EMBO/soda-nel huggingface dataset:
KLF10 promotes nonalcoholic steatohepatitis progression through transcriptional activation of zDHHC7 (2022)
Figure 5

Figure 5. zDHHC7 is required for KLF10-induced CD36 palmitoylation and plasma membrane localization.

(A-C) HepG2 cells were transfected with lentivirus vectors expressing shRNA targeting KLF10 or negative control (Ctrl). n=3. Results represent three independent experiments. (A) Protein level of KLF10, and its normalization to β-actin. (B) Protein levels of palmitoylated CD36. (C) CD36 protein expression the plasma membrane fractions, and its normalization to ATP1a1.

(D-G) HepG2 cells were treated with 2-BP and/or lentivirus vectors expressing KLF10. n=3. Results represent three independent experiments. (D) Protein levels of KLF10, and its normalization to β-actin. (E) Protein levels of palmitoylated CD36. (F) CD36 protein expression the plasma membrane fractions, and its normalization to ATP1a1. (G) BODIPY staining showing the lipid droplets. (H-L) HepG2 cells were transfected with lentiviruses expressing KLF10 and/or shRNA targeting zDHHC7. n=3 independent experiments. (H) Protein levels of KLF10 and zDHHC7, and its normalization to β-actin. (I) Protein levels of palmitoylated CD36. (J) Immunofluorescence staining of CD36 and Na+/K+-ATPase (left), and the quantitation of CD36 colocalization rate (right). (K) BODIPY staining for lipid droplets. (L) Cellular TG contents.

Data information: ** P < 0.01. Results are shown as mean ± SD. Student's t test (B, C). 1-way ANOVA (D-F, H-J, L).
KLF10 promotes nonalcoholic steatohepatitis progression through transcriptional activation of zDHHC7 (2022)
Figure 6

Figure 6. CD36 is required for the pathogenic role of KLF10 in promoting NASH. (A-G) 6-week-old male CD36 wild-type (WT) or knockout mice (CD36KO) were administered with AAV-GFP (Ctrl) or AAV-KLF10 (KLF10OE) through tail vein injection, and then kept on WD/CCl4 for 12 weeks. n=5 per group. (A) Protein levels of KLF10 and CD36 in the liver (left), and their normalization to β-actin (right). (B) Representative immunofluorescence staining of KLF10 and CD36 (left), and the quantitative results (right). (C) H&E and Sirius red staining of liver sections. (D) Quantitative results of Sirius red staining. (E) Body weights and liver/body weight ratio. (F) Hepatic TG contents. (G) Serum AST and ALT levels.

Data information: * P <0.05, ** P <0.01, *** P <0.001. Results are shown as mean ± SD. 1-way ANOVA (A, B, D-G).
KLF10 promotes nonalcoholic steatohepatitis progression through transcriptional activation of zDHHC7 (2022)
Figure 7

Figure 7. CD36 palmitoylation is required for KLF10-mediated NASH progression.

(A-I) 6-week-old male CD36 knockout mice were administered with AAV-TBG-wildtype-CD36, wildtype-CD36 plus KLF10, palmitoylation site mutant CD36, mutant-CD36 plus KLF10 through tail vein injection, and then kept on WD/CCl4 for 12 weeks. n=5 per group. (A) Serum AST and ALT levels. (B) Hepatic TG contents. (C) Body weights and liver/body weight ratio. (D) H&E and Sirius red staining of liver sections (left), and quantitation of Sirius red staining (right). (E) Immunofluorescence staining of CD36 and β-catenin. (F) Quantitation of CD36 positive area. (G) Co-localization rate of CD36 and β-catenin. (H) Protein levels of palmitoylated CD36 in mouse livers (left), and its quantitative results (right). (I) CD36 protein expression in the plasma membrane fractions, and its normalization to ATP1a1.

Data information: * P <0.05, ** P <0.01, *** P <0.001. Results are shown as mean ± SD. 1-way ANOVA (A-D, F-I).
KLF10 promotes nonalcoholic steatohepatitis progression through transcriptional activation of zDHHC7 (2022)
Figure 8

Figure 8. Liver-specific KLF10 deficient mice are resistant to NASH pathogenesis.

