EMBO/soda-nel huggingface dataset:
RANKL from Bone Marrow Adipose Lineage Cells Promotes Osteoclast Formation and Bone Loss (2021)
Figure 6

Figure 6. RANKL knockout from BM adipose lineage cells mediates rosiglitazone-induced bone loss.

(A-B) Three-dimensional CT analyses of BMD, BV/TV, Tb.N, and Tb.Sp in trabecular bone from Vehicle (n=5 independent biological replicates) and ROS (n=6 independent biological replicates) model of AdipoqCre; ranklfl/fl (Rankl-/-) and ranklfl/fl (Rankl+/+) mice. Scale bar=1 mm. Data were compared using an unpaired t‐test (** indicates P < 0.01), error bars are standard deviations.

D) TRAP staining and statistical analyses of numbers of osteoclasts in the trabecular bone from Vehicle (n=6 and 5 independent biological replicates) and ROS (n=6 and 6 independent biological replicates) treatment. Scale bar=100 µm. Data were compared using an unpaired t‐test (** indicates P < 0.01), error bars are standard deviations. n=5 independent microscopic vision fields in (D).

(E-F) OCN staining and statistical analyses of the number of osteoblasts in femurs from Vehicle (n=6 and 5 independent biological replicates) and ROS (n=6 and 6 independent biological replicates) treatment. Scale bar=50 µm. TB: trabecular bone. Data were compared using an unpaired t‐test (** indicates P < 0.01), error bars are standard deviations. n=5 independent microscopic vision fields in (F).
Mitochondrial Rab GAPs govern autophagosome biogenesis during mitophagy. (2014)
Figure 1.

TBC1D15 is dispensable for mitochondria and peroxisome morphologies.

(A) Total cell lysates prepared from the indicated HCT116 cell lines were analyzed by immunoblotting. For comparison, different amounts of proteins (1:3 ratio) were applied. An asterisk indicated non-specific crossreactive bands. (B) The indicated cell lines were analyzed by immunofluorescence microscopy using anti-Cytochrome c antibody for mitochondria and anti-PMP70 antibody for peroxisome staining. Images are displayed as z-stacks of 6 confocal slices. Magnified images are also shown for mitochondrial morphologies. Scale bars, 10 μm. (C) Quantification of mitochondrial morphologies in (B). Percentages of cells harboring fragmented, tubular, or elongated mitochondria are shown. Tubular and elongated denote normal tubular mitochondria seen in WT cells and highly connected mitochondrial network, respectively. The error bars represent ±SD from three independent replicates. Over 50 cells were counted in each of three replicate wells.

DOI:http://dx.doi.org/10.7554/eLife.01612.003
Mitochondrial Rab GAPs govern autophagosome biogenesis during mitophagy. (2014)
Figure 2.

Loss of Fis1 or TBC1D15 causes LC3 accumulation during mitophagy.

(A) The indicated cell lines stably expressing YFP-LC3 and mCherry-Parkin were treated with valinomycin for 3 hr and subjected to confocal immunofluorescence microscopy with anti-TOMM20 antibody. Scale bars, 10 μm. (B) Quantification of mCherry-Parkin translocation to mitochondria after 3 hr of valinomycin treatment. Partial or complete translocation to mitochondria in each cell was scored as separate phenotypes. Partial and complete denote that Parkin translocates to some of or all mitochondria, respectively. The error bars represent ±SD from three independent experiments. Over 100 cells were counted in each of three separate wells. (C) YFP-LC3 morphologies in (A) were quantified. Percentages of cells harboring diffuse, punctate or accumulated YFP-LC3 are shown. The error bars represent ±SD from three independent replicates. Over 100 cells were counted in each replicate. For the criteria of LC3 morphology, see Figure 2—figure supplement 1. (D) YFP-LC3 and mCherry-Parkin stably expressing TBC1D15−/− cells in the absence or presence of HA-tagged TBC1D15 WT or Δ221-250 mutant were treated with valinomycin for 3 hr. Cells were subjected to immunofluorescence microscopy with anti-HA antibody. Scale bars, 10 μm. (E) The YFP-LC3 morphology of cells in (D) was quantified. The error bars represent ±SD from three independent replicates. Over 50 cells were counted in each well. (F and G) YFP-LC3 and mCherry-Parkin stably expressing WT (F) and TBC1D15−/− (G) cells were treated with valinomycin for 3 hr and then subjected to immunoelectron microscopy with anti-GFP antibody. The square in panel a shows enlarged areas in panel b Scale bars, 500 nm.

