EMBO/soda-nel huggingface dataset:
Mitochondrial DNA that escapes from autophagy causes inflammation and heart failure (2012)
f2

Mice were analysed 2 days after TAC (a–c). a, Haematoxylin and eosin-stained heart sections. Scale bar, 100 &mgr;m. b, AZAN-Mallory-stained sections. Scale bar, 100 &mgr;m. c, Immunohistochemical analysis using antibodies to CD45, CD68, Ly6G and CD3. Scale bar, 100 &mgr;m.
Mitochondrial DNA that escapes from autophagy causes inflammation and heart failure (2012)
f3

Mice were analysed 2 days after TAC (a–e). a, Electron microscopic analysis. Images of mitochondria at higher magnification are shown in subsets. Scale bar, 1 &mgr;m. b, Autolysosome after incubation with anti-DNA antibody and 10 nm gold staining. Scale bar, 200 nm. Arrows indicate labelled DNA. Double staining of heart sections with EdU (green) and anti-LAMP2a antibody (red) (c), EdU (green) and anti-LC3 antibody (red) (d) or EdU and anti-TLR9 antibody (red) (e). Arrows indicate EdU-positive and LAMP2a-, LC3- or TLR9-positive structures. Scale bar, 10 &mgr;m.
Mitochondrial DNA that escapes from autophagy causes inflammation and heart failure (2012)
f4

a, Survival ratio of TAC-operated ODN-treated mice (n = 6–10 per group). b–d, Four days after TAC. b, Echocardiography. Scale bars, 0.2 s and 5 mm. c, Echocardiographic parameters. Open and closed bars represent ODN2088 control- and ODN2088-treated groups, respectively (n = 5–8 per group). d, Immunohistochemical analysis. Scale bar, 100 &mgr;m. TLR9-deficient mice were analysed 10 weeks after TAC (e, f). e, Scale bars, 0.2 s and 5 mm. f, Echocardiographic parameters (n = 6–10 per group). Data are mean ± s.e.m. *P  0.05 versus all other groups.
Essential role for Nix in autophagic maturation of erythroid cells (2008)
figf1

a, Polychromasia (Wright–Giemsa stain, indicated by arrows) and increased reticulocytes (brilliant cresyl blue stain) revealed by staining blood smears from 6-week-old wild-type (WT) or Nix-/- mice (scale bar, 20 &mgr;m). b, c, Ter119 (b) or Mitotracker deep red (c) versus CD71 staining of RBCs from 3- or 6-week-old mice. Cell counts were also calculated (mean ± s.e.m.; n = 3). *P  0.05; **P  0.01; ***P  0.001. d, e, Analyses of RBCs as in b by immunocytochemistry (d; scale bar, 10 &mgr;m) or transmission electron microscopy (e; scale bar, 0.5 &mgr;m).
Essential role for Nix in autophagic maturation of erythroid cells (2008)
figf2

a, Quantification of NHS-biotin-labelled RBCs or transferred CMFDA-labelled RBCs (n = 3). b, Western blot analyses of caspases in RBCs after in vitro culture. Asterisks denote processed caspases. FL, full length. c, The relative luminescence units (RLU) of caspase activities in cultured RBCs and the suppression of annexin V staining in Nix-/- RBCs after 12-h culture in the presence of qVD-oph or solvent control (DMSO) (n = 3). d, Mitotracker versus annexin V staining of RBCs after in vitro culture. e, Mean fluorescent intensity (MFI) of ROS staining in RBCs after in vitro culture, and caspase activities in Nix-/- RBCs after 24 h culture with solvent control, ROS scavengers or qVD-oph (n = 3). BHT, butylated hydroxytoluene; CAT, catalase; TEM, tempol. f, Haematoxylin and eosin (HE) staining of spleen sections of 9-week-old wild-type and Nix-/- mice. Arrows denote iron deposits within macrophage cytoplasm. Iron deposits were also stained with Prussian blue, followed by counterstain with nuclear-fast red. Scale bar, 20 &mgr;m. g, Real-time RT–PCR of erythropoietin (Epo) (n = 12). For all relevant panels, statistical significance of the data (mean ± s.e.m.) is: *P  0.05; **P  0.01; ***P  0.001.
Essential role for Nix in autophagic maturation of erythroid cells (2008)
figf3

