EMBO/soda-nel huggingface dataset:
Loss of TREM2 rescues hyperactivation of microglia, but not lysosomal deficits and neurotoxcity in models of progranulin deficiency (2021)
Figure 2

Figure 2 - Loss of TREM2 reduces the DAM signature of Grn-/- mice

A. Heatmap of 65 DAM-associated gene transcripts analyzed by NanoString in FCRLS- and CD11b-positive Grn-/- (n=6), Trem2-/- (n=5) and Double-/- (n=6) microglia in comparison to WT (n=7) microglia isolated from 6-month-old male mice. The expression corrected and housekeeping gene normalized RNA counts for each gene and sample were normalized to the mean value of WT followed by a log2 transformation.

B. Volcano plot presentation of the differently expressed transcripts in FCRLS- and CD11b-positive Grn-/- (n=6) in comparison to WT (n=6) microglia isolated from 6-month-old male mice. 35 out of 65 analyzed genes are significantly changed more than 20%, with 22 genes upregulated (purple) and 13 genes downregulated (blue).

C. Volcano plot presentation of the differently expressed transcripts in FCRLS- and CD11b-positive Trem2-/- (n=5) in comparison to WT (n=6) microglia isolated from 6-month-old male mice. 17 out of 65 analyzed genes are significantly changed more than 20%, with 7 genes upregulated (purple) and 10 genes downregulated (blue).

D. Volcano plot presentation of the differently expressed transcripts in FCRLS- and CD11b-positive Double-/- (n=6) in comparison to WT (n=6) microglia isolated from 6-month-old male mice. 21 out of 65 analyzed genes are significantly changed more than 20%, with 14 genes upregulated (purple) and 7 genes downregulated (blue).

E. Expression profiles of selected DAM genes, whose mRNA levels are rescued in Double-/- (n=6) vs. Grn-/- (n=6) microglia. mRNA expression normalized to the mean of the WT cohort. Data represent mean ± SEM.

Data information: For statistical analysis in B - D the unpaired, two-tailed student`s t-test was performed, and in E one-way ANOVA with Dunnett`s post hoc test was used to compare Grn-/-, Trem2-/- and Double-/- microglia. Statistical significance was set at **, p < 0.01; ***, p < 0.001; ****, p < 0.000; ns, not significant.
Loss of TREM2 rescues hyperactivation of microglia, but not lysosomal deficits and neurotoxcity in models of progranulin deficiency (2021)
Figure 4

Figure 4 - Antagonistic TREM2 antibodies enhance TREM2 shedding and reduce TREM2-dependent signaling and phagocytosis in PGRN deficient iPSC-derived human microglia (hiMGL)

A. Western blot of PGRN in whole-cell lysates of WT and GRN-/- hiMGL. Actin was used as loading control.

B. Western blot of TREM2 in whole-cell lysates of WT and GRN-/- hiMGL. Mature (mTREM) and immature TREM2 (imTREM2) are indicated. Actin was used as loading control.

C. Quantification of mTREM2 expression in whole-cell lysates of WT and GRN-/- hiMGL (data shown in B). mTREM2 levels were normalized to WT (n = 3, biological replicates).

D. ELISA-mediated quantification of sTREM2 in conditioned media of WT and GRN-/- hiMGL (n = 3, biological replicates).

E. ELISA-mediated quantification of sTREM2 in conditioned media of GRN-/- hiMGL upon treatment with Ab1 and Ab2 (20 μg/ml, 40 μg/ml) (n = 3, biological replicates). An isotype antibody (10 μg/ml) was used as a negative control.

F. AlphaLISA-mediated quantification of p-Syk levels in GRN-/- hiMGL upon treatment with Ab1 and Ab2 (5 μg/ml, 10 μg/ml) with liposomes (1 mg/ml) for 5 min. (n = 8, biological replicates).

G. Uptake assay for fluorescent myelin in WT hiMGL. Phagocytosis of myelin significantly decreased upon treatment with TREM2 antagonistic antibodies Ab1 and Ab2 (n=4, biological replicates).