(A-M) Albumin-cre mice were crossed with KLF10flox/flox to generate hepatocyte-specific KLF10 knockout mice (KLF10hep-/-). Then, 8-week-old male KLF10hep-/- mice and KLF10flox/flox were treated with WD/CCl4 for 12 weeks. KLF10flox/flox mice were used as the control (Ctrl). n=5 per group. (A-B) Protein levels of KLF10, zDHHC7 and TNF-α (A), and their normalization to β-actin (B). (C) Body weights. (D) Liver weights and liver/body weight ratio. (E) Hepatic TG contents. (F) Serum ALT and AST levels. (G) H&E staining and Sirius red staining of liver sections. (H) Quantitative results of Sirius red staining. (I) Immunofluorescence staining of CD36 and β-catenin. The arrows represent the colocalization of CD36 and β-catenin. (J) Quantitative results of co-localization rate of CD36 and β-catenin. (K) Protein levels of palmitoylated CD36 in mouse livers. (L) CD36 protein expression in plasma membrane fractions from mouse livers, and its normalization to ATP1a1. (M) Relative mRNA level of genes involved in the liver inflammation and fibrosis.

Data information: * P <0.05, ** P <0.01, *** P <0.001. Results are shown as mean ± SD. Student's t test (B-F, H, J-M).

Expanded View Figure Legends
GM-CSF suppresses antioxidant signaling and drives IL-1β secretion through NRF2 downregulation (2022)
Figure 1

Figure 1: GM-CSF induces NLRP3 dependent IL-1β secretion.

A. BMDMM were treated as indicated with either 60ng/mL ultra-pure (UP) LPS, 50ng/mL human derived recombinant GM-CSF, 40µM WEHD-FMK for 16 hours or 5µM nigericin for the last hour. Supernatants were then analysed for IL-1β secretion by ELISA.

B. HoxB8 macrophages were treated with the indicated dilutions of GM-CSF using supernatants from GM-CSF expressing cells. Shown are ELISA analysis of IL-1β secretion in supernatants and western blots indicating IL-1β expression in lysates..

C. HoxB8 macrophages of the indicated genotypes were treated as indicated with 60ng/mL UP-LPS, 1% GM-CSF containing supernatant (approximately 2ng/mL GM-CSF), 40µM WEHD.FMK, 0,5µM CRT-0066101 for 16 hours, 5µM nigericin was added for the last hour. Supernatants were analysed by ELISA for IL-1β. Inlay panels show western blots for levels of IL-1β expression in the indicated knockouts.

D. HoxB8 cells were treated with 60ng/mL of UP-LPS, 1% GM-CSF supernatant, 50mM KCl, and 5µM nigericin as indicated for 16 hours before supernatants were analysed for IL-1β by ELISA.

Data information: n≥ 3 biological replicates (every dot represents one biological replicate). For all panels, error bars are SEM. Significance was calculated using one way ANOVA with multiple comparisons or two-way ANOVA to compare across genotypes. p values are shown.
GM-CSF suppresses antioxidant signaling and drives IL-1β secretion through NRF2 downregulation (2022)
Figure 4

Figure 4: GM-CSF suppresses NRF2 upregulation to promote ROS activity.

A. BMDMM (left panel) and HoxB8 macrophages (right panel) were treated as indicated with either LPS or LPS+ GM-CSF. Cells were harvested and proteins extracted and analysed by western blot for levels of NRF2. Shown is the densitometric quantification of Nrf2 protein level from three independent experiments.

B. HoxB8 macrophages were treated as in A. and levels of IL-1β in the supernatant were analysed by ELISA

C. HoxB8 macrophages were treated for 12 hours with LPS or LPS+GM-CSF and RNA extracted and analysed by qPCR for the indicated targets.

D. Wild type and Tnf-/- HoxB8 cells were treated as indicated. Cells were harvested and proteins extracted and analysed by western blot for levels of NRF2 and HMOX1. For wild type cells, MitoTEMPO (50µM) was also added as indicated. Shown are the densitometric quantifications of NRF2 and HMOX1 protein levels of three independent experiments (right panel).

