence intensity of UVRAG was assayed. Scale bar, 10 μm. (C) In parallel, the total protein extracts were used for western blot analysis. (D,E) Pharmacological inhibition (for example, 3‐methyladenine (3‐MA), 10 mM) or genetic deletion (for example, ATG5−/−) of the autophagy pathway decreases the protein and mRNA expression of UVRAG and increases apoptosis at 12 h in HL60 cells treated with doxorubicin (Doxo) and UV irradiation (n=3, *P0.05). AU, arbitrary units; C‐PARP, cleaved poly‐ADP ribose polymerase; LC3, light chain 3; mRNA, messenger RNA; PI, propidium iodide; UV, ultraviolet; UVRAG, ultraviolet irradiation resistance‐associated gene.
UV irradiation resistance‐associated gene suppresses apoptosis by interfering with BAX activation (2011)
Figure 2

Inhibition of UVRAG expression increases tumour‐cell sensitivity to chemotherapy and radiation therapy in vivo and in vitro. (A–C) After transfection with UVRAG shRNA or control shRNA for 48 h, HL60 (A), HCT116 (B) and HeLa (C) cells were treated with doxorubicin (Doxo, 1 μg/ml), cisplatin (Cis, 10 μm) and UV irradiation (5 min after 50 mJ/cm2) for 12 h or starvation (HBSS) for 3 h, and then apoptosis and autophagy were assayed (n=3, *P0.05). (D) Analysis of LC3 processing by autophagy in the presence or absence of lysosomal protease inhibitors pepstatin A (10 μg/ml) and E64D (10 μg/ml) after doxorubicin (1 μg/ml) treatment for 12 h. *P0.05 compared with control shRNA group. (E) After transfection with UVRAG shRNA or control shRNA for 48 h, ATG5+/+ and ATG5−/− murine embryonic fibroblasts (MEFs) were treated with doxorubicin (1 μg/ml), with or without Z‐VAD‐FMK (20 μM) for 12 h, and then apoptosis was assayed (n=3, *P0.05). (F) Nude mice were inoculated with 4 × 106 HL60 tumour cells following transfection of control (shControl) or UVRAG (shUVRAG)‐specific shRNA and treated with doxorubicin (10 mg/kg) beginning at day 7. Tumours were measured twice weekly, and volumes were calculated for 21 days (n=10, *P0.05; shControl+Doxo compared with shUVRAG+Doxo). (G) On day 21 in the experiments described in (F), apoptosis and UVRAG expression in tumour samples were assayed by TUNEL or western blotting, respectively (n=10, *P0.05). BAX, BCL2‐associated X protein; HBSS, Hank's Buffered Salt Solution; LC3, light chain 3; shRNA, short‐hairpin RNA; TUNEL, terminal deoxytransferase uridine triphosphate nick end labelling; UV, ultraviolet; UVRAG, ultraviolet irradiation resistance associated gene.
UV irradiation resistance‐associated gene suppresses apoptosis by interfering with BAX activation (2011)
Figure 3

UVRAG inhibits BAX‐induced apoptosis. (A) Control shRNA or UVRAG shRNA cells were transfected with BAX plasmid, and apoptosis was assayed (n=3, *P0.05). (B) In parallel, the mitochondrial membrane potential, cytochrome c release from mitochondria, and caspase‐3 and ‐9 activation in HL60 cells were analysed (n=3, *P0.05). (C) After transfection with UVRAG vector or control vector for 48 h, cells were transected or treated with BAX (1 μg), BAD (1 μg), BID (1 μg), FAS antibody (10 μg/ml) or TRAIL (100 ng/ml) for 24 h, then cell apoptosis was analysed (n=3, *P0.05). (D) Knockdown of UVRAG in BAX−/− HCT116 cells restored the sensitivity to UV treatment‐induced apoptosis (n=3, *P0.05). AU, arbitrary units; BAX, BCL2‐associated X protein; shRNA, short‐hairpin RNA; UV, ultraviolet; UVRAG, ultraviolet irradiation resistance associated gene.
UV irradiation resistance‐associated gene suppresses apoptosis by interfering with BAX activation (2011)
Figure 4

