EMBO/soda-nel huggingface dataset:
Microglial phagocytosis of living photoreceptors contributes to inherited retinal degeneration. (2015)
Figure 6

Genetic ablation of microglia in the retina ameliorates photoreceptor degeneration in rd10 mice

Retinal microglia were depleted in rd10/CreDTA mice by the oral administration of tamoxifen (in corn oil) to activate microglia-specific Cre-mediated recombination and diphtheria toxin expression; control CreDTA littermates were administered corn oil without tamoxifen.

A–D Depletion of retinal microglia in the rd10 retina. Representative retinal section from a P28 control animal demonstrates Iba1+ microglia in the retina, including those infiltrating the ONL (A), while a tamoxifen-administered littermate (B) was substantially depleted of retinal microglia. Scale bar, 40 μm. Microglial cell counts in the entire retina (C) and in the ONL only (D) confirmed efficient depletion of infiltrating microglia following tamoxifen administration (n = 8 control and 11 depleted animals from four litters, two-sided unpaired t-test).

E–J Effect of microglial depletion on retinal degeneration at P28–29. ONL atrophy and thinning in control animals (E) was significantly more advanced relative to microglia-depleted littermates (F). Scale bar, 40 μm. Quantification of mean ONL thickness (G) and mean number of layers of ONL nuclei (H) at P28–29 demonstrate significantly greater ONL preservation in depleted retinas; the degree of ONL preservation correlated with the extent of microglia depletion (I). (J) The mean density of TUNEL+ nuclei in the ONL was not significantly decreased in depleted vs. control animals (n = 8 control and 11 depleted animals from four litters, two-sided unpaired t-test).

K–N Continuation of microglial depletion until P37–39 resulted in the persistence of morphological rescue (K–M), with a significant reduction in TUNEL+ nuclei density (N) (n = 6 control and nine depleted animals from two litters, two-sided unpaired t-test).

O–R Similar rescue effects as in (K–N) remained apparent when depletion was sustained until P50 (O–R), a time when rod degeneration in the rd10 model is relatively complete (n = 9 control and 10 depleted animals from three litters, two-sided unpaired t-test).

S, T Functional rescue of photoreceptors was evident following microglial depletion until P50 in significantly increased dark- (S) and light-adapted (T) responses in ERG testing in depleted animals (green lines) relative to control animals (black lines), in both a- and b-wave amplitudes across multiple flash intensities (n = 9 control and nine depleted animals, *P < 0.05 in one-way ANOVA with Sidak’s multiple comparison test).

Data information: Column heights (in C, D, G, H, J, K, L, N, O, P, R) and data points (in S, T) indicate mean, error bars indicate ± SEM.
Microglial phagocytosis of living photoreceptors contributes to inherited retinal degeneration. (2015)
Figure 7

Inhibition of microglial phagocytosis ameliorates photoreceptor degeneration in rd10 mice

A, B Effect of phagocytosis inhibition on retinal microglia ex vivo. Retinal explants acutely isolated from P21–23 rd10/CX3CR1GFP/+ mice were incubated in Ringer’s solution (control), or Ringer’s solution containing either the vitronectin receptor inhibitor cRGD peptide or its inactive analog cRAD (400 μM) for 1 h. GFP-labeled (green) ONL microglia in explants in cRGD transitioned from amoeboid morphologies containing multiple phagosomes to more ramified morphologies with fewer phagosomes. No morphological changes were detected in microglia incubated in cRAD (insets show morphologies at higher magnification). Scale bar, 40 μm. Quantifications of total number of phagosomes per 40× field (left) and the mean number of phagosomes per microglia (right) demonstrate significant reductions in phagocytic activity of cRGC, relative to cRAD, exposure (n = 8 imaging fields from in each condition).