H. Uptake assay for fluorescent myelin. GRN-/- hiMGL phagocytose significantly more myelin as compare to WT hiMGL. This is reversed upon treatment with TREM2 antagonistic antibodies Ab1 and Ab2 (n=4, biological replicates).

Data information: Data represent mean ± SEM. For statistical analysis in C and D the unpaired, two-tailed student`s t-test was performed, in E and F one-way ANOVA with Dunnett`s post hoc test, and in G and H one-way ANOVA with Tukey's post hoc was used to compare untreated, Ab1, and Ab2 (20 µg/ml and 40µg/ml) conditions to the isotype treated condition. Statistical significance was set at *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; and ns, not significant.
Loss of TREM2 rescues hyperactivation of microglia, but not lysosomal deficits and neurotoxcity in models of progranulin deficiency (2021)
Figure 5

Figure 5 - Antagonistic TREM2 antibodies reduce hyperactivation of PGRN deficient hiMGL

A. Expression of all analyzed gene transcripts in GRN-/- hiMGL treated with control isotype antibody, Ab1 or Ab2 in comparison to WT hiMGL. Data show the mean of four individual treatments and NanoString measurements. The mRNA counts for each gene were normalized to the mean value of all WT samples followed by a log2 transformation.

B. Volcano plot presentation of the differently expressed transcripts in GRN-/- hiMGL treated with isotype compared to WT hiMGL. Genes with more than 20% significantly changed expression are marked in purple (upregulated) or blue (downregulated).

C. Volcano plot presentation of the differently expressed transcripts in GRN-/- hiMGL treated with Ab1 comparison to WT hiMGL. Genes with more than 20% significantly changed expression are marked in purple (upregulated) or blue (downregulated).

D. Volcano plot presentation of the differently expressed transcripts in GRN-/- hiMGL treated with Ab2 comparison to WT hiMGL. Genes with more than 20% significantly changed expression are marked in purple (upregulated) or blue (downregulated).

E. Transcript levels of DAM gene transcripts significantly altered in GRN-/- hiMGL, treated with Ab1 or Ab2 in comparison to isotype treatment from the data set in A, normalized to the mean of the WT hiMGL samples (n=4, biological replicates).

F. Transcript levels of CTSD, NPC2 and CD68 of WT and GRN-/- hiMGL untreated, treated with isotype control, and Ab1 or Ab2 in comparison to WT hiMGL from the data set in A normalized to the mean of the WT hiMGL samples (n=4, biological replicates).

G. Catalytic activity of cathepsin D (CatD) in untreated WT and GRN-/- hiMGL or GRN-/- hiMGL treated with isotype control, Ab1 or Ab2 (20 μg/ml, 40 μg/ml), as measured by a CatD in vitro activity assay (n=3, biological replicates).

Data information: Data represent mean ± SEM. For statistical analysis in B - D the unpaired, two-tailed student`s t-test was performed, in E one-way ANOVA with Dunnett`s post hoc test was used to compare Ab1 and Ab2 (20 µg/ml and 40µg/ml) conditions to the isotype treated condition, and in F and G one-way ANOVA with Dunnett`s post hoc test was used to compare Ab1, Ab2 (20 µg/ml and 40µg/ml) and isotype treated condition to WT cells. Statistical significance was set at *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001, *****, p < 0.00001, and ns, not significant.
Loss of TREM2 rescues hyperactivation of microglia, but not lysosomal deficits and neurotoxcity in models of progranulin deficiency (2021)
Figure 6

Figure 6 - Abolishing TREM2 signaling does not rescue lysosomal dysfunction in Grn-/- mice

A. Western blot of CatD in total brain lysates from 14-month-old female WT, Grn-/-, Trem2-/-, and Double-/- mice. CatD maturation variants are indicated (sc: single chain; hc: heavy chain; n=3).

B., C. Quantification of CatD variants in A normalized to WT (n=3 per genotype).

D., E. Catalytic activity of CatD in brain lysates from female 6-month-old (n=4 per genotype) (D) or 14-month-old (n=3 per genotype) (E) Grn-/-, Trem2-/-, Double-/- mice normalized to WT.

