EMBO/soda-nel huggingface dataset:
Disease-causing mutations in parkin impair mitochondrial ubiquitination, aggregation, and HDAC6-dependent mitophagy. (2010)
Figure 1.

Disease-associated Parkin mutations are defective in mitophagy. (A) MEFs were transfected with GFP-tagged wild-type (WT) or mutant Parkin expression plasmid followed by an 18-h treatment of CCCP. Cells are immunostained with a Tom20 antibody to visualize mitochondria (red). GFP-parkin–transfected cells are marked by dotted lines. Arrows indicate parkin-positive mitochondria or mitochondrial aggregates. Bar, 10 µm. (B) The average percentages of mitochondria-free cells from three independent experiments from A are presented with standard deviation as error bar. **, P < 0.01 (C) MEFs were transfected and treated with CCCP as described in A, followed by an immunoblotting analysis with antibodies for cytochrome c, actin, and parkin. Note that levels of parkin R275W and R42P mutant were lower, as previously reported (Wang et al., 2005).
Disease-causing mutations in parkin impair mitochondrial ubiquitination, aggregation, and HDAC6-dependent mitophagy. (2010)
Figure 2.

CCCP-induced parkin-mitochondrial aggregate formation is an intermediate step for mitophagy. (A) MEFs expressing WT GFP-parkin were treated with CCCP or CCCP and nocodazole (NOC, 10 µM) for 8, 16, and 24 h as indicated. Cells were immunostained with anti-Tom20 to visualize mitochondria. GFP-parkin–positive cells are marked by dotted lines. Arrows indicate parkin-positive mitochondria or mitochondrial aggregates. Bar, 25 µm. (B) The average percentages of cells with mitochondrial aggregates or without mitochondria from three independent experiments from A are presented with standard deviation as error bar. **, P < 0.01.
Disease-causing mutations in parkin impair mitochondrial ubiquitination, aggregation, and HDAC6-dependent mitophagy. (2010)
Figure 3.

Parkin promotes mitochondrial ubiquitination. (A) MEFs were transfected with GFP wild-type (WT), A240R, and T415N mutant parkin followed by CCCP treatment for 8 h. Cells were immunostained with antibodies for cytochrome c (red) and ubiquitin (blue). Arrows indicate mitochondrial aggregates. Bar, 10 µm. (B) The average percentages of ubiquitin-positive mitochondrial aggregates from three independent experiments from A are presented with standard deviation as error bar. (C and D) MEFs were transfected and treated with CCCP as described in A. Cells were subjected to the fractionation to isolate crude mitochondria. Cytoplasmic and mitochondrial fractions were subjected to immunoblotting analysis using antibodies for ubiquitin, parkin, cytochrome c, and cytosolic Hsp90.
Disease-causing mutations in parkin impair mitochondrial ubiquitination, aggregation, and HDAC6-dependent mitophagy. (2010)
Figure 4.

Parkin-mediated ubiquitination recruits p62 and HDAC6. MEFs were transfected with WT, A240R, and T415N mutant GFP-parkin followed by CCCP treatment for 8 h. Cells were double immunostained with cytochrome c (red) and p62 antibody (blue) in A, and cytochrome c (red) and HDAC6 antibody in C. Arrows indicate mitochondrial aggregates. (B and D) The average percentages of p62- or HDAC6-positive mitochondrial aggregates from three independent experiments are presented with standard deviation as error bar. (E) Wild-type and HDAC6 knockout (KO) MEFs were transfected with parkin-GFP or cotransfected with a Flag-tagged HDAC6 followed by CCCP treatment for 16 h as indicated. Cells are immunostained with Tom20 (red) and Flag (blue) antibodies. Bar, 25 µm. (F) Wild-type MEFs were transfected with control or HDAC6 siRNA and parkin-GFP, and treated with or without CCCP for 16 h. Cell lysates were subjected to immunoblotting analysis using antibodies for Tom20, parkin, HDAC6, and actin.
Disease-causing mutations in parkin impair mitochondrial ubiquitination, aggregation, and HDAC6-dependent mitophagy. (2010)
Figure 5.

