EMBO/soda-nel huggingface dataset:
Connexins modulate autophagosome biogenesis (2014)
figf6

(a) Immunoblot for the indicated proteins in homogenates (Hom) and purified PM from fed (F) and 24 h starved mice (Stv). Starvation-induced changes in the PM content relative to fed levels were as follows: Vps15 (1.2 ± 0.2), Beclin-1 (0.7 ± 0.5), Vps34 (0.9 ± 0.1), Atg16 (1.1 ± 0.3), Atg14 (2.5 ± 0.2) and only Atg14 were significant for P 0.01. (b) Immunoblot of Cx43 immunoprecipitates (IP) in MEFs maintained in the presence or absence of serum. I, input; FT, flow through. GAPDH is shown as negative control for immunoprecipitation. (c) Immunoblot of Cx43 IP in MEFs from WT or Atg5−/− mice grown in serum-supplemented media. (d) Immunoblot of Cx43 IP in WT or Atg16 knockdown (KD) MEFs maintained in the presence/absence of serum for 4 h. (e) Co-staining for Atg14 and Cx43 in NRK cells maintained in the presence/absence of serum. Merged channels are shown and single channels and broad-field images are shown in Supplementary Fig. 5d. Insets: higher magnification. (f) Maximum-intensity confocal microscopy 3D reconstruction in HeLa cells expressing EBFP2–Cx43 (pseudocoloured in red) and EGFP–ATG14 followed by a single-plane time-lapse acquisition of the outlined area; cells were incubated in serum-deprived medium for 1 h before imaging. (g) Immunoblot of Atg14 IP in WT or Atg5−/− MEFs maintained in the presence of serum. (h) Immunoblot in total homogenates and PM purified from WT or Cx43−/− MOBs. (i) Immunofluorescence for e-cadherin and Vps34 in NRK cells WT or knocked down (KD) for Cx43. Single channel and merge (left) and z-stack surface reconstruction and higher magnification of the outlined region (right). (j) Immunostaining for Cx43 and e-cadherin in NRK cells expressing a tandem FYVE domain (2xFYVE) tagged to GFP and maintained in serum-free media for 4 h. Single channels (inverted black and white) and the merged image of the outlined area (colour). Inset: higher magnification image. Scale bars: 5 μm. Uncropped images of blots are shown in Supplementary Fig. 9.
Connexins modulate autophagosome biogenesis (2014)
figf7

(a) Co-immunostaining for Cx43 and the indicated Atgs in NRK cells maintained in the presence or absence of serum (top). Single and merged channels of the 3D reconstruction of the outlined regions are shown (bottom). Nuclei are highlighted with DAPI. (b,c) Immunoblot for the indicated proteins of immunoprecipitates (IP) of Cx43 in NRK cells maintained in the presence or absence of serum (b) or treated or not with 3-methyladenine (c). Atg5 is shown as the negative control for immunoprecipitation. (d) Co-staining for Atg9 and e-cadherin in control NRK cells and those knocked down for Cx43. Single and merged channels at higher magnification areas are shown. (e) Immunofluorescence for endogenous Atg9 and Cx43 in control NRK cells and those knocked down for Eps15. (f,g) Immunofluorescence for endogenous Cx43 (green) in control NRK cells or those knocked down for Atg14 (f) or Atg9 (g) maintained in the presence or absence of serum. (h) Immunofluorescence of endogenous Cx43 in control NRK cells or those knocked down for Atg14, treated (or not) with tamoxifen or lindane (left). E-cadherin staining (red) is shown to highlight PM in g and h. Full fields are shown in Supplementary Fig. 6h. Quantification of intracytoplasmic Cx43-positives vesicles (right; n = 3 wells, 3 independent experiments, >30 cells per experiment). Values are mean + s.e.m and significant for *P 0.01 and **P 0.001 using analysis of variance + Bonferroni test. Scale bars: 5 μm. Uncropped images of blots are shown in Supplementary Fig. 9.
Connexins modulate autophagosome biogenesis (2014)
figf8