F. Immunohistochemical analysis of lipofuscin (green) in coronal brain sections. Representative images of thalamus are shown. Scalebars = 50 μm.

G. Quantification of lipofuscin autofluorescence. Five images per mouse were taken, means were normalized to WT samples (n=3 per genotype, female).

Data information: Data represent mean ± SEM. For statistical analysis one-way ANOVA with Tukey's post hoc test of Grn-/-, Trem2-/- and Double-/- was used. Statistical significance was set at *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001, and ns, not significant.
Loss of TREM2 rescues hyperactivation of microglia, but not lysosomal deficits and neurotoxcity in models of progranulin deficiency (2021)
Figure 7

Figure 7 - Reduced TREM2 signaling does not rescue dysregulated lipids in Grn-/- mice

A-D. Volcano plot presentation of lipids and metabolites upregulated (purple) or downregulated (blue) in total brain homogenates from 6-month-old male Grn-/- (A, n=5), Trem2-/- (B, n=5), Double-/- (C, n=5) mice in comparison to WT (n=4), and Double-/- in comparison to Grn-/- mice (D). Counts for each sample were normalized to the mean value of WT followed by a log2 transformation (n=4-5 per genotype). Analyte values were adjusted with an FDR <10% to exclude type I errors in null hypothesis testing.

E-G. Abundance of BMP species and glucosylsphingosine (GlcSph) in total brain of 6-month-old Grn-/-, Trem2-/-, Double-/- and WT mice (n=4-5 per genotype).

H. Glucocerebrosidase (GCase) activity in whole brain lysates from 6-month-old male Grn-/-, Trem2-/-, Double-/- and WT mice. The linear increase of fluorescence signal was measured and then normalized to WT mice (n=3-6 per genotype).

Data information: Data represent mean ± SEM. For statistical analysis one-way ANOVA with Tukey`s post hoc test of Grn-/-, Trem2-/- and Double-/- was used. Statistical significance was set at ***, p < 0.001; ****, p < 0.0001, and ns, not significant.
Loss of TREM2 rescues hyperactivation of microglia, but not lysosomal deficits and neurotoxcity in models of progranulin deficiency (2021)
Figure 8

Figure 8 - Loss of TREM2 does not prevent synapse loss and activated microglia morphology

A. Immunohistochemical analysis of synaptophysin (SPH, pink) and VGAT (yellow) in coronal brain sections. Representative images of thalamus are shown. Scalebars = 50 μm.

B. Quantification of SPH positive area. Three images per mouse were taken, means were normalized to WT samples (n=3 per genotype, female).

C. Quantification of VGAT positive area. Three images per mouse were taken, means were normalized to WT samples (n=3 per genotype, female).

D. Western blot of SPH in RIPA lysates from 14-month-old female WT, Grn-/-, Trem2-/-, and Double-/- mice. Actin was used as loading control.

E. Quantification of SPH protein levels in D normalized to WT (n=3).

F. Morphological analysis of cortical microglia. Representative maximum intensity projections of confocal z-stack images showing IBA1+ microglial cells of female WT, Grn-/-, Trem2-/- and Double-/- mice (scalebar = 50 µm). Arrows point to individual microglia, which are shown as three-dimensional reconstruction, scalebar = 10 µm.

G. Morphological differences of cortical microglia from WT, Grn-/-, Trem2-/- and Double-/- mice shown by branch volume, sphericity score, branch length and the number of branch nodes. Statistical analysis of group difference for the morphological scores "Branch volume" (auc = 0.72), "Sphericity score" (auc = 0.82), "Branch length" (auc = 0.69) and "Number of branch nodes" (auc = 0.80) was performed using the Wilcoxon rank sum test with continuity correction and Bonferroni post-hoc correction for multiple testing in R (version 4.0.3). Two images per mouse (n=3 per genotype, female) were analyzed, each data point represents one microglia cell. Median and interquartile range are displayed.