a, Ter119+CD71+ reticulocytes sorted at day 3 or 6 after PHZ treatment were cultured for in vitro maturation for 0 (top row), 2 (middle row) or 4 (bottom row) days, followed by Mitotracker deep red and Ter119 staining. b, CD71+Ter119+ reticulocytes sorted at day 3 after PHZ treatment were cultured and stained with new methylene blue. Scale bar, 20 &mgr;m. c, Cells as in b were stained for LC3 and COX IV and analysed by deconvolution microscopy. Scale bar, 5 &mgr;m. The Pearson coefficiency for LC3 and COX IV co-localization at day 0 (mean ± s.e.m.) is: wild type, 0.81 ± 0.001; Nix-/-, 0.44 ± 0.036 (n = 35, P = 0.0006). d, Cells sorted as in b were analysed by electron microscopy. Scale bar, 0.5 &mgr;m.
Essential role for Nix in autophagic maturation of erythroid cells (2008)
figf4

a, Ter119+CD71+ reticulocytes were sorted from the peripheral blood of wild-type and Nix-/- mice at day 6 after PHZ treatment. The cells were cultured for in vitro maturation in the presence of 10 &mgr;M FCCP or 1 &mgr;M ABT-737, followed by staining with Mitotracker deep red and phycoerythrin-conjugated anti-Ter119. b, c, Reticulocytes were cultured with FCCP (b) or ABT-737 (c) for 24 h and analysed by transmission electron microscopy. Scale bar, 0.5 &mgr;m.
Am80-GCSF synergizes myeloid expansion and differentiation to generate functional neutrophils that reduce neutropenia-associated infection and mortality (2016)
Figure 1

Figure 1. Am80-GCSF alters transcription of RA-target genes that promote induction of large amounts of neutrophils with well-developed innate immunity in normal primary human hematopoietic specimens.

(A) Proliferation analysis of hematopoietic CD34+ precursors for up to 6 days (section i). Neutrophil morphologic differentiation, bacterial killing, and ROS production were assessed at day 6 (section ii-iv). Controls were without treatment. White arrows indicate neutrophil nuclear segmentation. A+G, Am80-GCSF combination; CL, chemiluminescence; RLU, relative light units; AUC, area under the curve. (B) qRT-PCR assessed RA-target gene expression after culturing CD34+ cells for 1, 2, and 6 days. (C) Flow cytometry analysis of CD66-CD18 co-expression after culturing CD34+ cells for 6 days, using anti-human CD66-PE and CD18-FITC antibodies (section i). Isotypes were used for controls (section i, top panel). CD66-CD18 co-expression was quantified in section ii. (D) Fresh peripheral blood (PB) collected from normal human donor. Bacterial killing was assessed in the presence or absence of PB neutrophils (section i). Bactericidal activities of neutrophils induced by Am80-GCSF from PB mononuclear cells were evaluated by ROS production (section ii) and bacterial killing assays (section iii). (E) Fresh PB neutrophils collected from normal human donor were tested for ROS production in the presence or absence of specific antibodies. Controls were with or without IgG. Normal IgG, φ; Ab, antibodies; Comb Ab, combined anti-CD18 and -CD11b antibodies. (F) Fresh PB mononuclear cells collected from normal human donors were treated for 3 days. Bacterial killing (sections i) and neutralization of bacterial killing in the presence of IgG or anti-CD18 antibody were assessed at day 3 (section ii). Data are shown as mean ± SD and represent at least 2 independent experiments with similar results. *: P < 0.05; **: P < 0.01; ***: P < 0.001. Numbers of P values are provided in Appendix Table S4.
Am80-GCSF synergizes myeloid expansion and differentiation to generate functional neutrophils that reduce neutropenia-associated infection and mortality (2016)
Figure 3

Figure 3. Significant induction of RARβ2 and C/EBPε by Am80-GCSF are associated with both production of functional neutrophils and growth inhibition in primary human leukemic specimens.