(a) Immunoblot for the indicated proteins in control MEFs or those knocked down (KD) for the three different Cx. (b) Immunofluorescence of LC3 in control MEFs (Ctr) or those knocked down (KD) for three different Cx maintained in the presence or absence of serum. Left: representative fields and higher magnification insets. Nuclei are highlighted with DAPI. Right: Quantification of the average number of LC3-positive puncta per cell (n = 3 wells, 3 independent experiments, >50 cells per experiment). (c) Immunoblot for LC3 in the same cells maintained in the presence of serum untreated (−) or treated with inhibitors of lysosomal proteases (PI) for the indicated times. GAPDH is shown as the loading control. (d) Representative images of control MEFs or those knocked down (KD) for the indicated proteins transfected with the mCherry–GFP–LC3 tandem reporter. Right: quantification of the average number per cell of puncta positive for GFP and mCherry (autophagosomes; APG) and those positive only for mCherry (autophagolysosomes; APL; n = 3 wells, 3 independent experiments, >50 cells per experiment). (e) Electron micrographs of control MEFs or those knocked down for the indicated Cx. Insets show autophagic vesicles. Bottom: Quantification of the average number of AV (left) and percentage of cytosolic area (right) occupied by AVs (n = 4 wells, 4 independent experiments, >10 micrographs per well). (f) Immunoblot for indicated proteins in immunoprecipitates (IP) for Cx26 or Cx32 (left) or Atg16 (right) from liver homogenates. Inp: input, FT: flow through. BIP is shown as the negative control for immunoprecipitation. All values are mean + s.e.m and significant for *P 0.01 (analysis of variance + Bonferroni test was used in b). Scale bars: 5 μm in fluorescence images; 1 μm and 0.1 μm in top panels and insets of e. Uncropped images of blots are shown in Supplementary Fig. 9.
Autophagy counterbalances endoplasmic reticulum expansion during the unfolded protein response. (2006)
Figure 1

ER Proliferation under UPR-Inducing Conditions

(A) Determination of ER abundance in control and UPR-induced cells. Representative cells are shown. The UPR was induced in wild-type cells by addition of DTT. Ultrastructure of control cells and UPR-induced cells was analyzed using ImageJ. The lower images show traces of cortical ER (represented in magenta) and the nuclear envelope (NE, in blue). Vacuoles, nuclei, and mitochondria are indicated as V, N, and M, respectively.

(B) Quantification of the ER proliferation during the UPR. UPR was induced and cells were collected for EM at the indicated time points. Length of the ER (as traced in [A]) was measured and divided by the area of the section. Data are plotted relative to time 0. Measurements for each time point correspond to the mean of 25 independent cell images.

(C) Expression of HAC1i was induced by addition of 100 μM DOC for 3 h. ER was quantified as described above in (B).
Autophagy counterbalances endoplasmic reticulum expansion during the unfolded protein response. (2006)
Figure 4

Fluorescence Visualization of an ER Marker after UPR Induction

(A) Cells treated with the UPR-inducing drug DTT (+DTT) or with no drug were visualized using a fusion protein between the translocon component Sec61 and the red-fluorescent protein “cherry.” Top panels show untreated cells, and bottom panels show representative UPR-induced cells.

(B) Representative images showing UPR-induced cells that contain ERAs (indicated by arrows).
Autophagy counterbalances endoplasmic reticulum expansion during the unfolded protein response. (2006)
Figure 5

Immunogold Labeling of ERAs with an Antibody Directed against an ER Membrane Marker

(A) Representative section of a cell immunolabeled against a myc-tagged Sec63, an integral ER membrane protein. As a primary antibody, we used a rabbit polyclonal anti-myc and, as a secondary, we used 15-nm gold particles–conjugated anti-rabbit antibody. Nucleus, nuclear envelope, ER, and ERA are indicated as N, NE, ER, and ERA, respectively.