Data information: Data represent mean ± SEM. For statistical analysis in B-C and E one-way ANOVA with Tukey's post hoc test of Grn-/-, Trem2-/- and Double-/- was used. Statistical significance was set at **, p < 0.01; ***, p < 0.001; ****, p < 0.0001, and ns, not significant.
Loss of TREM2 rescues hyperactivation of microglia, but not lysosomal deficits and neurotoxcity in models of progranulin deficiency (2021)
Figure 9

Figure 9 - Hyperactivation of microglia in Grn-/- mice is not deleterious

A. Immunoassay-based quantification of neurofilament-light chain (NfL) protein levels in CSF of 6-month-old Grn-/- (n=4), Trem2-/-(n=4), Double-/- (n=4) and WT (n=5) male mice.

B. Immunoassay-based quantification of NfL levels in CSF of 14-month-old Grn-/-, Trem2-/-, Double-/- and WT female mice (n=3 per genotype).

C. Neuropathology NanoString panel analysis of total brain mRNA expression of 6-month-old and 14-month-old Grn-/-, Trem2-/-, Double-/- and WT male mice based on NanoString advanced analysis R-script included in the panel (6-month-old mice: n=4, 14-month-old mice: n=3).

D. Transcript levels of all significantly changed genes in Double-/- vs. Grn-/- brain mRNA of 6-month-old and 14-month-old mice analyzed in C. Grin3b was under detection limit in the 14-month-old cohort. Transcript expression is normalized to the mean of the WT cohort.

E. The same cohort of mice scanned for TSPO-PET were additionally scanned for FDG-PET. Axial slices as indicated show %-FDG-PET differences between Grn-/-, Trem2-/- and Double-/- (all cold colour scales) when compared to WT at the group level. Images were adjusted to an MRI template.

F. Bar graph illustrates individual FDG-PET values derived from a whole brain volume of interest. Data represent mean ± SD. 8-15 female mice per group at an average age of 10.7 ± 1.5 months (Grn-/- n = 8, Trem2-/- n = 10, Double-/- n = 10, WT n = 15) were used.

Data information: Data in A, B and D represent mean ± SEM. For statistical analysis in A-B two-way ANOVA with Dunnett`s post hoc test was used the unpaired, in D, F two-tailed student`s t-test was performed. Statistical significance was set at *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001, and ns, not significant.

Expanded View Figure Legends
Upregulation of autophagy-related gene-5 (ATG-5) is associated with chemoresistance in human gastric cancer. (2014)
Figure 1

Representative images showing immunohistochemical staining for ATG-5 in non-tumorous and GC tumor tissues.

(A) ATG-5 staining in non-cancerous gastric tissues scored 285(×200); (B) ATG-5 staining in GC tumor tissues scored 50(×400); (C) ATG-5 staining in GC tumor tissues scored 270(×400); (D) ATG-5 staining in GC tumor tissues scored 400(×200).
Upregulation of autophagy-related gene-5 (ATG-5) is associated with chemoresistance in human gastric cancer. (2014)
Figure 2

Kaplane-Meier statistical analyses showing OS and DFS in different subgroups of GC patients.

(A and B) Patients were divided into four following subgroups based on ATG-5 immunostaining: scored 0–99 (curve a), scored 100–199 (curve b), scored 200–299 (curve c) and scored 300–400 (curve d). The difference among different subgroups was statistically significant as evaluated by overall Log-rank comparisons (OS: P<0.001, DFS: P = 0.003). Pairwise Log-rank comparisons showed that the subgroup D exhibited the poorest survival rates as compared to other subgroups (OS: P<0.05, DFS: P<0.01). (C and D) Patients were divided into four following subgroups based on MRP-1 immunostaining: scored 0–99 (curve a), scored 100–199 (curve b), scored 200–299 (curve c) and scored 300–400 (curve d). The difference among various subgroups was statistically significant by overall Log-rank comparisons (OS: P = 0.001, DFS: P = 0.018). Pairwise Log-rank comparisons showed that the subgroup A had the most favorable prognosis among the four subgroups (OS: P<0.05, DFS: P<0.001). (E and F) Patients were divided into four subgroups according to different TNM stages. The difference among different subgroups was statistically significant by overall Log-rank comparisons (OS: P<0.001, DFS: P<0.001). Pairwise Log-rank comparisons showed that subgroups III or IV exhibited poorer survival rates than that of subgroups IB and II (OS: P<0.01, DFS: P<0.001).
Upregulation of autophagy-related gene-5 (ATG-5) is associated with chemoresistance in human gastric cancer. (2014)
Figure 3

ATG-5 was upregulated in chemoresistant cells.