(A) Fresh BM mononuclear cells collected from AML patients were treated with 20 or 50 nM Am80 alone or combined with medium dose of GCSF (25 ng/ml) for up to 9 days. Dynamic changes in proliferation (left sections) and live cells on day 9 (right sections) were illustrated for each of specimens. Controls were without treatment. (B) Fresh PB or BM mononuclear cells collected from AML patients were treated with 20, 100, or 150 nM Am80 alone or combined with GCSF for up to 12 days. Dynamic changes in proliferation (left sections) and live cells on day 12 (right sections) were illustrated for each of specimens. (C) Fresh BM mononuclear cells collected from AML patients were treated with 150 nM Am80 alone or combined with GCSF. Proliferation (left sections) and bacterial killing (right sections) were analyzed on day 12 for each of samples. (D) Fresh BM mononuclear cells collected from AML patient were treated with 150 nM Am80 alone or combined with GCSF. Proliferation was assessed for up to 12 days (section i), whereas ROS production, bacterial killing, morphologic differentiation, and RA-target gene expression were assessed at day 12 (sections ii-v). White arrows indicate neutrophil nuclear segmentation. Data are shown as mean ± SD and represent at least 2 independent experiments with similar results. *: P < 0.05; **: P < 0.01; ***: P < 0.001. Numbers of P values are provided in Appendix Table S4.
Am80-GCSF synergizes myeloid expansion and differentiation to generate functional neutrophils that reduce neutropenia-associated infection and mortality (2016)
Figure 5

Figure 5. Am80-GCSF coordinates myeloid expansion with granulocytic differentiation to generate large amounts of functional neutrophils that reduce infection in CCIN mice.

(A) After 48 hr of CPA injection, mice were treated with low doses of Am80 and/or GCSF for 3 days. Mice were infected with 9 x 106 CFU of S. aureus through intravenous injection on day 4 and sacrificed 16 hr post-infection. Control mice without CPA. (B) Vetscan counting PB leukocytes. (C) Bacterial killing by PB neutrophils was evaluated 3 and 16 hr post-infection (sections i, ii), using blood agar analysis of extracellular viable bacteria. (D & E) Neutrophil recovery and morphologic differentiation induced by low doses of Am80 and/or GCSF in BM (panel D) and PB (panel E). White arrows indicate neutrophil nuclear segmentation. Data are shown as mean ± SD and represent 3 independent experiments with similar results. *: P < 0.05; **: P < 0.01; ***: P < 0.001. Numbers of P values are provided in Appendix Table S4.
Am80-GCSF synergizes myeloid expansion and differentiation to generate functional neutrophils that reduce neutropenia-associated infection and mortality (2016)
Figure 7

Figure 7. Am80-GCSF prevents myeloid overexpansion while generating sufficient numbers of functional neutrophils during ‘emergency’ granulopoiesis in CCIN mice.

(A & B) Neutrophil recovery and morphologic differentiation were assessed in BM (panel A) and PB (panel B) of CCIN mice surviving on day 9. White arrows indicate neutrophil nuclear segmentation. (C) PB neutrophil production and nuclear segmentation were assessed in control and Am80-GCSF mice surviving on day 13 (sections i, ii). The increased spleen size and weight on day 9 in Am80-GCSF mice dropped to the levels similar to those in control mice on day 13 (sections iii, iv). (D) Significant loss of body weight occurred in GCSF mice on day 9. (E) Ultrastructure analysis of neutrophils collected from mice surviving on day 11. Neutrophil from GCSF mice showed less granules (section i, images 1 vs. 2 or 3), more intracytoplasmic vacuoles (sections i, ii), and inner nuclear membrane dilation (sections i, iii). Pg: primary granules; Sg: secondary granules. Data are shown as mean ± SD and represent at least 2 independent experiments with similar results. *: P < 0.05; **: P < 0.01; ***: P < 0.001. Numbers of P values are provided in Appendix Table S4.
Protein quality control and regulated proteolysis in the genome-reduced organism Mycoplasma pneumoniae (2020)
Figure 2

Figure 2. Phenotypic characterization of Lon and FtsH conditional mutants in M. pneumoniae.