(B) High magnification of an electron micrograph of a section of ER. Quantification showed that there are 5 ± 2 gold particles per linear micrometer of ER.

(C) High magnification of ERAs. To predict how many gold particles one should expect in a particular ERA, we first calculated and averaged the amount of ER (expressed as length in linear micrometers) present in an ERA (similar to the ones shown in Figure 3B), and normalized the value for its area. These calculations determined that there are 20.8 ± 3.3 μm of ER per μm2 inside the ERAs. These values allowed us to predict how many gold particles would be expected over a section of an ERA if it were packed with ER membranes. Two representative ERAs are shown. The ERA shown in the middle picture should hold 2.4 μm of ER inside and, therefore, should have 12 gold particles. We counted 12 gold particles. The ERA on the right could contain 2.7 μm of ER and should contain14 gold particles; we counted 16 gold particles. The image on the right shows a representative view of a nucleoplasmic region.

(D) Quantification of gold-labeling density per area. To assess the signal-to-noise ratio of our immunogold-labeling procedure, we assessed background labeling by counting the number of gold particles over an areas of nucleoplasm (N) and over ERAs, and normalized the counts to the respective areas.
Autophagy counterbalances endoplasmic reticulum expansion during the unfolded protein response. (2006)
Figure 6

UPR-Induction of the Autophagy Marker GFP-Atg8

(A) Wild-type cells transformed with a plasmid containing GFP-Atg8 were grown for 4 h in synthetic media with no drug, with UPR-inducing conditions (+DTT and +TM), or under nitrogen starvation conditions (N starv), and then harvested for protein preparation. Protein extracts were analyzed by Western blotting using antibodies against GFP (top panel) or Hac1 (bottom panel). Total protein concentration was measured by BCA protein assay. Same concentration of protein was loaded in each lane, and transfer efficiency was checked by Ponceau staining. The identities of the different bands are indicated.

(B) Wild-type cells expressing GFP-Atg8 grown under the conditions described above were visualized by fluorescence microscopy.

(C) GFP-Atg8 was detected in extracts from untreated hac1Δ cells or cells expressing HAC1i (+DOC) by Western blotting using antibodies against GFP.

(D) Western blot using antibodies against GFP of extracts from hac1Δ, ire1Δ, or vps4Δ pep4Δ cells expressing GFP-Atg8. Mutant cells were grown under regular conditions, UPR-inducing conditions (+DTT), or nitrogen starvation conditions (N starv).
Autophagy counterbalances endoplasmic reticulum expansion during the unfolded protein response. (2006)
Figure 7

Localization of GFP-Atg8 during UPR Induction

Some of the DTT-treated cells shown in Figure 4B expressing GFP-Atg8 and Sec61-cherry (as an ER marker) were visualized using fluorescence microscopy. GFP-Atg8 localizes in close proximity to the ERAs detected by the ER marker.
Autophagy counterbalances endoplasmic reticulum expansion during the unfolded protein response. (2006)
Figure 8

Atg8 and Other <italic>ATG</italic> Genes Are Necessary during UPR Induction

Serial dilutions for wild-type, hac1Δ, atg1Δ, atg8Δ, atg9Δ, atg16Δ, and atg20Δ deletion cells and vps4Δ pep4Δ double deletion cells were grown on rich-media plates with no drug (YPD) or with different concentrations of tunicamycin (TM; 0.2 or 1.0 μg/ml). atg19Δ gave an identical result to the other autophagy genes shown here (unpublished data).
Endothelial Notch signaling controls insulin transport in muscle (2020)
Figure 1

Figure 1.

Endothelial Notch signaling regulates systemic glucose metabolism

A. Expression of endothelial Notch target genes in microvascular endothelial cells isolated from skeletal muscle of mice kept on control diet (CD, 10% fat, 70% carbohydrates) or high-fat diet (HFD, 60% fat, 20% carbohydrates) or high-fat and sugar diet (HFS, 60% fat, 20% carbohydrates and 42 g/l sucrose in drinking water). n=4, data represent mean ± SEM, unpaired t-test.