(A) The level of ATG5 was detected in cell lines using western blot analysis. β -actin was used as internal controls.(B) The IC 25, IC50 and IC75 of SGC-7901 and SGC-7901/DDP cells were tested using the MTT assays after DPP treatment.
Upregulation of autophagy-related gene-5 (ATG-5) is associated with chemoresistance in human gastric cancer. (2014)
Figure 4

Silencing ATG-5 sensitized chemoresistant cells to drug treatment.

(A) The mRNA level of ATG-5 was detected by real time PCR after treatment with siRNAs. GAPDH was used as internal controls. (B) The protein level of ATG-5 was detected by western blot after treatment with siRNAs. β -actin was used as internal controls. (C) The proliferation ability was tested using MTT assay 48 hours or 72 hours after different treatment.
Upregulation of autophagy-related gene-5 (ATG-5) is associated with chemoresistance in human gastric cancer. (2014)
Figure 5

Autophagy was involved in the drug resistant of DC cells.

(A) The autophagy was detected by immunofluorescence assay of LC3B in the cells 48 hours after different treatment. (B) The protein levels of LC3A and LC3B were tested by western blot. β-actin was used as internal controls.
NDP52 interacts with mitochondrial RNA poly(A) polymerase to promote mitophagy (2018)
Figure 1

Figure 1. Mitophagosome formation is reduced by NDP52 knockdown.

(A) HeLa cells stably expressing GFP-Parkin (HeLa-GFP-Parkin cells) were treated with 1 µM valinomycin and immunostained with anti-NDP52 and anti-Tom20 antibodies. Boxed areas in the images are shown in the next panels on the right. Colocalization of GFP-Parkin, NDP52, and Tom20 were determined using Line scan. Fluorescence intensities of each channels were measured along the dotted white arrow. Scale bars, 10 µm. Images are representative of three independent experiments.

(B) HeLa-GFP-Parkin cells were transfected with siNDP52 or scrambled siRNA. Seventy-two hours after transfection, cells were incubated with 1 µM valinomycin for the indicated times. Western blotting was performed using the indicated antibodies. Data are representative of three independent experiments.

(C) Mitophagy flux assay using Mtphagy dye. Increase in Mtphagy dye fluorescence intensity indicates the recruitment of mitochondria to lysosomes during valinomycin treatment. Results are a summary of three independent experiments. Values are the means ± SEM. *p < 0.05, **p < 0.01 compared with scrambled oligo-treated cells, determined with one-way ANOVA followed by the Student's t test.

(D) Fluorescence images of NDP52 KD and control cells expressing mCherry-LC3 and MTS-TagBFP. Arrows indicate mCherry-LC3 puncta colocalized with MTS-TagBFP. Scale bars, 10 µm. Boxed areas in images are shown in the next panels on the right. Images are representative of four independent experiments.

(E) The number of mCherry-LC3 puncta colocalized with MTS-TagBFP (representing mitophagosomes) per cell was counted. Results were from at least six microscopic fields of three independent experiments. Values are the means ± SEM. **p < 0.01 compared with scrambled oligo-treated cells, determined with one-way ANOVA followed by the Student's t test.