(A). Cell growth assessment of ΔIndLon (upper plot) and ΔIndFtsH (lower plot) mutants grown under inducing (blue) or depleting conditions (red). Growth was monitored by measuring DNA and protein biomass over time. The average from two independent biological replicates is shown.

(B). Protein aggregation in ΔIndLon (upper plot) and ΔIndFtsH (lower plot) mutants grown under inducing (blue) or depleting conditions (red, 48h and 72h of depletion for Lon and FtsH, respectively). Protein aggregates in Triton-X100 insoluble fractions of untreated or heat shock (15min at 45°C) treated cells were measured by thioflavin (ThT) staining. Bars represent the mean of three biological replicates (dots). Significance of comparisons was assessed by two-sided independent t-test (exact p-values are shown).

(C). Assessment of cell membrane integrity of ΔIndLon (upper plot) and ΔIndFtsH (lower plot) mutants grown under inducing (blue) or depleting conditions (red, 72h of depletion for both, Lon and FtsH). Membrane integrity of untreated cells or after exposure during 30 min to 0.001% Triton-X100 was assessed by trypan blue exclusion staining. Bars represent the mean of three biological replicates (dots). Significance of comparisons was assessed by two-sided independent t-test (exact p-values are shown).

(D). Role of Lon and FtsH under heat shock stress conditions. ΔIndLon and ΔIndFtsH mutants were grown under inducing (Lon+ or FtsH+) or depleting conditions (Lon+ or FtsH+, 60h of depletion for both, Lon and FtsH), and then exposed at 45°C during 0, 5, 10, 15 or 20 min. Then, growth after the heat treatment was monitored over time under inducing conditions by the 430/560 absorbance rate index that shows pH changes in the medium. The average from two independent biological replicates is shown for each condition.
Protein quality control and regulated proteolysis in the genome-reduced organism Mycoplasma pneumoniae (2020)
Figure 4

Figure 4. Validation of selected Lon and FtsH candidate substrates.

(A) Transcription profiles of ΔIndLon mutants grown in inducing (+) or depleting (-) conditions across the cell division and the MPN525 (DnaB) operons. The respective estimated protein copy numbers based on MS data are shown below.

(B) Time course experiments showing depletion levels of Lon over time and the corresponding accumulation of N-terminal FLAG-tagged derivatives of candidate substrates associated to cell division, including FtsA, FtsZ and DnaB. Protein levels of Lon and candidate substrates were assessed by immunoblot analysis using anti-Lon and anti-FLAG antibodies. LC, loading control.

(C) In vivo degradation assays of FtsA, FtsZ and DnaB. ΔIndLon mutants expressing the different N-terminal FLAG-tagged derivatives were grown in depleting conditions for 36h. Then, Lon expression was transiently induced for 3h before translation was blocked with gentamicin (Gm). Samples were taken at the indicated time points after gentamicin treatment, and processed for immunoblot analysis using anti-FLAG antibodies. LC, loading control. As controls, non-induced cells were also treated with gentamicin and processed at the same time points. Immunoblots are representative of two independent experiments.

(D) Estimated protein copy numbers based on MS data of components associated with the R-M system.

(E and F) Similar to panel B (E) and C (F) but for a C-terminal FLAG-tagged derivative of MPN201 as a representative of an unstable HsdS subunit.

(G) Estimated protein copy numbers based on MS data of FtsH candidate substrates.

(H) Similar to panel B but for C-terminal FLAG-tagged derivatives of FtsH candidate substrates. Protein levels of FtsH and candidate substrates were assessed by immunoblot analysis using anti-FtsH and anti-FLAG antibodies. LC, loading control.