DOI:http://dx.doi.org/10.7554/eLife.01612.005
Mitochondrial Rab GAPs govern autophagosome biogenesis during mitophagy. (2014)
Figure 3.

Tubular LC3 expands along microtubules.

(A) The indicated cells stably expressing YFP-LC3 and mCherry-Parkin were treated with valinomycin for 3 hr followed by immunofluorescence microscopy with anti-Cytochrome c antibody. Confocal images were acquired as z-stacks comprising 6 sequential sections with 0.8 μm z-intervals. Scale bars, 10 μm. (B) TBC1D15−/− cells prepared as in (A) were subjected to immunoelectron microscopy with anti-GFP antibody. White arrowheads indicate YFP-LC3-labeled tubules. High magnification image is shown in the lower panel. Scale bars, 500 nm. (C) The indicated cells prepared as in (A) were subjected to immunostaining with anti-Tubulin antibody. YFP-LC3 and Tubulin staining are merged in the right panels. Magnified images are shown for TBC1D15−/− cells. Scale bars, 10 μm. (D) The indicated cells stably expressing YFP-LC3 and mCherry-Parkin were treated with valinomycin in the presence or absence of nocodazole for 3 hr. YFP-LC3 and mCherry-Parkin images are merged in the right panels. Scale bars, 10 μm. (E) YFP-LC3 morphologies of cells in (D) were quantified. The error bars represent ±SD from three independent replicates. Over 50 cells were counted in each well.

DOI:http://dx.doi.org/10.7554/eLife.01612.012
Mitochondrial Rab GAPs govern autophagosome biogenesis during mitophagy. (2014)
Figure 4.

Loss of Fis1 or TBC1D15 impedes clearance of damaged mitochondria.

(A and B) YFP-Parkin stably expressing cells were treated with valinomycin for indicated times. Total cell lysates were subjected to immunoblotting. I and II denote cytosolic and lipidated LC3B, respectively. An asterisk indicates ubiquitinated TOMM20. (C) Indicated protein amounts as in (B) were quantified. The amount of protein without valinomycin treatment was set to 100%. The error bars represent ±SD from three independent experiments. (D) YFP-Parkin stably expressing WT HCT116 cells were treated with or without valinomycin for 40 hr. Total cell lysates were analyzed by immunoblotting.

DOI:http://dx.doi.org/10.7554/eLife.01612.015
Mitochondrial Rab GAPs govern autophagosome biogenesis during mitophagy. (2014)
Figure 5.

Effect of mitochondrial fission and starvation induction on LC3 accumulation in WT, <italic>FIS1−/−</italic> and <italic>TBC1D15−/−</italic> cells.

(A) WT, MFF−/− or DRP1−/− cells stably expressing YFP-LC3 and mCherry-Parkin were treated with valinomycin for 3 hr and subjected to immunofluorescence microscopy with anti-TOMM20 antibody. Scale bars, 10 μm. (B) Quantification of mCherry-Parkin translocation to mitochondria after 3 hr of valinomycin treatment. Partial and complete denote that Parkin translocates to some of and all mitochondria, respectively. The error bars represent ±SD from three independent replicates. Over 50 cells were counted in each replicate. (C) YFP-LC3 morphologies of cells in (A) were quantified. Percentages of cells harboring diffuse, punctate or accumulated YFP-LC3 are shown. The error bars represent ±SD from three independent replicates. Over 100 cells were counted in each replicate. (D) WT, FIS1−/−, and TBC1D15−/− cells stably expressing YFP-LC3 were grown in starvation media. Z-stacks of confocal images are shown. Scale bars, 20 μm. (E) The number of YFP-LC3 dots in cells under growth or starvation conditions was quantified. The error bars represent ±SD from three independent replicates. Over 50 cells were counted in each well. (F) Total cell lysates from cells grown in normal or starvation media for 6 hr were subjected to immunoblotting. LC3-I and LC3-II denote cytosolic and lipidated LC3B, respectively.