B. Expression of endothelial Notch target genes in microvascular endothelial cells isolated from skeletal muscle of mice fasted for 24 hours (Fasted group) and then refed for 4 hours (Fasted+Refed group) normalized to mice fed for 24 hours (Fed group). n=5, data represent mean ± SEM, unpaired t-test.

C. Blood glucose levels of control (n=5) and NICDiOE-EC (n=5) mice five weeks after recombination. Data represent mean ± SEM, unpaired t-test.

D. Plasma insulin levels of control (n=5) and NICDiOE-EC (n=5) mice five weeks after recombination. Data represent mean ± SEM, Welch's t-test.

E. Blood glucose levels for insulin tolerance test of control (n=7) and NICDiOE-EC control (n=7) mice. Data represent mean ± SEM, unpaired t-test.

F. Quantification of area under curve for insulin tolerance test in E. Data represent mean ± SEM, Welch's t-test.

G. Blood glucose levels for glucose tolerance test of control (n=6) or NICDiOE-EC (n=7) mice. Data represent mean ± SEM, unpaired t-test.

H. Quantification of area under the curve for glucose tolerance test in G. Data represent mean ± SEM, unpaired t-test.
Endothelial Notch signaling controls insulin transport in muscle (2020)
Figure 2

Figure 2.

Endothelial specific Notch deletion improves insulin sensitivity

A. Blood glucose levels of control (n=8) and Rbpji∆EC (n=8) mice five weeks after recombination. Data represent mean ± SEM, unpaired t-test.

B. Plasma insulin levels of control (n=8) and Rbpji∆EC (n=8) mice five weeks after recombination. Data represent mean ± SEM, unpaired t-test.

C. Blood glucose levels for insulin tolerance test of control (n=10) and Rbpji∆EC (n=7) mice. Data represent mean ± SEM, unpaired t-test.

D. Quantification of area under curve for insulin tolerance test in C. Data represent mean ± SEM, Welch's t-test.

E. Blood glucose levels for glucose tolerance test of control (n=6) or RbpjiΔEC (n=5) mice. Data represent mean ± SEM, unpaired t-test.

F. Quantification of area under the curve for glucose tolerance test in E. Data represent mean ± SEM, Welch's t-test.
Endothelial Notch signaling controls insulin transport in muscle (2020)
Figure 3

Figure 3.

Loss of Notch signaling improves endothelial insulin transport

A. Uptake of FITC-insulin and CAV1 expression in primary human umbilical venous ECs (HUVECs) upon Notch induction. Scale bar 20 µm.

B. Uptake of FITC-insulin and CAV1 expression in (HUVECs) upon Notch blockage. Scale bar 20 µm.

C. Representative western blot of AKT phosphorylation in gastrocnemius muscle 7 minutes after intravenous injection of PBS or 1.5 U/kg insulin in control (n=3) and Rbpji∆EC (n=3) mice five weeks after recombination.

D. Densitometric analysis of western blot in C. n=3, data represent mean ± SEM, unpaired t-test.

E. Representative western blot of AKT phosphorylation in liver 7 minutes after intravenous injection of PBS or 1.5 U/kg insulin in control (n=3) and Rbpji∆EC (n=3) mice five weeks after recombination.

F. Densitometric analysis of western blot in E. n=3, data represent mean ± SEM, unpaired t-test.
Endothelial Notch signaling controls insulin transport in muscle (2020)
Figure 4

Figure 4.

Cav1 dependent insulin transport across endothelial cells.

A. Schematic representation of experimental setup for trans-endothelial insulin transport assay.

B. AKT phosphorylation in myogenic C2C12 cells treated with different doses of insulin. n=3, data represent mean ± SEM, unpaired t-test.

C. Representative western blot of AKT phosphorylation in C2C12 cells upon Cav1 knockdown in mouse cardiac endothelial cells (MCECs).

