EMBO/soda-nel huggingface dataset:
Antagonizing miRNA-19a/b augments PTH anabolic action and restores bone mass in osteoporosis in mice (2022)
Figure 7

Fig 7. MiR-19a/b expression is increased in osteoporotic bones from mice, due to in vitro sex steroid-deficiency and in bones from humans with lower bone mass.

A, B Relative miR-19a (A) and miR-19b (B) expression in long bones of sham-operated (n=4) and ovariectomized (OVX) (n=4) mice.

C expression of Tgif1 mRNA in long bones of sham-operated (n=11) and OVX (n=6) mice.

D, E Relative expression of miR-19a (D) and miR-19b (E) in Ocy454 cells upon estrogen deficiency (ED) and control (C) treatment (n=6).

F, G Expression of Tgif1 (F) and PP2A-Cβ (G) mRNA in Ocy454 cells after control in response to ED (n=5-7).

H Expression of Rankl mRNA in Ocy454 cells transfected with scrambled (scr) control oligonucleotide or anti-miR-19a/b in the context of ED (n=9).

I Representative images of von Kossa-stained human bone samples obtained from non-fractured femoral heads of postmenopausal women after assignment to groups of relatively higher bone mass (HBM, n=4) and relatively lower bone mass (LBM, n=4).

J Histomorphometric analysis of bone mass (BV/TV, bone volume/tissue volume), Trabecular number (Tb.N/mm), trabecular thickness (Tb.Th) and trabecular separation (Tb.Sp) of the same human bone samples as in (I).

K, L Representative µCT images of 3D reconstructed human bone samples (K) and quantification of bone mineral density (BMD) (L). HBM, n=4, LBM, n=4.

M, N Relative expression of miR-19a (M) and miR-19b (N) in association with higher and lower bone mass, HBM, n=4, LBM, n=4.

O Expression of Tgif1 mRNA in human bone samples, HBM, n=4, LBM, n=4.

P Schematic model of the mechanism by which miR-19a/b and anti-miR-19a/b treatment affects bone remodeling.

Data information: Mean values ± SEM. In the box blots (J, L-O) the central band indicates median, and the whiskers indicate minimum and maximum values. One-way ANOVA followed by Tukey's post-hoc analysis was used for statistical analysis (H). Student's t-test was used for statistical analysis of all other experiments. *p<0.05, **p<0.01, ***p<0.001 vs. sham (A-C), vs. control (C) (D-G) vs. C; scr (H), vs. HBM (J-O); ##p<0.01 vs. ED; scr.

Extended View (EV) Figure legends
Somatically Mutated ABL1 is an Actionable and Essential NSCLC Survival Gene (2015)
Figure 2

Figure 2. Drug mediated effects on cell viabilities are specific to ABL1 inhibition. A -C. Drug-resistant ABL1-T315I rescues dasatinib- and imatinib-induced cytotoxicity. Western blot analysis of (A) transient and (B) tetracycline-inducible overexpression of ABL1-T315I in H1915 cells treated with dasatinib or imatinib. C. CFA with dasatinib or imatinib for H1915 cells with or without tetracycline-inducible expression of ABL1-T315I. Error bars represent mean ±S.E.M., n=9, *P<0.001 by two-tailed unpaired Student’s t-test (control, DMSO vs dasatinib P=4.3×10-12 vs imatinib P=6×10-6; dasatinib, control vs tetracycline, P=9×10-7; imatinib, control vs tetracycline P=9.7×10-5). D-F. Knockdown of mutated ABL1 reduces viability of lung cancer cells. Top, western blot analysis of ABL1 knockdown in (D) Beas-2B, (E) H1915 and (F) H2110 cells upon transfection with three ABL1 siRNAs, negative control (scrambled) and CyclophilinB positive control. Bottom, MTT assay showing viability of (D) Beas-2B, (E) H1915 and (F) H2110 cells in response to ABL1 knockdown. Error bars represent mean ±S.E.M, n=9, *P<0.005, **P<0.001 by two-tailed unpaired Student’s t-test (Beas-2B, scrambled vs siABL1-3 P=0.002; H1915, scrambled vs siABL1-1 P=0.004, vs siABL1-2 P=0.001, vs siABL1-3 P=0.002; H2110 scrambled vs siABL1-1 P=5×10-6, vs siABL1-2 P=2×10-5, vs siABL1-3 P=4.2×10-6).
Somatically Mutated ABL1 is an Actionable and Essential NSCLC Survival Gene (2015)
Figure 3