C–N Structural effects of in vivo inhibition of microglial phagocytosis. P20 rd10 mice were injected intravitreally in one eye with the phagocytosis inhibitor cRGD, and in the control contralateral eye with the inactive analogue, cRAD. At P23, prominent microglia infiltration in the ONL observed in control-injected eyes (C) was decreased in the contralateral cRGD-injected eyes (D). ONL microglia in control eyes demonstrated more numerous phagosomes (inset, arrows) (E) compared with cRGD-injected eyes (F). Pairwise comparisons of control- vs. cRGD-injected eyes demonstrated that phagocytosis inhibition significantly reduced the densities of infiltrating microglia (G) and microglial phagosomes (H), and mean phagosome number per microglia (I). ONL atrophy in control eyes (J) was more advanced compared to cRGD-injected eyes (K), with significantly greater mean ONL thickness (L), and ONL nuclear layers (M) in cRGD-injected eyes. Mean density of TUNEL+ ONL nuclei was lower, with marginal significance (N) (n = 13 animals; measurements in cRGD-injected eyes normalized to contralateral control eyes, two-sided paired t-test). Scale bar, 40 μm.

O–T Functional effects of in vivo inhibition of microglial phagocytosis. Representative dark-adapted ERG recordings at P26 (following intravitreal injections at P20 and P23) in a cRGD-injected eye (red) relative to the contralateral control eye (black) (O) showing that mean a- (P) and b-wave (Q) amplitudes were significantly greater in the cRGD-injected vs. control eyes at higher flash intensities. Representative light-adapted responses (R) illustrating that while mean light-adapted a-wave amplitudes (S) were slightly but not significantly greater in cRGD-injected eyes, mean b-wave amplitudes (T) were significantly greater at higher flash intensities (n = 13 animals, two-sided paired t-test, *P < 0.05, with exact values alongside).

Data information: Column heights (in B, G, H, I, L, M, N) and data points (in P, Q, S, T) indicate mean, error bars indicate ± SEM.
Microglial phagocytosis of living photoreceptors contributes to inherited retinal degeneration. (2015)
Figure 8

Upregulation of IL-1β expression in infiltrating microglia potentiates photoreceptor apoptosis

Microglia infiltrating the rd10 retina upregulate IL-1β expression during degeneration. Iba1+ microglia (green) were immunonegative for IL-1β (red) at P18 but become prominently IL-1β immunopositive at P22 (arrows). Scale bars, 25 μm.

Depletion of infiltrating microglia in the rd10 retina decreases IL-1β levels. Cytokine levels in rd10/CreDTA mouse retinas were assayed following tamoxifen-induced microglial depletion (from P21 to P37–50, green bars; n = 9 animals) and compared with their untreated littermate controls (white bars, n = 8 animals; values normalized to control animals in the same litter). IL-1β protein levels were significantly lowered following microglial depletion, but IL-6, CCL2, or TNFα were not significantly changed.

Inhibition of microglial phagocytosis results in decreased microglial IL-1β expression. Eyes of rd10 animals were treated with intravitreal injections of cRGD at P20, and analyzed at P23 by immunohistochemistry for Iba1 (green) and IL-1β (red), and compared to uninjected eyes and with eyes injected with inactive analog, cRAD (n = 3 eyes in each category). Immunopositivity was high in infiltrating microglia (indicated by arrows) for untreated and cRAD-injected control eyes and significantly reduced in microglia in cRGD-injected eyes (upper panel), as demonstrated by the quantification of IL-1β expression (area of immunopositivity as a fraction of the ONL) (lower left panel). ONL thickness correlated negatively with the extent of IL-1β immunopositivity (lower right panel). Scale bar, 25 μm.

IL-1R signaling during rod degeneration was inhibited by intravitreal injections of recombinant IL-1 receptor antagonist, anakinra (from P20–22 to P26–27) in one eye of each rd10 animal, while the contralateral eye was injected with PBS as a control. Upper panels show representative retinal sections from control PBS-injected and IL-1RA-injected eyes from the same animal. ONL atrophy in control eyes was more advanced compared to IL-1RA-injected eyes, with greater mean ONL thickness and lower mean density of TUNEL+ ONL nuclei in IL-1RA-injected eyes (lower panels) (n = 15 animals; measurements in IL-1RA-injected eyes normalized to contralateral control eyes, paired t-test). Scale bar, 25 μm.