E. HoxB8 cells of the indicated genotypes were treated with LPS for 8 hours followed by staining with mitoSOX red. The fluorescent signal was analyzed using flow cytometry. Shown are average geometric mean fluorescence intensity of mitoSOX red.

F. Tnf-/- HoxB8 macrophages were treated with LPS with or without 50ng/ml recombinant TNF for 16 hours. Cells were harvested and proteins extracted and analysed by western blot for levels of NRF2 and HMOX1 (upper panel). Shown are the densitometric quantifications of indicated protein levels of three independent experiments (lower panel).

Data information: n≥ 3 biological replicates (every dot represents one biological replicate). For all panels, error bars are SEM. Significance was calculated using one way ANOVA with multiple comparisons, or two-way ANOVA to compare across genotypes. For the western blot quantification, significance was calculated using unpaired t-test. p values are shown.
GM-CSF suppresses antioxidant signaling and drives IL-1β secretion through NRF2 downregulation (2022)
Figure 5

Figure 5: GM-CSF does not activate NLRP3 inflammasomes.

A. HoxB8 macrophages were seeded in an 8-well Ibidi chamber and treated as indicated for 12 hours. Cells were fixed and stained for ASC (Red) and with DAPI (blue). For nigericin treatments nigericin was added for only 30 minutes. Example cells containing ASC Specks are shown with white arrows. Scale bars represent 50µM.

B. HoxB8 cells were treated as indicated with LPS or LPS+GM-CSF. The cross linker DSS was added to the cells as described in methods. Samples were analysed by western blot for ASC oligomerization. Black vertical lines indicate that non-relevant lanes were removed during figure preparation but samples were run on the same gel.

C. Wild type and caspase-1-/- HoxB8 macrophages were treated as indicated for 16 hours. Cell lysates were made and analysed by western blot for levels of caspase-1 and gasdermin D cleavage.

D. HoxB8 macrophages were treated as indicated with LPS or LPS+GM-CSF for 12 hours. Cells were lysed and NLRP3 was immune precipitated using a magnetic bead coupled antibody. As a negative control, beads without NLRP3 antibody were used. Beads were boiled and analysed for recruitment of NLRP3, and caspase-1. * indicates light chain from the antibody. Molecular weight markers shown in middle of NLRP3 blot were embedded in image using Chemostar imaging software (INTAS) at time of image acquisition.

E. HoxB8 macrophages were incubated with combinations of LPS, GM-CSF, nigericin +/- Z-VAD.FMK as indicated. Proteins were extracted and analysed by western blot for caspase-1 processing.

F. HoxB8 macrophages were seeded in a 96 well microplate and co-treated with either 10µM or 50µM z-VAD with LPS+GM-CSF for 16 hours. Then 100nM of Sytox Green was added to the cells. The number of sytox green+ cells per image was quantified.

Data information: Microscopy pictures and westerns are representative images from 3 biological replicates. Significance was calculated using one way ANOVA with multiple comparisons. Every dot represents one biological replicate. P values are shown.
GM-CSF suppresses antioxidant signaling and drives IL-1β secretion through NRF2 downregulation (2022)
Figure 7

Figure 7: GM-CSF induced ROS promotes IL-1β ubiquitylation which drives IL-1β secretion.

A. HoxB8 macrophages were treated for 16 hours with LPS +/- GM-CSF as indicated. Proteins were extracted and analysed for levels of IL-1β by western blot.

B. HoxB8 macrophages were treated with LPS or LPS+GM-CSF as indicated for 16 hours. Cells were lysed in buffer containing FLAG tagged K63 TUBE and K63 ubiquitylated proteins were precipitated using anti FLAG pull down. Pulled down proteins were analysed for IL-1β and ubiquitin by western blot.

C. IL-1β-/- hoxB8 macrophages were infected with lentivirus expressing IL-1β-FLAG-6xHis or the indicated mutants from the IL-1β promoter. Cells were treated as indicated with LPS or LPS+GM-CSF or LPS+Nigericin and levels of secreted IL-1β were analysed by ELISA (top panel). GM-CSF treated cells were also lysed in urea buffer and nickel NTA affinity pull down was performed. Nickel NTA pull down was also performed on supernatants (bottom panel). Pulled down proteins form the His tag pull down were analysed for the presence of IL-1β by western blot. Molecular weight markers shown in middle of blot were embedded in image using Chemostar imaging software (INTAS) at time of image acquisition.