Figure 3. Imatinib reduces tumor growth in vivo. A-C. Average tumor volumes of xenografts derived from H1915 (A), H650 (B) and (C) H1915 cell line with doxycycline inducible ABL1 gate-keeper mutation (H1915-ABL1T315I) in mice treated with either vehicle or imatinib (100mg/kg). Error bars represent ±S.D., n=6-10 mice per group, *P=0.0016 by Mann-Whitney U test.
Somatically Mutated ABL1 is an Actionable and Essential NSCLC Survival Gene (2015)
Figure 4

Figure 4. ABL1 mutations lead to intermediate increase in kinase activity. A and C. Root mean square deviation (RMSD) analysis of the movement of (A) αC-helix and (C) DFG motif in the active and inactive conformation of ABL1 kinase with R351W mutation (orange) compared to the wild type (WT) kinase (black). B and D. Time-lapse images show movement within the αC-helix (B) and DFG motif (D) of the inactive structure of the WT (black outline) and R351W (orange outline) constructs. E. left, western blot analysis of transient overexpression of wild type ABL1 (WT) and ABL1-R351W, compared to empty vector control (EV) in HEK293T cells. Right, quantification of p-CRKL signal based on densitometry analysis of blots from 6 independent experiments. Error bars represent ±S.D., n=6, *P=0.0004 by two-tailed unpaired Student’s t-test. F. Overexpression of mutant ABL1 constructs with mutations identified in selected primary lung tumors (Ding et al, 2008) in H157 cells. Values indicate quantified band intensity for p-CRKL, relative to α-tubulin and HA-tagged ABL1.
Somatically Mutated ABL1 is an Actionable and Essential NSCLC Survival Gene (2015)
Figure 5

Figure 5. ABL1 mutations increase cytosolic retention of the mutated ABL1 proteins. A. Immunofluorescence imaging of endogenous ABL1 in the panel of NSCLC cell lines; scale bars=20 μm. B and C Quantification of nuclear versus cytoplasmic localization of (B) endogenous ABL1 in NSCLC cell lines and (C) tetracycline-inducible expression of ABL1-WT, ABL1-R351W and ABL1-G340L in Beas-2B cells. Error bars represent ±S.D., n=6-10 images, *P<0.005 by two-tailed unpaired Student’s t-test (Beas-2B vs H1915 P=6.6×10-5, vs H2110 P=0.0001; WT vs R351W P=0.003, vs G340L P=0.002).
ZSCAN4+ embryonic stem cells have an oxidative and flexible metabolic profile (2020)
Figure 1

Figure 1. arg2 expression in Zscan4 ESCs metastate

(A) Transgenic ESZscan4_Emerald cells were sorted and total RNAs were extracted and subjected to RT-qPCR. The graph shows the mRNA expression level of Arg2 in ES subpopulations.

(B)Transgenic ESZscan4_Emerald cells were sorted and total proteins were extracted and subjected to immunoblotting. The image is a representative of three independent biological western blotting analysis on arg2 expression in sorted ESZscan4_Emerald and control cells.

(C) Schematic illustration of Arg2 silencing and magnetic separation of the Zscan4+ cells. The modified ESZscan4_LNGFR cells were transfected with a siRNA that specifically target mouse Arg2. 96h after siRNA transfection, cells were harvested and incubated with a LNGFR magnetically labeled antibody. Positive fractions were collected through autoMacs Separator. All the analyses were performed on Zscan4+ siArg2 and Zscan4+ siCtrl cells.