Data information: Column heights (in B, C, D) indicate mean, error bars indicate ± SEM.
Ketogenic HMG-CoA lyase and its product β-hydroxybutyrate promote pancreatic cancer progression (2022)
Figure 1

Figure 1: βOHB promotes PDA aggressiveness

A Tumor weight in KIC mice treated with βOHB (100mg/kg/day, i.p.) or 0.9% NaCl (i.p.) (n=13 mice for NaCl and n=15 mice for βOHB). Data are expressed as mean ± SEM. Significance was defined by Mann-Whitney test. *: p<0.05.

B Kinetic of spheroid area from PK4a cells cultured during 12 days in medium alone (untreated) or with βOHB 1 or 10mM (n=4, and 5 respectively). Data are expressed as mean ± SEM. Significance compared to untreated cells was defined by two-way ANOVA followed by a Dunnett's multiple comparisons test. **p<0.01, **** p<0.0001.

C Representative images of mouse PDA and human PDA primary cells-derived organoids after 7 days of culture in medium alone (untreated) or supplemented with 1 or 10mM of βOHB. Scale bar: 1000 µm.

D, E Immunostaining of MCT2 and SMCT1 in tumors of KIC mice treated with βOHB (100mg/kg/day, i.p.) or 0.9% NaCl (i.p.) (n=5 mice/group). Areas of MCT2 (upper panel) and SMCT1 (lower panel) stainings (D) are expressed as mean of percentage of total tissue area ± SEM. Significance was defined by Mann-Whitney test. *: p<0.05, **p<0.01. Representative images of MCT2 and SMCT1 stainings (E) in tumors from KIC mice treated with βOHB or NaCl. Scale bar: 100 µm.

F Histological characterization of liver and spleen of KIC mice treated with βOHB (100mg/kg/day, i.p.) or 0.9% NaCl (i.p.) (n=14 mice/group). Number of mice displaying metastatic liver or spleen in each experimental group is reported.

G Schematic showing isotopomer transition from [U-13C]βOHB to label TCA-cycle intermediates, glutamate and proline. Grey filled circles indicate 13C carbon derived from labeled βOHB. Empty circles illustrate unlabeled 12C-species.

H [U-13C]βOHB tracing into the TCA intermediate: citrate in poorly differentiated PDA explants from KIC mice (n=5). Data are expressed as mean ± SEM.

I Schematic showing ketone metabolism pathway (left panel). Reversible enzymes involved in the production of acetyl-CoA, acetoacetyl-CoA, acetoacetate, βOHB, mT, SCOT1/2, and BDH1/2 are indicated in bold case. Immunoblots of ketone metabolic enzymes (BDH1/2, SCOT1/2, mT) (right panel) in pancreatic tissues from 7-week old KI (n=3 for BDH1/2, SCOT1/2, and n=4 for mT) and KIC mice (n=4).

Source data are available online for this figure.
Ketogenic HMG-CoA lyase and its product β-hydroxybutyrate promote pancreatic cancer progression (2022)
Figure 3

Figure 3: HMGCL targeting limits oncogenic properties of pancreatic tumor cells

A Quantification of colony forming area of sg-CTRL and sg-HMGCL (#2, #3 and #4 clones) PANC-1 cells. Colony area is expressed as mean fold change relative to sg-CTRL ± SEM (n=3 independent experiments). Significance was defined by one-tailed Student's t-test. *** p<0.001.

B Quantification of wound healing capacity of sg-CTRL and sg-HMGCL (#2, #3 and #4 clones) PANC-1 cells. Wound healing capacity is expressed as mean of percentage of restored area at 72hr ± SEM (n=4 independent experiments). Significance was defined by one-tailed Student's t test. ***p<0.001.

C, D Quantification of migratory (C) and invasive (D) sg-CTRL and sg-HMGCL (#2, #3 and #4 clones) PANC-1 cells. Migratory or invasive cell numbers are expressed as mean fold change relative to sg-CTRL cells ± SEM (n=3 independent experiments). Significance was defined by one-tailed Student's t test. ***p<0.001.

E Representative images of spheroid formation from sg-CTRL and sg-HMGCL (#2, #3 and #4 clones) PANC-1 cells after 4 or 15 days of culture. Photos are representative of n=3 independent experiments.

F Representative images of spheroid invasion assay from sg-CTRL and sg-HMGCL (#2, #3 and #4 clones) PANC-1 cells after 10 days of culture in presence of Matrigel. Photos are representative of n=3 independent experiments.