D. HoxB8 macrophages were treated as in A with addition of 50µM mitoTEMPO or 10mM NAC as indicated. Proteins were extracted and analysed for IL-1β by western blot.

E. Wild type and TNF-/- HoxB8 macrophages were treated as indicated with LPS or LPS+GM-CSF. Proteins were extracted and analysed for IL-1β by western blot. Black vertical lines indicate that non-relevant lanes were removed during figure preparation but samples were run on the same gel.

F. HoxB8 macrophages were treated as in A but with the addition of 25µM Punicalagin throughout the 16 hour time course. Proteins were extracted and analysed by western blot (left panel). Supernatants were also analysed by ELISA for secreted IL-1β (right panel).

Data information: n≥ 3 biological replicates (every dot represents one biological replicate). For all panels, error bars are SEM. Significance was calculated using one way ANOVA with multiple comparisons, or two-way ANOVA to compare across genotypes. p values are shown.
GM-CSF suppresses antioxidant signaling and drives IL-1β secretion through NRF2 downregulation (2022)
Figure 8

Figure 8: IL-1β secretion occurs independently of pores formation.

A. Wild type and GsdmD-/- HoxB8 macrophages were treated as indicated for 16 hours. Supernatants were analyzed for IL-1β secretion by ELISA.

B. HoxB8 macrophages were seeded in an 8-well Ibidi chamber and treated for 14 hours with LPS and GM-CSF. Cells were fixed and stained for GasderminD (green), IL-1β (red) and DAPI (blue). Images were taken at the Zeiss LSM880 Confocal microscope

C. HoxB8 macrophages were co-stimulated with three different PEG 3mM for 16 hours as indicated. Supernatants were analyzed for IL-1β secretion by ELISA.

D. HoxB8 macrophages were seeded in 96 well microplate and treated as indicated. Before treatment, sytox green was added and cells were imaged in an incucyte incubator microscope every hour for 16 hours. Shown is the sytox green signal normalized to the total cell area.

E. Wild type and GsdmD-/- HoxB8 macrophages were treated as indicated for 16 hours. Proteins were extracted and analysed for IL-1β ubiquitination by western blot.

Data information: n≥ 3 biological replicates (every dot represents one biological replicate). For all panels, error bars are SEM. Significance was calculated using one way ANOVA with multiple comparisons, or two-way ANOVA to compare across genotypes. p values are shown. Microscopy pictures and westerns are representative images from 3 biological replicates. Scale bars in microscopy images represent 10µM.

Expanded View figure legends
Proteome Profiling in Cerebrospinal Fluid Reveals Novel Biomarkers of Alzheimer's Disease (2020)
Figure 3

Figure 3. CSF proteome alterations across the three cohorts

A-B) On the left, number of proteins that differ significantly (p-value < 0.05 in A; q-value < 0.05 in B) in abundance by AD status within each cohort. On the right, number of proteins thereof that have a consistent directionality of either elevated or reduced abundance in AD CSF in pairwise comparisons of cohorts.

C-E) Correlation of protein AD/non-AD fold changes in pairwise combinations of two cohorts each. Combinations are Sweden vs Magdeburg/Kiel (C), Sweden vs Berlin (D), and Magdeburg/Kiel vs Berlin (E). Proteins included differ significantly (p < 0.05) and consistently in abundance by AD status in both cohorts each.

F-G) Proteins that differ significantly (p-value < 0.05 in E; q-value < 0.05 in F) in abundance by AD status across all three cohorts. Z-scored abundances of proteins in the AD and non-AD groups of all cohorts shown by the heat map (see Material and Methods). Hierarchical clustering separates AD from non-AD groups. Pyruvat kinase PKM (PKM) was quantified in two isoforms and UniProt IDs are given in parenthesis. Black frames highlight proteins with consistent AD/non-AD fold changes across cohorts.
Proteome Profiling in Cerebrospinal Fluid Reveals Novel Biomarkers of Alzheimer's Disease (2020)
Figure 4

Figure 4. Protein correlation with t-tau measurements and analysis of annotation term enrichment

A-C) Correlation of proteins with ELISA-measured t-tau concentration across samples within the Sweden (A), Magdeburg (B) and Berlin (C) cohorts. Proteins with a q-value below 0.05 are labeled in yellow. Proteins of the 40 protein signature are colored in red for those with higher abundance in AD CSF and in blue for those with higher abundance in non-AD CSF.