Cortactin is required for parkin-CCCP–induced mitochondrial clearance. MEFs were transfected with control or cortactin RNAi and parkin-GFP, and incubated with or without CCCP for 16 h as indicated. (A) Cells were subjected to immunostaining with antibodies for Tom20 and parkin. Bar, 10 µm. (B) Cell lysates were subjected to immunoblotting with antibodies for Tom20, parkin, cortactin, and actin.
Activation of the ISR is a vulnerability for multidrug resistant FBXW7-deficient cells (2022)
Figure 3

Fig. 3. Targeting mitochondrial function is preferentially toxic for FBXW7-deficient cells.

(A) Percentage of viable FBXW7+/+ (green) and FBXW7-/- (red) DLD-1 cells 72h after being treated with the indicated antibiotics. The culture started with a 1:3 ratio of FBXW7+/+ and FBXW7-/- cells (dashed line). The experiment was repeated three times, and a representative example is shown. Error bars indicate SD, two biological replicates. Cell percentages were quantified by flow cytometry and the data for each drug normalized to that of its solvent (see Methods). The dashed line represents the average percentage in the control.

(B-D) Normalized viability of FBXW7+/+ and FBXW7-/- DLD-1 (B), A2780 (C) and HeLa (D) cells upon treatment with increasing doses of tigecycline for 72h. Cell nuclei were quantified by high-throughput microscopy (HTM) upon staining with DAPI. The experiment was repeated three times, and a representative example is shown. Data from 2 independent FBXW7-/- clones is shown for each cell line. Error bars indicate SD, two biological replicates.

(E) Normalized viability of FBXW7+/+ and FBXW7-/- DLD-1 cells upon treatment with increasing doses of oligomycin for 72h. Cell nuclei were quantified by high-throughput microscopy (HTM) upon staining with DAPI. The experiment was repeated three times, with 2 biological replicates per experiment, and a representative example is shown. Data from 2 independent FBXW7 -/- clones is shown. Error bars indicate SD.

(F) Percentage of viable FBXW7+/+ (green) and FBXW7-/- (red) DLD-1 cells 7 days after being transfected with esiRNAs targeting the indicated mitochondrial factors or luciferase (RLUC) as control. The culture started with a 1:3 ratio of FBXW7+/+ and FBXW7-/- cells. The experiment was repeated three times, with two biological replicates per experiment and a representative example is shown. Error bars indicate SD. Cell percentages were quantified by flow cytometry. The dashed line represents the average percentage in the control (RLUC) condition.

(G) Tumour growth (in mm3) of FBXW7+/+ and FBXW7-/- xenografts in nude mice (n=10 animals per group). Treatment with either vehicle, paclitaxel (1,5mg/kg) or tigecycline (50mg/kg) started at day 6 post-tumour-injection, and was administered three times per week. Error bars indicate SEM.

(H) Representative images of the xenografts defined in (G) at day 15.

Data information: n.s. p>0.05, *p<0.05, **p<0.01, ***p<0.001. (A, F) t-test, (B-E, G, two-way ANOVA)
TGF-β1 secreted by Tregs in lymph nodes promotes breast cancer malignancy via up-regulation of IL-17RB (2017)
Figure 3

Figure 3. Up-regulation of Il-17rb contributes to the aggressive malignancy phenotypes of breast cancer cell derived from tumor-draining lymph node

(A) Gene expression profiles were shown at 4T1PT to 4T1LN cells. Five genes encoding cell surface proteins were identified among up-regulated genes.

(B) mRNA expression of each candidate gene in 4T1PT and 4T1LN cells was determined by RT-qPCR. Gapdh was used as an internal control.

(C and D) Il-17rb, Gpr56, and Scara5 expression in 4T1PT and 4T1LN cells were examined by Western blotting analysis.

(E) Western blotting and RT-qPCR analysis of Il-17rb, Gpr56, and Scara5 expression in 4T1 cells transduced with Il-17rb, Gpr56, Scara5, or control LacZ shRNA lentivirus respectively.

(F) Soft-agar colony forming activity was examined in lentivirus transduced shIl-17rb, shGpr56, shScara5, or shLacZ 4T1 cells (5x102 cells/well, n = 6 wells per group).