G Volcano plot illustrating proteins significantly under- or over-represented in sg-CTRL and sg-HMGCL PANC-1 spheroids (n=3 independent experiments). Significance was defined by one-tailed Student's t-test. Protein levels with a q-value<0.05 (horizontal axis) and a fold change <-1.5 or >+1.5 (vertical axis) are considered as significantly down or up-regulated in sg-HMGCL PANC-1 spheroids.

Source data are available online for this figure.
Ketogenic HMG-CoA lyase and its product β-hydroxybutyrate promote pancreatic cancer progression (2022)
Figure 4

Figure 4: HMGCL and βOHB promote pancreatic tumor aggressiveness

A, B Quantification of volume with representative images (A) and weight (B) of sg-CTRL (n=15 for A and n=9 for B), sg HMGCL #2 (n=19 for A and n=8 for B), #3 (n=7 for A and B) pancreatic tumors. Data are expressed as mean of tumor volume or weight ± SEM. Significance was defined by Mann-Whitney test. *: p<0.05, **p<0.01, ***p<0.001.

C Extracellular matrix quantification following trichrome staining in sg-CTRL and sg-HMGCL #2 pancreatic tumors sections (n=8 and 9 mice/group respectively, left panel). Data are expressed as mean of percentage of total tissue area ± SEM. Significance was defined by Mann-Whitney test. **: p<0.01. Representative images of trichrome staining in sg-CTRL and sg-HMGCL pancreatic tumors. Scale bar: 100 µm (right panel).

D, E Quantification of volume (D) and weight (E) of sg-CTRL pancreatic tumors treated with 0.9% NaCl (i.p.) (n=4), and pancreatic tumors from two different clones of sg-HMGCL treated with 0.9% NaCl (i.p.) or βOHB (100mg/kg/bi-weekly, i.p.) (n=10/group). Data are expressed as mean of tumor volume or weight ± SEM. Significance was defined by Mann-Whitney test. ns: not significant, **p<0.01.

F Histological characterization and representative picture of liver from mice orthotopically xenografted with sg-CTRL or sg-HMGCL PANC-1 cells and treated with 0.9% NaCl (i.p.) or βOHB (100mg/kg/bi-weekly, i.p.). Number of mice displaying healthy or metastatic liver in each experimental group is reported. Metastatic area (orange star) is separated from liver (green circle) by dotted lines. Scale bar: 100µm (inset images scale bar: 20µm).

G Schematic showing isotopomer transition from [U-13C]βOHB to label TCA-cycle intermediates, glutamate, and proline. Grey filled circles indicate 13C carbon derived from labeled βOHB. Empty circles illustrate unlabeled 12C-species.

H [U-13C]βOHB tracing into TCA intermediate: citrate in sg-CTRL and sg-HMGCL #2 and #3 PANC-1 cells cultured in indicated glucose concentrations. Data are expressed as mean ± SEM. (n=2 independent experiments). Significance was defined by one-way ANOVA followed by a Bonferroni's multiple comparisons test, only significances between sg-HMGCL #2, #3 PANC-1 cells and sg-CTRL PANC-1 cells under the same culture condition are mentioned. ***p<0.001.

Source data are available online for this figure.
Higher-order assemblies of oligomeric cargo receptor complexes form the scaffold of the Cvt vesicle (2016)
Figure 1

Figure 1: S. cerevisiae prApe1 higher-order organization and Ch. thermophilum mApe1 crystal structure fitted into S. cerevisiae mApe1 low-resolution EM map.

(A) Negative stain electron micrograph showing the tendency of S. cerevisiae prApe1 (sc-prApe1) dodecamers to form higher-order assemblies or chains (black arrowheads).

(B) Negative stain electron micrograph of S. cerevisiae mApe1 (sc-mApe1) showing monodisperse dodecameric particles (circles) as well as hexameric particles (white arrowheads).

(C) Bar plot showing the occurrence of single, double, triple or multiple aggregates of sc-prApe1 in comparison with sc-mApe1. Insets show single, double, triple or multiple dodadecamers. Data information: n=3,186 particles were counted in the case of sc-prApe1 and n=3,107 particles were counted in the case of sc-mApe1. Particles were picked from 20 different micrographs. ***P < 2.2x10-16 (Pearson’s Chi-squared test).