(D) Transcript (left panel) and protein expression (right panel) levels of Arg2 in ESZscan4_LNGFR cells after transfection with a non-silencing siRNA (siCtrl) or siRNA against Arg2 (siArg2).

(E) Imaging flow cytometry analysis on Zscan4+ siArg2 and Zscan4+ siCtrl cells. Samples were stained by labelling histone H3 protein. The intensity of H3 fluorescent signal was measured in the nuclear region by creating a mask defined by DAPI staining. The texture feature of fluorescent signal was analysed by using the modulation method (for details see Materials and Methods). Two cell populations (R1 and R2) correspond to two different patterns of fluorescent signal: homogenous and clustered distribution, respectively.

Data information: Student's t-test was used for the statistical analysis in panels A,B, D and E. Bars values are means ± SD of n=3 independent biological replicates; *, P < 0.05; **, P < 0.01 based on NEJM decimal format.
ZSCAN4+ embryonic stem cells have an oxidative and flexible metabolic profile (2020)
Figure 3

Figure 3. Morpho-functional imaging of Zscan4- and Zscan4+ cells.

(A) Representative confocal microscopy analysis of cells loaded with MitoTracker Green (pictures on the left); digital enlargements and false colors rendering of details shown as white contoured squares (pictures on the right).

(B) Electron microscopy images of sorted ESZscan4_Emerald and control cells. Scale bar 1μm

(C) Representative confocal microscopy analysis of cells loaded with dichlorofluorescein-diacetate converted in dichlorofluorescein (DCF) (pictures on the left); digital enlargements and false-color rendering of details shown as white contoured squares (pictures on the right); histogram on the left, averaged fluorescence intensity/cell ± SEM of n=3 independent biological replicates.

Data information: Student's t-test was used for the statistical analysis in panel C; bar values are means from three independent biological replicates for each of which the fluorescence/cell was averaged from at least ten randomly selected different fields each imaging about 30 cells
A subdomain of the endoplasmic reticulum forms a cradle for autophagosome formation (2009)
figf1

(a) Electron micrographs of the IM (black arrowheads) associated with the ER (black arrows) from Atg4BC74A-expressing NIH 3T3 cells. The IM appears to expand between the two ER cisternae and forms an autophagosome. The white arrowhead indicates vesicular clusters visible in the vicinity of the IM. The white arrow indicates the autophagosome. (b) The ratio of ER-associated autophagic structures (ER–IM complexes) to total autophagic structures. Data are expressed as the mean ± s.d. of triplicates from representative experiments. (c) Localization of GFP–DFCP1 with Atg16L1 in both control and Atg4BC74A-expressing cells. Insets show enlarged images of boxed regions. (d–g) Gold-enhanced immunogold electron microscopy revealed that GFP–DFCP1 localized to ER–IM complexes in both control (d, e) and Atg4BC74A-expressing cells (f). Immunogold particles are indicated by small arrows. No specific labelling was observed on ER–IM complexes in cells expressing GFP (g). Large arrows and arrowheads indicate the ER and the IM, respectively. Scale bars, 200 nm in a and d–g, an d 10 μm in c.
A subdomain of the endoplasmic reticulum forms a cradle for autophagosome formation (2009)
figf2

Atg4BC74A-expressing cells (for Atg16L1) or control cells (for PDI, COP II and β-COP) were labelled with specific antibodies against the indicated markers. The left and right anti-Atg16L1 and anti-PDI images are from two different ER–IM complexes. Arrows, arrowheads and open arrowheads indicate the ER, IMs and VTCs, respectively. Small arrows in the image from the anti-COP II experiment indicate immunogold particles. Scale bars, 200 nm.
Targeting of Early Endosomes by Autophagy Facilitates EGFR Recycling and Signalling (2019)
Figure 1

Figure 1 - Loss of Autophagy Perturbs EGFR Endocytic Trafficking.

The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2).