(G) Western blotting analysis was used to detect ectopic expression of Il-17rb in 4T1 cells infected with either lentiviruses carrying Il-17rb cDNA or empty vector.

(H) Soft-agar colony forming activity was examined using control or Il-17rb over-expressing 4T1 cells (5x102 cells/well, n = 6 wells per group).

(I) Western blotting analysis was used to examine Il-17rb in a representative Il-17rbDel 4T1 clone derived from Il-17rb knocked-out 4T1 cells using a CRISPR/Cas9 system.

(J and K) Tumorigenesis assays were determined in BALB/c mice (n = 3 mice per group) orthotopically injected with 5x102 4T1 cells from WT or Il-17rbDel clone. Tumor mass (J) and weight (K) were measured on day 28. Scale bar: 1 cm.

(L and M) BALB/c mice (n = 3 mice per group) were injected with 5x105 4T1 cells from WT or Il-17rbDel clone via tail-vein. Lung colonization was examined by lung morphology (L) and the numbers of tumor nodule (M) on day 21. Scale bar: 1 cm.

Data information: All experimental data were verified in at least two independent experiments. All values are presented as mean ± SD. In (B), Data were presented as means ± SD (triplicate measurement). In (C, D, E, G, and I), the intensity of each band was quantified using the Image J software, and Gapdh was used as a loading control. Relative expression (RE) of protein levels in each figure is indicated. In (F), **P = 0.00216 for the shIl-17rb 4T1 cells. In (H), **P = 0.0000009. In (K), **P = 0.00012. In (M), **P = 0.032862. Level of significance was determined using two-tailed unpaired t-test.
TGF-β1 secreted by Tregs in lymph nodes promotes breast cancer malignancy via up-regulation of IL-17RB (2017)
Figure 4

Figure 4. Tregs in the tumor-draining lymph node microenvironment mainly contribute to the up-regulation of Il-17rb in breast cancer cells.

(A) Schematic diagram of the in vitro co-culture system using 4T1 cells and total cells isolated from tumor-draining lymph nodes.

(B and C) 4T1-injected BALB/c mice were sacrificed at the indicated week after initial injection. Total cells isolated from inguinal lymph node tissues were transwell co-cultured with 4T1 cells. Inguinal lymph node tissues came from un-injection BALB/c mice as control. After 5-day co-culture, 4T1 cells at lower well were examined in the RT-qPCR (B) or Western blotting (C) analyses of Il-17rb expression. Gapdh was used as an internal control or as a loading control.

(D) Soft-agar colony forming activity was examined using co-cultured 4T1 cells at lower well (5x102 cells/well, n = 4 wells per group).

(E and F) 4T1-injected BALB/c mice were sacrificed at three weeks post initial injection. Each lymphocyte subsets isolated from inguinal lymph node tissues by FACS sorting were transwell co-cultured with 4T1 cells. After 5-day co-culture, 4T1 cells at lower well were used for the RT-qPCR (E) or Western blotting (F) analyses of Il-17rb expression. Gapdh was used as internal control or as a loading control.

(G) Percentage of CD4+Foxp3+ Tregs of total CD4+ cells in inguinal LN, spleen and peripheral blood of BALB/c mice (n = 3 mice per group) injected with 4T1 (Blue) or γ-irradiated 4T1 (Red) cancer cells was analyzed by FACS.

(H) 4T1 cells co-cultured with the indicated lymphocyte populations isolated from inguinal LN of 4T1 tumor-bearing mice by FACS sorting. After 5-day co-culture, 4T1 cells at lower well were analyzed for Il-17rb expression by RT-qPCR analysis. Gapdh was used as internal control.

Data information: All experimental data were verified in at least two independent experiments. All values are presented as mean ± SD. In (B, E, and H), values were presented as mean ± SD of triplicate measurement. In (C and F), the intensity of each band was quantified using the Image J software, and Gapdh was used as a loading control. Relative expression (RE) of Il-17rb levels in each figure is indicated. In (D), **P = 0.0069 and **P = 0.000015 for the week 2 LNs co-cultured 4T1 cells and week 3 LNs co-cultured 4T1 cells, respectively. In (G), **P = 0.000233 and **P = 0.001717 for the percentage of CD4+Foxp3+ Tregs at week 2 and week 3, respectively. Level of significance was determined using two-tailed unpaired t-test.
TGF-β1 secreted by Tregs in lymph nodes promotes breast cancer malignancy via up-regulation of IL-17RB (2017)
Figure 5

Figure 5. Depletion of Tregs in the tumor-draining lymph node microenvironment abolishes Il-17rb induction and enhanced malignancy in breast cancer cells.