(I) Similar to panel C, but for FtsH candidate substrates. In this case, C-terminal FLAG-tagged derivatives were grown in depleting conditions for 60h before transient expression of FtsH for 3h and gentamicin treatment.
Protein quality control and regulated proteolysis in the genome-reduced organism Mycoplasma pneumoniae (2020)
Figure 5

Figure 5. Mutational analysis to identify Lon degrons in Lon substrates.

(A) Amino acid sequence of the last 50 residues of FtsA (MPN316), FtsZ (MPN317) and DnaB (MPN525) proteins and their mutant derivatives. Hydrophobic motifs that can potentially act as Lon degrons are highlighted in bold. Mutations performed in these putative Lon degrons are shown in red.

(B) Protein stability assessment of Lon substrates containing multiple or single mutations in putative Lon degrons. N-terminal FLAG-tagged derivatives with mutations shown in panel A were expressed in the ΔIndLon mutant. Protein levels were then determined by immunoblot using anti-Lon and anti-FLAG antibodies comparing inducing (+) or depleting (-) conditions (48h of depletion). LC, loading control.
Protein quality control and regulated proteolysis in the genome-reduced organism Mycoplasma pneumoniae (2020)
Figure 6

Figure 6. Role of Lon as a protein quality control protease.

(A) Protein stability of an unstable firefly luciferase variant (Fluc_F89E) assessed by anti-Fluc antibodies in ΔIndLon mutants grown in inducing (+) or depleting (-) conditions (48h of depletion). LC, loading control.

(B) Lon surveillance of truncated protein variants. As an example, an N-terminal FLAG-tagged derivative of MPN304, which encodes a truncated variant of ArcA (ArcA-Nt), was expressed in the ΔIndLon mutant, and its accumulation monitored over time under non-inducing conditions using anti-FLAG antibodies. Anti-Lon antibodies were used to monitor Lon depletion along the time course experiment (western blot figure on left). MPN304 stability was also assessed by in vivo degradation experiments in Lon depleted mutants (36h of depletion), in which protein expression was blocked with gentamicin (Gm) during the indicated time points after transient induction of Lon for 3h. As control, samples after Gm treatment were also taken from non-induced samples. LC, loading control (western blot figure on right). Immunoblots are representative of two independent experiments. A schematic representation of the arginine deiminase pathway, which is inactive in M. pneumoniae, and the estimated protein copy number of its components are also shown for the ΔIndLon mutant grown in inducing (+) or depleting (-) conditions.

(C) MS analysis showing the distribution of log2 fold-changes of the Triton X-100 insoluble fraction compared to the whole cell lysate, under Lon depleting conditions. Proteins associated with the terminal organelle (TO proteins), known to be enriched in the insoluble fractions, are shown as reference. Lon substrate candidates exhibit larger log2 fold-changes compared to the other proteins (Mann-Whitney-Wilcoxon two-sided test, ****p=0.0003), showing enrichment in the insoluble fraction under Lon depleting conditions.

(D) Lon enrichment in protein insoluble fractions during heat stress conditions. Lon levels in total (T) and Triton-X100 soluble (S) and insoluble (I) fractions were assessed by immunoblot analyses in untreated wild-type cells or exposed at 45°C during the indicated time points. As controls, the Triton-X100 insoluble protein HMW1 and the soluble ribosomal protein RL-7 were also analysed by using antibodies raised against these proteins.

(E) Protein quality control of terminal organelle assembly mediated by Lon. Top of panel E, a schematic representation of the genetic organization at the P65 operon after deleting the N-terminal region of the hmw2 gene in the ΔIndLon mutant. The expected PCR products for the deletion are also indicated. Below, an agarose electrophoresis gel showing the PCR validation of the intended deletion, and SDS-PAGE and immunoblot analyses monitoring the expression of specific terminal organelle proteins, whose stability depend on HMW2, for ΔIndLon or ΔIndLon_Δhmw2Nt cell lysates grown in inducing (+) or depleting (-) conditions (48h of depletion). LC, loading control.
mGluR-Long Term Depression regulates GluA2 association with COPII vesicles and exit from the endoplasmic reticulum (2016)
Figure 1

Figure 1. DHPG treatment reduces surface GluA1 levels, and increases surface GluA2 levels in an ER Ca2+ dependent manner.