(D) Negative stain electron micrograph of GraFixed sc-mApe1 showing dodecameric particles (circles).

(E) Class averages of sc-mApe1 dodecamers including views of two-fold and three-fold symmetry.

(F) Fourier shell correlation of the EM reconstruction of the sc-mApe1 dodecamer indicates a resolution of 24 Å.

(G) C. thermophilum mApe1 crystal structure fitted into electron microscopy (EM) map of sc-mApe1 dodecamer. Each of the 12 chains is depicted by a different color. A black sphere represents the N-terminus where the propeptide would emanate from the dodecamer. From left to right: two-fold axis view from the edge, three-fold axis view from the facet and three-fold axis view from the vertex.
Higher-order assemblies of oligomeric cargo receptor complexes form the scaffold of the Cvt vesicle (2016)
Figure 2

Figure 2. Cryo-EM structure of S. cerevisiae Ams1.

(A) Electron micrograph of negatively stained Ams1 with particles and higher-order assemblies (arrows). Class averages exhibit two distinct two-fold views consistent with D2 symmetry. Scale bar 10 nm.

(B) Electron cryomicrograph of Ams1 with particles highlighted (circles).

(C) Selected particles (top row) are shown with corresponding class averages (bottom row). Scale bar 10 nm.

(D) Fourier shell correlation indicates a resolution of 6.3 Å according to the 0.143 criterion.

(E) Top view of the cryo-EM structure, four Ams1 molecules were segmented and colored (dark blue, beige, red, light blue).

(F) Molecular architecture of Ams1 monomer. Primary structure, two map segments with fitted pseudo-atomic models are shown. Left. The N-terminal portion was built from distant homology models: a four-helix bundle (17 – 27, 209 – 271) in yellow and a jelly-roll fold (45 – 203) in red. Right. The α-mannosidase core (287 – 1083) with an α/β barrel (287 – 573) in blue, a three-helix bundle (574 – 671) in green, and the β-sandwich domain (672 – 1083) in purple.

(G) Density section through the Ams1 monomer with three-helix bundle in the center.

(H) Complete atomic model of Ams1 with active site indicated as yellow sphere fitted into the cryo-EM map. Corresponding 3D structure in Movie EV1.

(I) Schematic representation of Ams1 domain distribution highlighting the molecular interfaces within the tetramer.

(J) The β-sandwich domain (purple) makes up the transverse interface between chain A and chain B.

(K) The N-terminal four-helix bundle (orange) mediates the longitudinal interface. Asterisk denotes position of W234E mutation between chain A and C/D.

(L) Negatively stained W234E Ams1 mutant reveals predominantly smaller particles when compared with wild-type tetramers. Representative class averages show a two-lobed structure consistent with reprojections of chain A and B (dark blue and beige) dimers depicted in ribbon and surface representation below. Scale bar 10 nm.
Higher-order assemblies of oligomeric cargo receptor complexes form the scaffold of the Cvt vesicle (2016)
Figure 3

Figure 3. prApe1/Atg19 complex.

(A) SEC-MALS profile of Atg19 yields a molecular weight estimate of 142 kDa (measured in triplicate).

(B) Right. In vitro pull-down binding assay and subsequent SDS-PAGE. Amylose beads are used as a bait to demonstrate interaction of purified prApe1 dodecamers with MBP-Atg19 (lane 1) whereas purified mApe1 dodecamers are not capable of interacting with MBP-Atg19 (lane 3). As controls, MBP does not bind prApe1 and mApe1 (lanes 2 and 4). Left. SDS-PAGE showing input of the corresponding pull-down assay.

(C) Co-expression of MBP-Atg19 and prApe1 in Sf21 cells; SDS-PAGE of eluate from amylose beads.

(D) Size-exclusion chromatography profile (Superose 6 column) of the prApe1/Atg19 complex after anti MBP-Atg19 purification and cleavage of the MBP tag (top). SDS-PAGE of peak fractions of the gel filtration profile (bottom). Samples were run on two separate gels.