D) Three cohort summary of proteins significantly correlating with ELISA-measured t-tau. Protein names given for the 29 proteins out of the 40 protein signature with significant (p < 0.05) correlation in each of the three cohorts. Pyruvat kinase PKM (PKM) was quantified in two isoforms and UniProt IDs are given in parenthesis.

E) Overlap of proteins significantly differing by AD status with proteins significantly correlating to ELISA-measured t-tau.

F) Annotation enrichment in the AD versus non-AD fold change dimension. Terms with positive enrichment means are enriched in AD CSF over non-AD CSF. Conversely, terms with enrichment means below zero are enriched in non-AD compared with AD CSF. Annotations filtered for significance of enrichment (p-value < 0.0.05) and term size (10-100 proteins per term) in all three cohorts.

G-H) Protein abundance distribution of CSF (G) and brain (H) showing the abundances of AD-modulated CSF proteins. Proteins of our 40 protein signature are highlighted in red (elevated abundance in AD) and blue (elevated abundance in non-AD). Proteins linked to glucose metabolism are highlighted in purple and labelled.
Mpl Traffics to the Cell Surface Through Conventional and Unconventional Routes (2014)
Figure 1

Ratios of mature:immature Mpl in normal platelets and cell lines expressing wildtype or mutant JAK2

A) Western blot analysis of Mpl in healthy donors' (HD) platelets shows two distinct bands representing the mature glycoprotein (top band) and the ER-core glycosylated form (bottom band). The ratio is ~1. B) Western blot analysis of HEL whole cell lysate after transfection with cDNA coding for JAK2WT or JAK2V617F, alone or cotransfected with cDNA coding for Mpl. C). Summary of the ratios of mature:immature Mpl for at least three independent (2 for the JAK2V617F transfection) blotting experiments under the protocol shown in (B). Based on two-tailed Student t-test statistical analysis, there is a significant difference (p?=?0.0001) in the ratio of Mpl isoforms in cells transfected with JAK2WT by comparison with control cells. Differences were also statistically significant in HEL cells transfected with either JAK2V617F or JAK2L611V/V617F by comparison to controls (p?=?0.0159 and p?=?0.0009, respectively). The mature:immature ratio is also significantly higher in cells transfected with JAK2WT than in cells transfected with JAK2V617F or JAK2L611V/V617F (p?
Mpl Traffics to the Cell Surface Through Conventional and Unconventional Routes (2014)
Figure 2

In JAK2WT cells, Mpl is detected at the cell surface and colocalizes with JAK2

Confocal fluorescent images of HEL cells (JAK2V617F+/+) after immuno-fluorescence labeling of endogenous Mpl (A), or after transient transfection with cDNA coding for a chimeric Mpl protein fused to mOrange2 (B). C) Confocal fluorescence image of K562 cells (JAK2WT+/+) transiently transfected with cDNA coding for MplmOrange2. Red arrow in this image points to plasma membrane localization. D and E) Confocal fluorescent images obtained after transient expression of MplmOrange2 and immuno-fluorescent labeling of endogenous JAK2 in K562 (D) or HEL (E) cells. Profiles of fluorescence (across individual cells and indicated by a white arrow) are showing a good colocalization of Mpl and JAK2WT in K562 cells at the plasma membrane (black arrow heads) but no colocalization have been observed between MplmOrange2 and JAK2V617F in HEL cells. F and G) Confocal fluorescent images obtained after transient coexpression of MplmOrange2 and either JAK2WTmCitrine (F) or JAK2V617FmCitrine (G) in K562 cells. Profiles of fluorescence (white arrows) are showing a very good colocalization of MplmOrange2 with JAK2WTmCitrine, especially at the plasma membrane (black arrow heads), but a very poor colocalization with JAK2V617FmCitrine (grey arrow heads). Nuclei are labeled with Hoechst and scale bars?=?5?µm. R: Pearson's correlation coefficient.
Mpl Traffics to the Cell Surface Through Conventional and Unconventional Routes (2014)
Figure 3

Mpl and Jak2 association is not limited to the plasma membrane.