D. Densitometric analysis of western blot in C. Data represented as fold change over control. n=3, data represent mean ± SEM, unpaired t-test.

E. Proximity ligation assay showing the association of CAV1 and INSR (insulin receptor) in MCECs upon stimulation with insulin. n=86-120 cells from 8 microscopic high power fields per time point, data represent mean ± SEM, unpaired t-test, *** means p<0.001.
Endothelial Notch signaling controls insulin transport in muscle (2020)
Figure 5

Figure 5.

Notch signaling controls insulin transport by expression of caveolar genes.

A. Quantitative RT-PCR detection of CAV1, CAV2 and CAVIN1 in primary human umbilical venous endothelial cells (HUVECs) upon Notch blockade (dnMAML) and induction (NICD). n=3, data represent mean ± SEM, unpaired t-test.

B. Representative western blot of CAV1 expression in HUVECs upon Notch manipulation.

C. Densitometric analysis of western blot in B. Data represented as fold change over GFP control. n=3, data represent mean ± SEM, unpaired t-test.

D. Representative western blot of CAVIN1 expression in HUVECs upon Notch induction.

E. Densitometric analysis of western blot in D. Data represented as fold change over LacZ control. n=5, data represent mean ± SEM, unpaired t-test.

F. Expression of CAV1 in HUVECs upon Notch manipulation and HEY1 induction. n=3, data represent mean ± SEM, unpaired t-test.

G. Expression of Cav1, Cav2, Cavin1 in primary microvascular ECs freshly isolated from skeletal muscle of RbpjiΔEC mice compared to control mice. n=5, data represent mean ± SEM, unpaired t-test.

H. Expression of Cav1, Cav2, Cavin1 in primary microvascular ECs freshly isolated from skeletal muscle of NICDiOE-EC mice compared to control mice. n=4, data represent mean ± SEM, unpaired t-test.

I. Expression of Cav1, Cav2, Cavin1 in microvascular endothelial cells isolated from skeletal muscle of mice kept on control diet (CD, 10% fat, 70% carbohydrates) or high-fat diet (HFD, 60% fat, 20% carbohydrates) or high-fat and sugar diet (HFS, 60% fat, 20% carbohydrates and 42 g/l sucrose in drinking water). n=4, data represent mean ± SEM, unpaired t-test.

J, K. Quantification of number of caveolae attached to apical membrane in EC of cardiac capillaries from (H) NICDiOE-EC mice (6 segments of 1000 nm length from 3 hearts per genotype) or (I) RbpjiΔEC (6 segments of 1000 nm length from 8 hearts per genotype) based on electron microscopy. Box plot depicts median and percentiles (10th, 25th, 75th, 90th), unpaired t-test, * means p<0.05.

L, M Representative immune-histochemistry (J) and confocal images (K) of Cav1 staining in heart sections from control and RbpjiΔEC mice. n=3, scale bar 50 µm.
Endothelial Notch signaling controls insulin transport in muscle (2020)
Figure 6

Figure 6.

Endothelial specific Notch modulation alters glucose uptake in muscle

A. Schematic illustration of 2-DG uptake assay protocol for control and NICDiOE-EC mice.

B. Blood glucose levels of control and NICDiOE-EC mice during the 2-DG uptake assay. n=5, data represent mean ± SEM, unpaired t-test.

C. Plasma insulin levels of control and NICDiOE-EC mice during the 2-DG uptake assay. n=5, data represent mean ± SEM, unpaired t-test.

D. 2-DG uptake levels in skeletal muscle of control and NICDiOE-EC mice. n=5, data represent mean ± SEM, unpaired t-test.

E. 2-DG uptake levels in visceral white adipose tissue (vWAT) of control (n=4) and NICDiOE-EC (n=5) mice. Data represent mean ± SEM, unpaired t-test.

F. Schematic illustration of 2-DG uptake assay protocol for control and RbpjiΔEC mice.

G. Blood glucose levels of control and RbpjiΔEC mice during the 2-DG uptake assay. n=5, data represent mean ± SEM, unpaired t-test.