DOI:http://dx.doi.org/10.7554/eLife.01612.016
Mitochondrial Rab GAPs govern autophagosome biogenesis during mitophagy. (2014)
Figure 6.

Identification of TBC1D17 as a Fis1 and TBC1D15 binding protein.

(A) TBC1D15−/− cells and those stably expressing HA-TBC1D15 WT and HA-TBC1D15 (Δ221-250), and FIS1−/− cells stably expressing HA-TBC1D15 WT were subjected to immunostaining with anti-TOMM20 and anti-HA antibodies. Scale bars, 10 μm. (B) HA-TBC1D15 WT or HA-TBC1D15 (Δ221-250) with or without Fis1 was transiently overexpressed (OE) in HeLa cells. Cells were subjected to immunostaining with anti-HA and anti-Fis1 antibodies. Scale bars, 20 μm. (C) YFP-TBC1D17 together with pcDNA vector or Fis1 was transiently overexpressed (OE) in HeLa cells. Cells were subjected to immunostaining with anti-Fis1 and anti-Cytochrome c antibodies. Scale bars, 20 μm. (D) YFP or YFP-Fis1 was co-overexpressed with HA-TBC1D15 or HA-TBC1D17 in HEK293 cells. The cell extracts were subjected to pull down assays with GFP-Trap. 5% input and bound fractions were analyzed by immunoblotting with anti-HA (upper panel) and anti-GFP (lower panel) antibodies. (E) YFP, YFP-TBC1D15, or YFP-TBC1D17 was co-overexpressed with HA-TBC1D15 (upper panel) or HA-TBC1D17 (lower panel) in HEK293 cells. The cell extracts were subjected to pull down assays with GFP-Trap. 5% input and bound fractions were analyzed by immunoblotting with anti-GFP and anti-HA antibodies. (F) HA-TBC1D15 (Δ221-250) together with Fis1 and YFP-TBC1D15 WT or YFP-TBC1D17 WT were transiently overexpressed (OE) in HeLa cells. Cells were subjected to immunostaining with anti-HA and anti-Fis1 antibodies. Images of HA and Fis1 staining were merged in the right panels. Scale bars, 20 μm. (G) Schematic model of Fis1, TBC1D15, and TBC1D17 binding. Homo- or hetero-dimer of TBC1D15 can interact with Fis1 dimer on the mitochondrial outer membrane (OMM).

DOI:http://dx.doi.org/10.7554/eLife.01612.017
Mitochondrial Rab GAPs govern autophagosome biogenesis during mitophagy. (2014)
Figure 7.

TBC1D15 binds ATG8 family proteins.

(A) The indicated cells transiently expressing YFP-GABARAPL1 (green) and mCherry-Parkin (red) were treated with valinomycin for 3 hr. Scale bars, 10 μm. (B) YFP-TBC1D15 overexpressed in HEK293 cells was subjected to binding assays with GST-fused proteins (GABA, L1, and L2 represent GABARAP, GABARAPL1, and GABARAPL2, respectively). 5% input and bound fractions were analyzed by immunoblotting with anti-GFP antibody (upper panel). Coomassie brilliant blue (CBB) staining shows GST-fusion proteins in bound fractions (lower panel). (C) Binding assay carried out as in (B) with GST-GABARAPL1 WT (GST-WT) or its Y49A/L50A mutant (GST-YL). Immunoblotting with anti-GFP antibody (upper panel) and CBB staining (lower panel) are shown. (D) Cell extracts from HEK293 overexpressed HA-TBC1D15 and YFP, YFP-GABARAPL1 (YFP-WT), or its Y49AL50A mutant (YFP-YL) were subjected to pull down assays with GFP-Trap. 5% input and bound fractions were analyzed by immunoblotting with anti-HA (upper panel) and anti-GFP (lower panel) antibodies. (E–H) The indicated YFP-tagged TBC1D15 full-length, truncated, or point-mutant protein or YFP-Fis1 overexpressed in HEK293 cells were subjected to binding assays with recombinant GST-GABARAPL1. 5% input and bound fractions were analyzed by immunoblotting with anti-GFP antibody (upper panel) and CBB staining (lower panel). (I) Summary of binding abilities of truncated or point-mutated TBC1D15 constructs. –, +, ++, and +++ indicates binding of recombinant GST-GABARAPL1 to less than 1%, 1–5%, 5–10%, and over 10%, respectively of the total YFP-TBC1D15 fragment. Yellow boxes indicate YFP tags. (J) YFP-LC3 and mCherry-Parkin stably expressing TBC1D15−/− cells in the presence of HA-tagged TBC1D15 WT or F280A mutant were treated with valinomycin for 3 hr. Cells were subjected to immunofluorescence microscopy with anti-HA antibody. Scale bars, 10 μm. (K) The YFP-LC3 morphology of cells in (J) was quantified. The error bars represent ±SD from three independent replicates. Over 50 cells were counted in each well.