(F, G) Electron micrographs of NDP52 KD and control cells. Asterisks indicate mitochondria sequestered by autophagosomes (mitophagosomes, yellow dotted line). The ratios of mitophagosomes per mitochondrial area are shown in (G). Ten cells from three independent experiments were counted. Values are the means ± SD. *p < 0.05 compared with scrambled-oligo treated cells, determined with one-way ANOVA followed by the Student's t test.
NDP52 interacts with mitochondrial RNA poly(A) polymerase to promote mitophagy (2018)
Figure 2

Figure 2. SKICH and LIM-L domains of NDP52 are both required for mitophagy. (A, C) Images of HeLa GFP-Parkin cells expressing mCherry-NDP52 (A) or mCherry-OPTN (C) under valinomycin treatment. The images are from Movie EV 2 and 3, respectively. Cyan, GFP-Parkin; magenta, mCherry-NDP52 or OPTN. Scale bars, 10 µm. Images are representative of five independent experiments.

(B) Time-lapse images of HeLa GFP-Parkin cells expressing mCherry-NDP52 under valinomycin treatment. The images are representative of five independent experiments and correspond to the boxed regions of Figure 2A and Movie EV 2. Cyan, GFP-Parkin; magenta, mCherry-NDP52. The white arrowheads indicate colocalization of GFP-Parkin and mCherry-NDP52. The yellow arrowheads indicate mCherry-NDP existing inside GFP-Parkin ring structures.

(D) mCherry-NDP52 or OPTN signals colocalized with Parkin-ring structures and existing inside of Parkin-ring structures (irrupted) were quantified. Data were from five independent experiments in cells incubated with valinomycin for 2 h. Values are the means ± SEM. **p < 0.01 compared with mCherry-OPTN expressing cells, determined with one-way ANOVA followed by the Student's t test.

(E) Mitophagic flux in NDP52 KD cells expressing siRNA-resistant NDP52 or NDP52 mutants. Cells were incubated with valinomycin for 3 h. Results are the summary of three independent experiments. Values are the means ± SEM. *p < 0.05, **p < 0.01 vs scrambled oligo-transfected cells; †p < 0.05, ††p < 0.01 vs empty vector-transfected cells, determined with one-way ANOVA followed by the Tukey-Kramer post hoc test.

(F) HeLa-GFP-Parkin cells expressing mCherry-NDP52 mutants were incubated with 1 µM valinomycin and 100 nM bafilomycin A1 for 3 h. Cell lysates were subjected to immunoprecipitation using anti-RFP magnetic beads. Immunoprecipitates were analyzed by western blotting. Data are representative of three independent experiments.

(G) Images of HeLa GFP-Parkin cells expressing mCherry-NDP52∆LIM-L or mCherry-NDP52∆SKICH treated with valinomycin for 2h. The images are from Movie EV 4. Cyan, GFP-Parkin; magenta, mCherry-NDP52 mutant. Scale bars, 10 µm. Images are representative of three independent experiments.
NDP52 interacts with mitochondrial RNA poly(A) polymerase to promote mitophagy (2018)
Figure 3

Figure 3. Effects of TBK1 knockdown on Parkin-mediated mitophagy.

(A) Mitophagic flux in cells transfected with siNDP52, siTBK1, siOPTN or a combination of siRNAs. Results are the summary of three independent experiments. Values are the means ± SEM. **p < 0.01 vs scrambled oligo-transfected cells; †p < 0.05 vs siNDP52-transfected cells; §p < 0.05, §§p < 0.01 vs siTBK1; NS: not significant, determined with one-way ANOVA followed by the Tukey-Kramer post hoc test.

(B) HeLa-GFP-Parkin cells transfected with siNDP52, siOPTN, siTBK1 or scrambled siRNA were incubated with 1 µM valinomycin for the indicated times. Cell lysates were subjected to SDS-PAGE or Phos-tag-PAGE prior to western blotting using the indicated antibodies. Data are representative of two independent experiments. The position of phosphorylated forms of NDP52 or OPTN are indicated by arrowheads.

(C) Images of TBK1 KD cells expressing mCherry-NDP52 or mCherry-OPTN under valinomycin treatment from Movie EV 5. Cyan, GFP-Parkin; magenta, mCherry-NDP52. Images are representative of two independent experiments. Scale bars, 10 µm.

(D) TBK1 KD and control cells expressed mCherry-NDP52. Lysates were subjected to immunoprecipitation using anti-RFP beads. Immunoprecipitates were analyzed by western blotting. Data are representative of three independent experiments.