A: Spinning disc confocal live cell imaging of Alexa 555-EGF (555-EGF) shown as vesicle tracking with time represented as a colour spectrum. Tracking started 5 min after addition of 20 ng/mL 555-EGF for the indicated durations. Scale bar: 10 μm.

B: Immunofluorescence staining of EGFR following 5 or 15 min stimulation with 20 ng/mL EGF. Scale bar: 20 μm.

C: Quantification of EGFR vesicles in a perinuclear region (within 30 μm diameter of the centre of the nucleus) (in B).

D: Cells were stimulated with 20 ng/mL 555-EGF for 15 or 30 min before immunofluorescence staining against EGFR. Scale bar: 10 μm.

E: Quantification of percentage of total EGFR vesicles that colocalise with 555-EGF (in D).

F: Cells stably expressing mCherry-Rab5 were stimulated with 20 ng/mL EGF for indicated times before immunofluorescence staining against EGFR. Scale bar: 10 μm.

G: Pearson's colocalisation coefficient between mCherry-Rab5 and EGFR (in F).

Data information: Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: NS p>0.05, * p<0.05, ** p<0.01.
Targeting of Early Endosomes by Autophagy Facilitates EGFR Recycling and Signalling (2019)
Figure 2

Figure 2 - Autophagy Inhibition Increases PI(3)P-Positive Early Endosomes.

The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying.

A: Control, sgAtg7, or sgAtg16l1 cells were treated with 2 ng/mL EGF for 15 min. Cells were then processed for staining using anti-EEA1 antibodies and PI(3)P probe (Alexa 488-labelled 2XFYVE domains). To ensure the specificity of the probe, control cells were pre-treated with 5 mM 3'MA for 30 min. Scale bar: 10 μm.

B: Quantification of PI(3)P+ label intensity per cell (in A).

C: Pearson's colocalisation coefficient between PI(3)P and EEA1 (in A).

D: Control and sgAtg7 cells were stimulated 20 ng/mL EGF for 5 or 15 min before immunofluorescence staining against EEA1 and EGFR. Scale bar: 10 μm.

E: Pearson's colocalisation coefficient between EEA1 and EGFR (in D).

F: Endogenous VPS34 was immunoprecipitated from control and sgAtg7 cells that were treated with 2 ng/mL EGF for 15 min then lysed in CHAPS-containing detergent buffer and binding partners detected by western blotting.

G: Densitometry analyses of proteins coimmunoprecipitated with endogenous VPS34 (in F).

Data information: Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: NS p>0.05, * p<0.05, ** p<0.01, *** p<0.001.
Targeting of Early Endosomes by Autophagy Facilitates EGFR Recycling and Signalling (2019)
Figure 3

Figure 3 - Early Endosomes Stain Positively for Gal8 Upon Autophagy Inhibition or Monensin Treatment.

The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2).

A: Cells transiently expressing YFP-Galectins (YFP-Gal3, -Gal8, or -Gal9) were stimulated with 2 ng/mL EGF for 15 min before fixation and immunofluorescence staining against EEA1. Scale bar: 10 μm.

B: Quantification of percentage of total EEA1 vesicles that colocalise with YFP-Galectins (in A).

C: Cells transiently expressing YFP-Gal8 were stimulated with 20 ng/mL Alexa 555-EGF (555-EGF) for 15 min. Scale bar: 10 μm.

D: Quantification of the percentage of YFP-Gal8-labelled vesicles that colocalise with 555-EGF in control or ATG7-deficient cells (in C).

E: Cells transiently expressing YFP-Gal8 were treated 100 μM monensin for 1 hr and stimulated with 2 ng/mL EGF for 15 min before fixation and immunofluorescence staining against EEA1. Scale bar: 10 μm.

F: Quantification of percentage of total EEA1 vesicles that colocalise with YFP-Gal8 upon monensin treatment (in E).

Data information: White arrows indicate colocalisation. Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: NS p>0.05, ** p<0.01, *** p<0.001.
Targeting of Early Endosomes by Autophagy Facilitates EGFR Recycling and Signalling (2019)
Figure 4

Figure 4 - Autophagy Machinery Targets Early Endosomes.