(E) Negative stain electron micrograph corresponding to the size-exclusion chromatography peak at 10 ml retention volume of (D). Several prApe1 dodecamers possess extra density corresponding to Atg19 and are highlighted in the insets.

(F) Comparison between class averages of purified prApe1 dodecamers with class averages of prApe1 dodecamers that were co-purified with Atg19.

(G) Bar plot of occurrence of single, double, triple and multiple dodecamers from the prApe1 and the prApe1/Atg19 sample.

Data information: n=3,186 particles were counted in the case of prApe1 and n=3,340 particles were counted in the case of prApe1/Atg19. Particles were picked from 20 different micrographs. ***P < 2.2x10-16 (Pearson’s Chi-squared test).

H) Epifluorescence microscopy images of the prApe1-GFP/Atg19-mCherry/ypt7Δ, prApe1-GFP/Atg19Δ/ypt7Δ S. cerevisiae cells that were used to analyze the fluorescence intensity of all prApe1-GFP spots. BF represents bright-field images.

I) Bar plot showing the ratio between the GFP intensity of prApe1 spots in prApe1-GFP/Atg19-mCherry/ypt7Δ versus prApe1-GFP/Atg19Δ/ypt7Δ S. cerevisiae cells. The intensity of Ape1-GFP was normalized over the average fluorescence intensity of Nuf2-GFP measured in kinetochores as performed in [36]. Data information: a total of 54 prApe1-GFP spots and 80 Nuf2-GFP spots were analyzed in prApe1-GFP/Atg19Δ/ypt7Δ cells, whereas 52 prApe1-GFP spots and 98 Nuf2-GFP spots were analyzed in prApe1-GFP/Atg19-mCherry/ypt7Δ control cells. Error bars indicate standard error of the mean (SEM). *P = 0.03 (Z test).
Higher-order assemblies of oligomeric cargo receptor complexes form the scaffold of the Cvt vesicle (2016)
Figure 4

Figure 4: Quantification of the Ape1, Ams1, Atg19 protein abundances in living cells.

(A) Epifluorescence microscopy images of the prApe1-GFP/Atg19-mCherry/ypt7Δ, Ams1-GFP/Atg19-mCherry/ypt7Δ, Atg19-GFP/Ape1-mCherry/ypt7Δ S. cerevisiae cells that were used to quantify the fluorescence intensity of GFP spots. Only co-localizing GFP and RFP spots were considered. BF represents bright-field images.

(B) Bar plot showing the number of molecules of prApe1, Ams1 and Atg19 found in Cvt vesicles in living cells.

Data information: data are representative of two independent experiments in which a total of 217 prApe1-GFP, 181 Ams1-GFP and 233 Atg19-GFP spots were analyzed. Error bars indicate standard deviation (SD). ***P < 0.001; *P = 0.0176 (Z test).
Higher-order assemblies of oligomeric cargo receptor complexes form the scaffold of the Cvt vesicle (2016)
Figure 5

Figure 5: Correlative light and electron microscopy of S. cerevisiae Cvt vesicles from resin-embedded cell sections.

(A) Fluorescence microscopy image of a resin section of prApe1-GFP/Atg19-mCherry/ypt7Δ cells, merge of green (prApe1-GFP) and red (Atg19-mcherry) channels. Tetraspeck beads are detected in the blue channel.

(B) Electron tomography slice of the boxed area in (A). The outer and inner dashed green circles of 160 and 60 nm radius represent estimated 50% and 90% localization accuracy, respectively. Insets show close-up views of a double (top) and single (bottom) membrane. Corresponding tomogram Movie EV2.

(C) Plot of the x and y dimensions of the Cvt vesicles obtained from localization correlation. The average dimensions give rise to spheroids with an average ellipticity of 11 % (shown in dark grey). Both prApe1-GFP and prApe1-GFP/Atg19-mCherry datasets were included. The cases representing autophagosomes were excluded.

(D) Dense Cvt aggregate that is not surrounded by membrane. Corresponding tomogram Movie EV3.

(E) Cvt aggregate that is partially enwrapped by double membrane. Corresponding tomogram Movie EV4.

(F) Cvt vesicle fully enclosed by double membrane. Bottom: schematic representation. White arrowheads indicate the double membrane. Corresponding tomogram Movie EV5.