DOI:http://dx.doi.org/10.7554/eLife.01612.021
Mitochondrial Rab GAPs govern autophagosome biogenesis during mitophagy. (2014)
Figure 8.

Rab7 is involved in autophagosome fusion during mitophagy.

(A) Total cell lysates from HCT116 treated with control (NTC) or RAB7A siRNA were analyzed by immunoblotting. (B) The indicated cells stably expressing YFP-LC3 (green) and mCherry-Parkin were treated with control (NTC) or Rab7_#5 siRNA. After 3 hr valinomycin treatment, cells were analyzed by immunofluorescence microscopy with anti-TOMM20 antibody (red). Z-stacks of confocal images are shown. Magnified images are also shown. Scale bars, 10 μm. (C) YFP-LC3 morphologies of cells in (B) were quantified. Percentages of cells harboring diffuse, punctuate or accumulated/tubulated YFP-LC3 are shown. Data and error bars were obtained from at least 50 cells in each of three independent replicates. (D) The indicated cells stably expressing YFP-LC3 (green), mCherry-Parkin, and 2HA-Rab7 (Red) were treated with or without valinomycin for 3 hr and analyzed by immunofluorescence microscopy with anti-HA antibody. Magnified images are also shown. Scale bars, 10 μm. (E) YFP-LC3 and mCherry-Parkin stably expressing TBC1D15−/− cells in the presence of HA-tagged TBC1D15 WT or the D397A mutant were treated with valinomycin for 3 hr. Cells were subjected to immunofluorescence microscopy with anti-HA antibody. Scale bars, 10 μm. (F) The YFP-LC3 morphology of cells in (E) was quantified. The error bars represent ±SD from three independent replicates. Over 50 cells were counted in each replicate.

DOI:http://dx.doi.org/10.7554/eLife.01612.024
Macrophages induce malignant traits in mammary epithelium via IKKε/TBK1 kinases and the serine biosynthesis pathway (2019)
Figure 1

Fig 1: Macrophage-conditioned medium promotes anchorage-independent growth and invasiveness of MCF10A cells.

A) Macrophage-conditioned medium induces MCF10A colony formation in soft agar as compared to control medium. Colonies ≥ 50 μm were counted at 5 weeks. Error bars represent mean ± SEM from 10 independent experiments (n=3 per condition). Data are shown also in Fig. 4A and 6A.

B-I) 16-days old MCF10A spheroids grown in matrigel/collagen mix were stimulated for 24h with either macrophage-conditioned or control medium.

B) Representative images of MCF10A spheroids categorized into 4 groups according to the filling of the lumen with cell nuclei (clear, partially filled, almost filled and filled). Spheroids stained for DNA (Hoechst 33342 in blue) and F-actin (Phalloidin in red).

C) Macrophage-conditioned medium induces filling of the spheroid lumen with cell nuclei compared to control. Error bars represent mean ± SEM from 11 independent experiments (n=2 per condition; 50 spheroids each). Data are shown also in Fig. 4D and 6D.

D) Representative images of Ki67 positive nuclei of MCF10A cells. Spheroids stained for DNA (Hoechst 33342 in blue), F-actin (Phalloidin in red) and Ki67 (green).

E) Quantitative analysis of Ki67 positive nuclei per spheroid indicates enhanced proliferation of MCF10A cells upon stimulation with M1 macrophage-conditioned media. Error bars represent mean ± SEM from 3 independent experiments (20-30 non-empty spheroids per condition).