(A) 4T1-injected BALB/c mice were treated with control IgG, anti-CD4 or anti-CD25 neutralizing antibody at the day 15 and day 18 after initial injection. Mice were sacrificed at three weeks after initial injection. Depletion of CD4+Foxp3+ Tregs in inguinal LNs was confirmed by FACS analysis.

(B) Western blotting analysis of Il-17rb expression was performed in 4T1LN or 4T1PT cells collected from control IgG-, anti-CD4, or anti-CD25 antibody-treated 4T1 tumor-bearing mice.

(C and D) BALB/c mice were orthotopically injected with 5x102 4T1LN or 4T1PT cells collected from control IgG-, anti-CD4, or anti-CD25 antibody-treated 4T1 tumor-bearing mice. (n = 3 mice per group). Tumor mass (D) and tumor weight (E) were measured on day 28. Scale bar: 1 cm.

(E) NSG mice (n = 5 mice per group) were injected with 5x102 4T1LN or 4T1PT cells collected from control IgG-, anti-CD4, or anti-CD25 antibody-treated 4T1 tumor-bearing mice. Distant organ metastasis was examined by bioluminscent images on day 21. The bioluminescent signal (pseudocolor) was recorded as photons per second per centimeter squared per steradian (p/s/cm2/sr) and the luminescent image was overlaid on the photographic image.

Data information: In (A-D), all experimental data were verified in at least two independent experiments. All values are presented as mean ± SD. In (B), the intensity of each band was quantified using the Image J software, and Gapdh was used as a loading control. Relative expression (RE) of Il-17rb levels in each sample to 4T1PT cells derived from control IgG-treated 4T1 tumor-bearing mice is indicated. In (D), **P = 0.000209. Level of significance was determined using two-tailed unpaired t-test.
TGF-β1 secreted by Tregs in lymph nodes promotes breast cancer malignancy via up-regulation of IL-17RB (2017)
Figure 6

Figure 6. TGF-β1 secreted from Tregs induces Il-17rb expression of breast cancer cells via Smad2/3/4 signaling.

(A) CD4+CD25+ Tregs were isolated from inguinal LN of BALB/c mice injected with 4T1 cells for three weeks, and were transwell cocultured with 4T1 cells in the presence of control IgG or neutralizing antibodies against IL-10 or TGF-β1. After 5-day coculture, 4T1 cells at lower well were analyzed for Il-17rb expression by RT-qPCR analysis. Gapdh was used as internal control.

(B and C) Western blotting analyzed Il-17rb in 4T1 cells treated with recombinant TGF-β1 (B) or IL-10 (C) proteins for 5 days.

(D) Western blotting analyzed Il-17rb in shLacZ or shTgfbr1 4T1 cells treated with recombinant TGF-β1 for 5 days.

(E) 4T1-injected BALB/c mice were sacrificed at three weeks post initial injection. Total cells isolated from inguinal lymph node tissues were transwell co-cultured with 4T1 cells. After 5-day coculture, soft-agar colony forming activity was determined in co-cultured shLacZ or shTgfbr1 4T1 cells at lower well. (5x102 cells/well, n = 6 wells per group).

(F-H) Western blotting analyzed Il-17rb in shLacZ, shSmad2 (F), shSmad3 (G), or shSmad4 (H) 4T1 cells treated with recombinant TGF-β1 for 5 days.

(I and J) WT 4T1 (I) or Il-17rbDel 4T1 (J) cells were treated with or without recombinant TGF-β1 for 5 days. After treatment, nuclear translocation of NF-κB p65 was assayed by Western blotting in cells treated with recombinant Il-17b for 2 hours. Nuclear Matrix Protein p84 was used as a loading control of nuclear extracts.