The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2).

A: Cells stably expressing GFP-LC3 were treated with 100 μM monensin in the presence or absence of bafilomycin A1 (20 nM) for 1 hr and stimulated with 2 ng/mL EGF for 15 min before fixation and immunofluorescence staining against EEA1. White arrows indicate colocalisation. Scale bar: 10 μm.

B: Quantification of percentage of total EEA1 vesicles that colocalise with GFP-LC3 (in A).

C: Cells were treated for 1 hr with 100 μM monensin then stimulated with 2 ng/mL EGF for 15 min. LAMP2 and EEA1 were then detected by immunofluorescence staining. White arrows indicate colocalisation. Scale bar: 10 μm.

D: Quantification of the percentage of total EEA1 vesicles that colocalise with LAMP2 (in C).

E: Cells were treated with 2 ng/mL EGF for 15 min before immunofluorescence against endogenous ATG16L1 and EEA1. Scale bar: 10 μm.

F: Quantification of percentage of total EEA1 puncta that colocalise with ATG16L1 (in E).

G: Cells stably expressing Flag-S-ATG16L1 were treated for 1 hr with either 100 μM monensin or 30 μM Dynasore, then stimulated with 2 ng/mL EGF for 15 min. Cells were then stained by immunofluorescence against EEA1 and Flag tag. Scale bar: 10 μm.

H: Quantification of percentage of total EEA1 vesicles that colocalise with Flag-S-ATG16L1 (in G).

I: Untreated control or sgAtg7 cells, or control cells pre-treated with 100 μM monensin 1 hr, stably expressing Flag-S-ATG16L1 were stimulated 20 ng/mL Alexa 555-EGF (555-EGF) for 15 min before fixation and immunofluorescence staining against Flag tag and EEA1. Cells were then imaged by structured illumination microscopy (SIM) and images were reconstructed in Nikon Elements software. Scale bar: 10 μm.

J: Quantification of the percentage of EGF-EEA1 colocalised vesicles that stained triple-positive with ATG16L1 by SIM (in I). Due to the low throughput nature of this assay, the following cell numbers were counted: control untreated (9 cells), sgAtg7#1 (10 cells), sgAtg7#2 (9 cells), and control + monensin: (11 cells).

K: Cells were treated with 2 ng/mL EGF for 15 min before immunofluorescence against endogenous WIPI2 and EEA1. Scale bar: 10 μm.

L: Quantification of percentage of total EEA1 puncta that colocalise with WIPI2 (in K).

Data information: White arrows indicate colocalisation. Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: * p<0.05, ** p<0.01, *** p<0.001.
Targeting of Early Endosomes by Autophagy Facilitates EGFR Recycling and Signalling (2019)
Figure 5

Figure 5 - TBK1 and Gal8 are Required for the Recruitment of Autophagy Machinery to Early Endosomes.

The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2).

A: Cells were treated for 15 min with 2 ng/mL EGF before fixation and immunofluorescence staining against EEA1 and p-TBK1. White arrows indicate colocalisation. Scale bar: 10 μm.

B: Quantification of percentage of total EEA1 vesicles that colocalise with p-TBK1 (in A).

C: Cells stably expressing Flag-S-ATG16L1 were pre-treated for 1 hr with TBK1 inhibitors (100 μM MRT68601 or 5 μM momelotinib). Control cells were also treated 100 μM monensin for 1 hr as indicated. All cells were stimulated for 15 min with 2 ng/mL EGF followed by fixation and immunofluorescence staining against Flag tag and EEA1. Scale bar: 10 μm.

D: Quantification of total EEA1 vesicles that colocalise with Flag-S-ATG16L1 (in C).

E: Western blotting of shNf-1/shTp53 glial cells expressing gRNA sequences targeting Gal8.