UVRAG interacts with Bax. (A,B) HL60 and HCT116 cells were treated with UV irradiation for 24 h and then assayed for protein expression levels as indicated by co‐immunoprecipitation (RIPA or CHAPS buffer) or western blotting. (C) Proteins in the pellet after GST pull‐down assays in HCT116 cell lysate were analysed by SDS–PAGE and immunodetected with UVRAG or GST antibodies. (D) UVRAG C2 domain deletion terminates BAX binding. HCT116 cells were transfected with Flag‐UVRAG or its mutants (ΔC2, ΔCCD), and then assayed for protein expression levels as indicated by IP or western blot. (E) Apoptosis was assayed in indicated HCT116 cells after treatment with doxorubicin (Doxo, 1 μg/ml), UV irradiation (5 min after 50 mJ/cm2) or transfected with BAX (1 μg) for 24 h. BAX, BCL2‐associated X protein; CCD, coiled‐coil domain; CHAPS, 3‐[(3‐cholamidopropyl)‐dimethylammonio]‐1‐propane sulphonate; GST, glutathione‐S‐transferase; IB, immunoblotting; IgG, immunoglobulin G; IP, immunoprecipitation; RIPA, radioimmunoprecipitation assay; SDS–PAGE, SDS–polyacrylamide gel electrophoresis; UV, ultraviolet; UVRAG, ultraviolet irradiation resistance associated gene.
UV irradiation resistance‐associated gene suppresses apoptosis by interfering with BAX activation (2011)
Figure 5

UVRAG inhibits mitochondrial translocation of BAX. After transfection with indicated shRNA or cDNA for 48 h, HL60 cells were treated with doxorubicin (Doxo) and UV irradiation for 12 h, and then BAX in cytosol (Cyt) and mitochondria (Mit) was assayed by western blotting (A–C) and activation of BAX (D–F) was assayed by western blotting after IP using BAX monoclonal antibody (clone 6A7) *P0.05. AU, arbitrary units; BAX, BCL2‐associated X protein; CHAPS, 3‐[(3‐cholamidopropyl)‐dimethylammonio]‐1‐propane sulphonate buffer; Cyt c, cytochrome c; IP, immunoprecipitation; shRNA, short‐hairpin RNA; UV, ultraviolet; UVRAG, ultraviolet irradiation resistance associated gene.
Loss of autophagy in hypothalamic POMC neurons impairs lipolysis (2011)
Figure 1

POMC neuron‐selective loss of autophagy. (A) Immunofluorescence for POMC (green) and Cre (red; n=4), (B) POMC (green) and Atg7 (red; n=4) and (C) POMC (green) and p62 (red; n=6) in MBH sections from Con and Atg7F/F‐POMC‐Cre (KO) mice. (D) TUNEL‐positivity (green; n=4) and (E) number of POMC‐positive neurons (red) in MBH sections from Con and KO mice (n=6). Values are mean±s.e.m. P values are as compared with diet‐ and age‐matched controls. *P0.05, ***P0.001. Nuclei are blue (DAPI). Scale inset: 10 μm. White arrows indicate Atg7, Cre, p62 or POMC signal. Yellow arrows indicate merged signal. Con, control; DAPI, 4′‐6‐diamidino‐2‐phenylindole; KO, knockout; MBH, mediobasal hypothalamic; NS, not statistically significant; POMC, proopiomelanocortin; TUNEL, TdT‐mediated dUTP nick end labelling.
Loss of autophagy in hypothalamic POMC neurons impairs lipolysis (2011)
Figure 2

Loss of atg7 in POMC neurons promotes adiposity. (A) Body wt of 4‐mo‐ and 12‐mo‐old Con and KO mice on RD (n=9–13) or HFD (n=5–7). (B) Total body fat and (C) lean mass of 4‐mo‐old Con and KO mice on RD (n=7) and 12‐mo‐old Con and KO mice on HFD (n=6–8). (D) eWAT wt, (E) fat pads and (F) liver wt of 4‐mo‐old Con and KO mice on RD (n=6–8). (G) Immunoblots for POMC and ACTH, and (H) NPY in MBH from RD‐fed Con and KO mice (n=3–4). (I) Immunofluorescence for POMC (green) and α‐MSH (red), and (J) α‐MSH (green) in MBH from Con and KO mice on RD (n=5). Values are mean±s.e.m. P values are as compared with diet‐ and age‐matched controls. *P0.05, **P0.01, ***P0.001. Nuclei are blue (DAPI). Scale inset: 10 μm. Yellow arrows indicate merged signal. ACTH, adrenocorticotrophic hormone; Con, control; DAPI, 4′‐6‐diamidino‐2‐phenylindole; eWAT, epididymal fat; HDF, high‐fat diet; KO, knockout; MBH, mediobasal hypothalamic; mo, month; MSH, melanocyte‐stimulating hormone; NPY, neuropeptide Y; POMC, proopiomelanocortin; RD, regular chow; wt, weight.