F) NSC23766 (Rac1 inhibitor; 50 μM) reduces the filling of the spheroid lumen with cell nuclei upon macrophage-conditioned medium stimulation compared to control. Filling of the spheroid lumen with cell nuclei categorized into 4 groups (clear, partially filled, almost filled and filled). Error bars represent mean ± SEM from 3 independent experiments (n=2 per condition; 50 spheroids each). Partially filled, almost filled and filled MCF10A spheroids were combined together (non-empty spheroids) for statistical analysis. All the data shown without the use of NSC23766 are also included in Fig. EV3D.

G) Invasive protrusions of spheroids into matrigel/collagen mix marked with a white arrow.

H) 3D structures of spheroids stained for DNA (Hoechst 33342 in blue), F-actin (Phalloidin in red) and laminin V (green). Laminin V staining indicates the loss of basal membrane continuity at invasive protrusions. Invasive protrusions are marked with a white arrow.

I) Macrophage-conditioned medium induces invasive protrusions in MCF10A spheroids compared to control. Error bars represent mean ± SEM from 11 independent experiments (n=2 per condition; at least 15 spheroids each from at least 2 fields of view). Data are shown also in Fig. 4E and 6E.

Macrophage donors are indicated as D1-D16. M1D - M1 differentiated, M1A - M1 activated, M2D - M2 differentiated, M2A - M2 activated macrophages. *p < 0.05 as measured by one-way ANOVA with uncorrected Fisher's LSD post hoc test (exact P values are shown in Table EV3). Scale bar: 50 μm.
Macrophages induce malignant traits in mammary epithelium via IKKε/TBK1 kinases and the serine biosynthesis pathway (2019)
Figure 3

Fig 3: IKKε expression in breast cancer is associated with immune infiltration.

A) IKKε expression was assessed semi-quantitatively using a 0-3 grade scale ("0": no staining; "1": weak; "2": moderate; "3": strong) on human breast cancer sections using immunohistochemistry. Representative images according to the IKKε staining grade are shown. Scale bar: 100 μm.

B) Distribution table of IKKε expression in the cohort of 66 human breast carcinomas according to the staining grade.

C) Correlation between IKKε expression and immune cell infiltration ("0": no inflammatory cells; "1": weak; "2": moderate; "3": strong; "4": very strong). Bubble plot of IKKε and immune infiltration grades, size shows the number of tumours falling in the category. Spearman's rho coefficient and significance of difference from slope = 0 (p) are shown.

D) Correlation between IKBKE mRNA levels (log2 ratio) and immune signature (Yoshihara et al, 2013) in the METABRIC transcriptomic dataset from 1981 breast cancer patients (Curtis et al, 2012). Pearson's correlation rho coefficient and significance of difference from slope = 0 (p) are shown.

E) IKBKE gene copy number and mRNA levels (log2 ratio) from the METABRIC transcriptomic dataset (see D) are plotted. Estimated +1 copy number values are shown by the dotted vertical line. No significant correlation by Pearson's correlation rho coefficient and significance of difference from slope = 0 (p) was detected.
Macrophages induce malignant traits in mammary epithelium via IKKε/TBK1 kinases and the serine biosynthesis pathway (2019)
Figure 5

Fig 5: PSAT1 expression in breast cancer is associated with IKKε expression and immune infiltration.

A) PSAT1 expression was assessed semi-quantitatively using a 0-3 grade scale ("0": no staining; "1": weak; "2": moderate; "3": strong) on human breast cancer sections using immunohistochemistry. Representative images according to the PSAT1 staining grade are shown. Scale bar: 50 ­μm.

B) Distribution table of PSAT1 expression in the cohort of 41 human breast carcinomas according to the staining grade.

C) Correlation between PSAT1 and IKKε expression ("0": no staining; "1": weak; "2": moderate; "3": strong). Bubble plot of PSAT1 and IKKε staining grades, size shows the number of tumours falling in the category.

D) Correlation between PSAT1 and and immune cell infiltration ("0": no inflammatory cells; "1": weak; "2": moderate; "3": strong; "4": very strong). Bubble plot of PSAT1 and immune infiltration grades, size shows the number of tumours falling in the category.