Data information: All experimental data were verified in at least two independent experiments. In (A), values were presented as means ± SD of triplicate measurement. In (B, C, D, F, G, H, I, and J), the intensity of each band was quantified using the Image J software. Gapdh, α-tubulin, or p84 was used as a loading control. Relative expression (RE) of protein levels in each sample to control 4T1 cells or Il-17rbDel 4T1 clone is indicated. In (E), Data were presented as means ± SD. **P = 0.000615. Level of significance was determined using two-tailed unpaired t-test.
TGF-β1 secreted by Tregs in lymph nodes promotes breast cancer malignancy via up-regulation of IL-17RB (2017)
Figure 7

Figure 7. IL-17RB expression is correlated with the elevated prevalence of regulatory T cells in the tumor-draining lymph nodes of breast cancer patients. (A and C) Representative IHC images of IL-17RB (A) and Foxp3 (C) were taken in breast specimens from primary tumors and their paired LN metastasis specimens. Scale bar: 10 μm.

(B) Dot plot showed the results of composite IHC score (staining intensity × percentage of positively stained cell) of IL-17RB from primary tumors (PT; n=60) and their paired LN metastasis (LN; n=60) specimens.

(D) The correlation of IL-17RB expression (composite IHC score) and Foxp3+ Tregs (percentage of total cells) in LN metastasis specimens (n=60). Statistical analysis was performed with the Pearson"s correlation test.

Data information: In (B), **P = 0.0015. Level of significance was determined using Mann-Whitney U test statistic analysis. In (D), **P = 0.001. Statistical analysis was performed with the Pearson"s correlation test.
TGF-β1 secreted by Tregs in lymph nodes promotes breast cancer malignancy via up-regulation of IL-17RB (2017)
Figure 8

Figure 8. Elevated IL-17RB expression in cancer cells derived from tumor-draining lymph nodes in breast cancer patients correlates with aggressive growth nature.

(A) Freshly collected human breast cancer (hBC) specimens (PT, primary tumors; LN, paired LN metastasis from the same breast cancer patient) were digested with collagenase-containing buffer overnight. Depletion of hematopoietic cells by CD45 Dyna beads (Invitrogen) enriched the EpCAM+ cells for following xenografts. Percentages of EpCAM+ cells in human primary breast cancer cells were confirmed by FACS analysis (Left panel: before enrichment; Right panel: after enrichment). All of the histograms were shown that they were EpCAM expressing (black line), compared to isotype control (gray filled).

(B) Tumorigenesis assay were determined in NOD/SCID/IL2Rγnull mice orthotopically injected with hBCPT and hBCLN cells. Representative data on day 180 were shown.

(C) Dot plot illustrated the tumor volumes of hBCPT and hBCLN cells-injected NOD/SCID/IL2Rγnull mice on day 180 (n = 11). All tumors were confirmed by pan-keratin IHC staining of fat pad sections.

(D and E) H&E stainings and IHC stainings of pan-keratin were shown in the representative cases of fat-pad sections from (B). Scale bar: 1 mm (left panel), 100 μm (right panel).

(F) IHC stainings of IL-17RB were shown in the representative cases of fat-pad sections from (B). Scale bar: 10 μm.

(G) The correlation of tumor volumes and IL-17RB expression (composite IHC score) in hBCLN cells-injected NOD/SCID/IL2Rγnull mice.

Data information: In (C), **P = 0.0279. Level of significance was determined using Mann-Whitney U test statistic analysis. In (G), Statistical analysis was performed with the Pearson"s correlation test.
Higher CSF sTREM2 and microglia activation associate with slower rate of beta-amyloid accumulation (2020)
Figure 1

Figure 1: Association between baseline amyloid and amyloid PET change:
A, B Regression plot showing the association between baseline AV45 PET (x-axis) and annual AV45 PET change (y-axis) across the entire sample (N=300). The vertical dashed line represents the Aβ-positivity threshold of AV45 PET SUVR > 0.079 (A). Regression plot for the association between baseline AV45 PET and annual AV45 PET change stratified by baseline CSF sTREM2 levels (B). Source data are available in the supplementary (Fig1_source_data.xlsx)
Higher CSF sTREM2 and microglia activation associate with slower rate of beta-amyloid accumulation (2020)
Figure 2

Figure 2: Rate of change in Florbetaben PET vs baseline GE180 PET:

A, B Boxplots (central band = median, boxes = Quartiles, whiskers = 1.5 * interquartile range), illustrating the differences in GE180 PET (A) and the rate of change in Florbetaben PET (B) compared between wild type C57BL/6 mice (n=43) and APPNL-G-G (n=15).