F: Control and sgGal8 cells stably expressing Flag-S-ATG16L1 were treated 100 μM monensin for 1 hr and stimulated with 2 ng/mL EGF for 15 min before fixation and immunofluorescence staining against EEA1 and Flag tag. White arrows indicate colocalisation. Scale bar: 10 μm.

G: Quantification of percentage of total EEA1 vesicles that colocalise with Flag-S-ATG16L1 in sgGal8 cells (in F).

Data information: Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: * p<0.05, ** p<0.01, *** p<0.001.
Targeting of Early Endosomes by Autophagy Facilitates EGFR Recycling and Signalling (2019)
Figure 6

Figure 6 - Autophagy Loss Disrupts Rab11-Mediated Recycling of EGFR.

A: Glial shNf-1/shTp53 control and sgAtg7 cells were serum starved 4 hr then stimulated with 20 ng/mL TexasRed-EGF (TxRed-EGF) for 30 min. Scale bar: 10 μm.

B: Quantification of TxRed-EGF puncta per cell, relative to the number taken up by control cells (in A).

C: Endocytosis rate of wild type (WT) EGFR was assayed in control, sgAtg7, or sgAtg16l1 glial shNf-1/shTp53 or EGFRvIII-expressing cells by cell surface biotinylation and one application of 2 ng/mL EGF for 15 min. Monensin treatment was added as indicated.

D: Plasma membrane recycling rate of WT EGFR in control, sgAtg7, or sgAtg16l1 glial shNf-1/shTp53 or EGFRvIII-expressing cells was assayed by cell surface biotinylation and successive applications of 2 ng/mL EGF treatments for 15 min (detailed in Fig EV4H). Monensin treatment was added as indicated.

E: Glial shNf-1/shTp53 control and sgAtg7 cells stably overexpressing mCherry-Rab11 were serum starved 4 hr then stimulated with 20 ng/mL EGF for 15 min then fixed and stained by immunofluorescence against EGFR. White arrows indicate colocalisation. Scale bar: 10 μm.

F: Quantification of Pearson's colocalisation coefficient between mCherry-Rab11 and EGFR (in E).

Data information: Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: NS p>0.05, * p<0.05, *** p<0.001.
Targeting of Early Endosomes by Autophagy Facilitates EGFR Recycling and Signalling (2019)
Figure 7

Figure 7 - Autophagy is Required for the Maintenance of EGF-Mediated Signalling and Survival.

A-C: Glial shNf-1/shTp53 sgAtg7 (A), sgAtg16l1 (B), or sgGal8 (C) cells were either left untreated (+serum) or serum starved (-serum) 4 hr then stimulated 2 ng/mL EGF for 15 min before analyses by western blotting.

D: Glial shNf-1/shTp53 control and sgAtg7 cells were serum starved 4 hr then stimulated with 2 ng/mL EGF for the indicated times before analysis of EGFR signalling pathway activity by western blotting.

E: Phosphorylated:total protein ratios for EGFR and AKT calculated from densitometry analyses of western blots were made relative to their respective points of maximal stimulation (100%) (for EGFR: t=5 min in control cells and for AKT: t=7 min in control cells) (in C).

F: Glial shNf-1/shTp53 control and sgAtg7 cells were serum starved 4 hr with or without 20 ng/mL EGF 30 min stimulation before subcellular fractionation and analysis by western blotting.

G: Radar chart indicating the relative levels of pAKT (pink line), pEGFR (light blue line), or total EGFR levels (dark blue line), in autophagy-deficient cell lines relative to their control counterparts. The following knockouts were introduced: glial cells (sgAtg7), HT29 (crATG7), MCF-7 (sgATG5), A549 (sgATG5), U2OS (sgATG5), and MEFs (Atg16l1-/-).

H: Control or sgAtg7 glial shNf-1/shTp53 cells were serum starved for 24 hr in the presence or absence of 20 ng/mL EGF. Quantification of the percentage of propidium iodide (PI)-positive cells is shown.

Data information: Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: NS p>0.05, * p<0.05, ** p<0.01.

Expanded View Figure Legends