H. Plasma insulin levels of control and RbpjiΔEC mice during the 2-DG uptake assay. n=5, data represent mean ± SEM, unpaired t-test.

I. 2-DG uptake levels in skeletal muscle of control and RbpjiΔEC mice. n=4, data represent mean ± SEM, unpaired t-test.

J. 2-DG uptake levels in vWAT of control and RbpjiΔEC mice. n=5, data represent mean ± SEM, unpaired t-test.
Endothelial Notch signaling controls insulin transport in muscle (2020)
Figure 7

Figure 7.

Endothelium-specific deletion of Rbpj improves glucose tolerance in obese mice.

A. Schematic illustration of feeding and recombination protocol.

B. Blood glucose levels for glucose tolerance test (GTT) of control (n=4) or RbpjiΔEC (n=5) mice kept on high-fat diet (HFD). Data represent unpaired t-test, mean ± SEM.

C. Quantification of area under curve (AUC) for GTT in B. Data represent mean ± SEM, unpaired t-test.

D. Blood glucose levels for insulin tolerance test (ITT) of control (n=4) or RbpjiΔEC (n=3) mice kept on HFD. Data represent unpaired t-test, mean ± SEM.

E. Quantification of AUC for ITT in D. Data represent mean ± SEM, unpaired t-test.

F. HbA1c levels of control (n=4) or RbpjiΔEC (n=6) mice kept on HFD seven weeks after recombination. Data represent mean ± SEM, unpaired t-test.

G. Schematic illustration of 2-DG uptake assay protocol for control and RbpjiΔEC mice on HFD.

H. Blood glucose levels of control and RbpjiΔEC mice on HFD during 2-DG uptake assay. n=5, data represent mean ± SEM, unpaired t-test.

I. Plasma insulin levels of control and RbpjiΔEC mice on HFD during 2-DG uptake assay. n=5, data represent mean ± SEM, unpaired t-test.

J. 2-DG uptake levels in skeletal muscle of control (n=5) and RbpjiΔEC (n=4) mice on HFD. Data represent mean ± SEM, unpaired t-test.

K. 2-DG uptake levels in vWAT of control and RbpjiΔEC mice. n=4, data represent mean ± SEM, unpaired t-test.

Expanded View Figure legends
Calcium signaling induces a partial EMT (2021)
Figure 1

Figure 1: Ca2+ signaling is enriched in P-EMT cells

Representative calcium signature enriched in P-EMT tumors (comparing ECAD- vs. ECAD+ tumor cells). NES, nominal P-value, and FDR are shown. Statistical analysis performed in GSEA.

HOMER motif analysis comparing P-EMT ECAD- versus ECAD+ tumor cells. The five most enriched motifs are shown. Statistical analysis performed in HOMER.

Representative image of tumor cells transduced with GCaMP6 (Left). Yellow arrows (mesenchymal cells), red arrows (epithelial cells), scale bar = 50μm. GCaMP6 mean fluorescent intensity (MFI) in ECADHIGH or ECADLOW cells cultured in indicated medium (0 Ca2+ or 2 Ca2+) for 1h (Right). Representative of 3 independent murine PDA cell lines run in triplicate. Statistical analysis by Student's unpaired t-test (*, p<0.05; non-significant (NS); ±SD).

Flow cytometry analysis of ECADHIGH or ECADLOW cells loaded with Indo-1 and cultured in indicated medium (0 Ca2+ or 2 Ca2+) for 1h. Representative of 2 independent cell lines (n indicates the number of cells analyzed in each group, ECADHIGH 0 Ca2+ n=4963, ECADLOW 0 Ca2+ n=14, ECADHIGH 2 Ca2+ n=6575, ECADLOW 2 Ca2+ n=29). Representative of 2 independent murine PDA cell lines run in triplicate. Statistical analysis by Student's unpaired t-test (****, p<0.0001; non-significant (NS); ±SD).