A. Labeling of non-permeabilized (top) and permeabilized (bottom) cells demonstrate that surface labeling does not label internal proteins. GluA2 labeling (green) is visible both on the surface and internally, while DARPP32 labeling (red) is only visible internally. B. Top. Representative images of surface GluA2 and DARPP32 expression in striatal neurons from co-cultures with either no treatment or 20 min 2APB and dantrolene. Bottom. Quantified expression of GluA2 under both conditions, n = 9 replicates of 10 cells each, Student’s t test. C. Representative images of surface GluA1 and DARPP32 expression in striatal neurons from co-cultures with either no treatment, 30 min DHPG, or 2APB and dantrolene pretreatment followed by DHPG. D. Quantified expression of GluA1 under each condition, n = 9 replicates of 10 cells, p < 0.001, ANOVA. E. Representative images of surface GluA2 and DARPP32 expression in striatal neurons from co-cultures under the same conditions as in C. F. Quantified expression of GluA2 under each condition, n = 12 replicates of 10 cells each, p < 0.001, ANOVA. G. Representative images of surface myc-GluA2, internal myc-GluA2, and DARPP32 expression in striatal neurons from co-cultures under the same conditions as in C. H. Quantified expression of surface myc:total myc under each condition, n = 4 replicates of 10 cells each, p < 0.05, ANOVA. All error bars represent s.e.m. Abbr. A1 = GluA1, A2 = GluA2, D = DARPP32, s-myc = surface myc, i-myc = internal myc, m = merge.
mGluR-Long Term Depression regulates GluA2 association with COPII vesicles and exit from the endoplasmic reticulum (2016)
Figure 2

Figure 2. Blocking Ca2+ release from the ER but not extracellular Ca2+ entry into the cell prevents the DHPG mediated increase of surface GluA2 levels.

A. Left. Representative images of surface GluA2 and DARPP32 expression in striatal neurons from co-cultures treated either for 30 min with DHPG (DHPG) or for 30 min with DHPG and pretreated with Xestospungin C and S107 (XeC/S107 + DHPG). Right. Quantified expression of surface GluA2 under each condition, n = 3 replicates of 10 or 20 cells each, p < 0.0001, Student’s t test. B. Left. Representative images of surface GluA2 and DARPP32 expression in striatal neurons from co-cultures treated either for 30 min with DHPG (DHPG), or for 30 min with DHPG and pretreated with S107 (S107 + DHPG), or for 30 min with DHPG and pretreated with Xestospungin C (XeC + DHPG). Right. Quantified expression of surface GluA2 under each condition, n = 4 replicates of 10 cells each, ANOVA. C. Top. Representative images of surface GluA2 and DARPP32 expression in striatal neurons from co-cultures under control or 10 min Thapsigargin (TG) treatment. Bottom. Quantified expression of surface GluA2 under each condition, n = 3 replicates of 10 cells each, Student’s t test. D. Top. Representative images of surface GluA2 and DARPP32 expression in striatal neurons from co-cultures with treatment with either no DHPG or with DHPG for 30 min, after 15 min of Thapsigargin. Bottom. Quantified expression of surface GluA2 under each condition, n = 4 replicates of 10 cells each, Student’s t test. E. Top. Representative images of surface GluA2 and DARPP32 expression in striatal neurons from co-cultures with either no DHPG or with DHPG for 30 min, after 5 min of BAPTA. Bottom. Quantified expression of GluA2 under each condition, n = 3 replicates of 10 cells each, p < 0.001, Student’s t test. F. Top. Representative images of surface GluA2 and DARPP32 expression in striatal neurons from co-cultures with either no DHPG or with DHPG for 30 min after 5 min of BAPTA-AM. Bottom. Quantified expression of GluA2 under each condition, n = 3 replicates of 10 cells each, Student’s t test. All error bars represent s.e.m.