C The regression plot shows lower rate of change in Florbetaben PET to be associated with lower GE180 PET at baseline, i.e. at 5 months of age (C). The shaded area corresponds to the 95% CI of the regression line. Source data are available in the supplementary (Fig2_source_data.xlsx)
Higher CSF sTREM2 and microglia activation associate with slower rate of beta-amyloid accumulation (2020)
Figure 3

Figure 3. Brain rendering of GE180 and Florbetaben PET by group and time point:

A-C Coronal (top row) and axial (bottom row) slices of average 18-F GE-180 TSPO (microglia) tracer and 18F-florbetaben amyloid-PET split up by group (A - C): C57BL/6 (A), APPNL-G-F mice subgroup with a low (< median) GE-180 PET (B), and the APPNL-G-F mice subgroup with a high (> median) GE-180 PET (C). APPNL-G-F mice with low baseline GE180 PET levels showed faster increase in Florbetaben PET between 5 and 10 months (B) compared to those at high baseline level of GE-180 PET (C).
Higher CSF sTREM2 and microglia activation associate with slower rate of beta-amyloid accumulation (2020)
Figure 4

Figure 4: TREM2 vs. tau-PET:
Effects of sTREM2 on AV1451-PET-assessed tau pathology stratified by Braak-stages shown as boxplots (central band = median, boxes = Quartiles, whiskers = 1.5 * interquartile range). Source data are available in the supplementary (Fig4_source_data.xlsx)
Muscle-specific Cand2 is translationally upregulated by mTORC1 and promotes adverse cardiac remodeling (2021)
Figure 1

Figure 1. Identification of the mTORC1-dependent cardiac translatome.

(A) Experimental design of translational profiling of mTOR-dependent mRNAs in vitro in NRCMs. Polysomal fractions were isolated on sucrose gradient and ribosomal footprints were recovered by ribonuclease digestion. NRCMs were treated with vehicle or Torin 1 (100nM for 3h) to block the mTOR pathway.

(B) Scatter plot of ribosome occupancy in Ribo-seq of cultured cardiomyocytes in response to PE and Torin 1 treatment (blue dots - mRNAs upregulated after Torin 1, red dots - downregulated mRNA. RPKM - Reads per kilobase million.

(C) Percentage of IRES (Internal Ribosomal Entry Site) and TOP or TOP-like containing mRNAs in total and Torin 1-sensitive mRNA pools.

(D) Transcriptional (RNA-seq) vs. translational (Ribo-seq) control of mTOR-regulated mRNAs.

(E) Network of enriched GO categories (biological process) among mTOR-sensitive genes into several functional groups represented by circles of different colors. Each node represents one enriched GO category with the size of the node is proportional to the number of transcripts. Edges indicate similarity (between the two connected GO categories).

(F) Cumulative fraction of mRNAs relative to their fold change of Ribo-seq and RNA-seq between (G) all and mTOR-dependent transcripts two days after TAC surgery. Kolmogorov-Smirnov-Wallis test.

(H) Immunoblot and RT-qPCR (I) representing levels mTOR-dependent targets with defined 5'TOP motifs 3 hours and 2 days after TAC. n=3 biological replicates per condition.

(J) Immunoblots and quantifications of protein expression of mTOR targets two days after TAC surgery and after mTOR inhibition with Torin 1.* p≤ 0.05 vs sham surgery, # p≤ 0.05 vs TAC 2d surgery. t-test, n=5 biological replicates per group.

(K) RT-qPCR for indicated mRNAs 2 days after TAC for indicated groups, n=5 biological replicates per group.

Data information: Error bars indicate means ± SEM