Proximity ligation assay (Duolink) in HEL, K562 and K562Mpl-mKO2 cells. A) K562 cells labeled live for 30?min with an anti-CD235a-Atto655 (glycophorin A) antibody before treatment with the Duolink reagents. B) K562Mpl-mKO2 cells non-treated with the Duolink reagents. C and D) HEL cells primed live for 30?min with an anti-CD235a-Atto655 antibody before treatment with the Duolink reagents. E and F) K562Mpl-mKO2 cells submitted to the Duolink reaction. White arrow heads are pointing at example of Duolink positive reactions at the plasma membrane, while green arrow heads are pointing at positive Duolink reactions within the cytoplasm. Nuclei are labeled with Hoechst and scale bars?=?5?µm. R: Pearson's correlation coefficient.
Mpl Traffics to the Cell Surface Through Conventional and Unconventional Routes (2014)
Figure 4

JAK2 is critical for Mpl subcellular localization.

HEL (A) and K562 (B) cells treated with non-targeting (NT) or anti-JAK2 siRNA analyzed by western blot. C) Confocal live cell (upper panel), or fixed cells (lower left panel), imaging of K562 cells expressing MpleGFP or MplmKO2 treated with NT or anti-JAK2 siRNA showing a difference of distribution of intracellular Mpl in the siRNA treated cells compare to the control cells. After treatment with anti-JAK2 siRNA, Mpl strongly colocalizes with the ER marker, calnexin. This colocalization in treated vs non-treated cells is quantified and shown in Figure C lower right panel. Nuclei are labeled with Hoechst and scale bars?=?5?µm.
Mpl Traffics to the Cell Surface Through Conventional and Unconventional Routes (2014)
Figure 5

Intracellular Mpl pools partially colocalize with ER markers and colocalize with autophagic and lysosomal markers in K562 and HEL cells. A–M) Confocal fluorescent images obtained after transient expression of MplmOrange2 or MplmKO2 and indicated labeling in K562 (A–F) or HEL (G–M) cells. Plots at right of each image show fluorescence intensities for each emission spectrum at points along the line drawn across each image. Black arrows in the plots point to overlap between MplmKO2 and either ER‐tracker (C,I), or Sec31a (B,J) that marks ER exit sites. MplmOrange2 did not colocalize with markers for the Golgi (Golgin‐97) or early endosomes (EEA1) in either cell line. However, MplmKO2 colocalized greatly with autophagic (LC3) (E,L) and lysosomal (LAMP1) (F,M) markers. Nuclei are labeled with Hoechst and scale bars = 5 µm. R: Pearson's correlation coefficient.
Mpl Traffics to the Cell Surface Through Conventional and Unconventional Routes (2014)
Figure 6

MiniSOG‐tagged Mpl reveals that Mpl enters the autophagic pathway from specialized ER exit sites.

Confocal fluorescent images obtained after transient expression of Mpl tagged with mKO2 and miniSOG in K562 (A) and HEL (B) cells. Electron micrographs of K562 (C) and HEL (D) cells transiently expressing MplmKO2-miniSOG. Dark precipitate indicates the presence of the miniSOG-tagged Mpl protein after photo-precipitation of DAB. White boxes indicate original location of higher magnification images with corresponding letters. EG) High magnification electron micrograph of K562 cells treated in the same conditions as Figure C, showing MplmKO2-miniSOG dark precipitate into autophagic carrier vesicles (black arrows, Figure E). Osmiophilic reaction products also heavily stain mature AP in these preparations (black arrows, Figure G). H) High magnification electron micrograph of HEL cells treated in the same conditions as Figure D, showing different stage of formation of APs (black arrows numbered from stages 14). Scale bars?=?5?µm (AD) or 100?nm (EH).