E-G) Correlation of mRNA levels of the SBP enzyme genes PSAT1 (E), PHGDH (F), PSPH (G) with the immune signature (Yoshihara et al, 2013) in the METABRIC transcriptomic dataset from 1981 breast cancer patient (Curtis et al, 2012).

Spearman's (C,D) or Pearson's (E-G) correlation rho coefficient and significance of difference from slope = 0 (p) are shown.
Macrophages induce malignant traits in mammary epithelium via IKKε/TBK1 kinases and the serine biosynthesis pathway (2019)
Figure 7

Fig 7: Amlexanox delays tumour formation in a diet-induced obesity/PyMT-MMTV mouse model.

A-J)Ten per group female C57Bl/6 mice heterozygous for the PyMT-MMTV started high fat diet (HFD) when 6-7 weeks old (prior of mammary tumour development) together with control mice on normal diet (ND; n=10). Mice were administered amlexanox (17 mM) or vehicle control daily by oral gavage (Reilly et al, 2013). A control group (wild-type mice) was also included to confirm the efficacy of the diet and to control for possible unpredicted interactions of the MMTV-PyMT gene.

A) Schematic representation of combined genetic mouse model of breast cancer and high fat diet-induced obesity used to study the effects of amlexanox in tumour formation.

B) Wild-type mice on HFD gain more weight compared to ND mice. *p < 0.05 by one-tailed students t-test.

C) Average adipocytes size (major diameter) in mammary fat pads is larger in HFD mice compared to ND mice based on the analysis of at least 100 adipocytes per mice on H&E stained sections. *p < 0.05 by two-tailed students t-test.

D) The number of F4/80+ macrophages in mammary fat pads is increased in HFD mice compared to ND mice. The average of F4/80+ macrophages in at least 10 fields of view per mouse is shown. *p < 0.05 by two-tailed students t-test.

E) PyMT-MMTV+/- mice on HFD gain more weight compared to ND mice. *p < 0.05 by one-tailed students t-test.

Amlexanox does not affect weight gain either in mice on F) ND or G) HFD.

H) Mammary tumours are developing earlier in PyMT-MMTV+/- mice on HFD compared to ND mice.

I) Amlexanox does not affect tumour development in PyMT-MMTV+/- mice on ND, but delays tumour formation in J) HFD mice.

H-J) P value was calculated using log-rank test.

Error bars represent mean and SD. Exact P values are shown in Table EV3.

Table EV1

Human cytokine array profile.

Media conditioned by human primary macrophages or MCF10A cell line was analysed.

Macrophage donors are D19, D22 and D25. M1D - M1 differentiated, M1A - M1 activated, M2D - M2 differentiated, M2A - M2 activated macrophages.

Data show mean± SEM from 3 independent experiments (n=2).

Table EV2

Characteristics of breast carcinoma patients and analysed tissues.

IKK
ATR inhibition potentiates ionizing radiation-induced interferon response via cytosolic nucleic acid-sensing pathways (2020)
Figure 1

Figure 1. ATR inhibition significantly potentiates ionizing radiation (IR)-induced inflammatory signals.

A, Hierarchical clustering analysis of RNA-sequencing data from cells treated with DMSO, 20 Gy IR, 250nM AZD6738, or AZD6738 + IR (n=2 biological replicates).

B, Top enriched pathway analysis of differentially expressed genes in each cluster as shown in A.

C, Heat map of 33 representative inflammatory genes regulated by AZD6738 + IR.

D, MCF10A WT cells were pretreated with DMSO or AZD6738(250 nM) for 2 hours, and then irradiated with 20 Gy, cells were harvested at indicated time points and immunoblotting was performed with indicated antibodies. h, hours; pSTAT1, pSTAT1(Y701); pIRF3, pIRF3(S396); pTBK1, pTBK1(S172).

E, MCF10A WT cells were pretreated with DMSO or AZD6738 (250 nM) for 2 hours, and then irradiated with indicated dose. 72 hours later, cells were harvested for immunoblotting with indicated antibodies.

F, Same condition as E except the irradiation dose with 20 Gy, mRNA levels of inflammatory genes as indicated were assessed by real-time quantitative PCR, presented as mean + standard error of the mean of three biological replicates.