EMBO/soda-nel huggingface dataset:
A genome-wide RNAi screen reveals essential therapeutic targets of breast cancer stem cells (2019)
Figure 3

Figure 3. Identification and validation of compounds that modulate bCSC-fate. A. Table summarizing the different target genes/targeted therapies identified for each subnetworks. B. Comparison of bCSC variation after gene knockdown by ALDEFLUOR phenotyping (right) and tumor SFE (left) using the 11 siRNAs pool (3 siRNAs/pool) against the selected target genes and one scrambled siRNA pool used as control. Results for each siRNA pool (from top to bottom) are represented as opposite bars (n=3). Correlations are measured using Spearman rank correlation (R). C-D. Examples of dose-dependent ALDHbr cells variation in response to drug treatments. ALDHbr cell proportion in response to Tazemetostat in SUM159 (n=10) (C) and JQ1 in SUM149 (n=10) (D). The horizontal red dashed-line corresponds to a reduction of 50% of the ALDHbr cell proportion in the treated conditions compared to the control and the vertical red dashed-line corresponds to the target dose predicted by the K-score. On the bottom panel, corresponding flow charts for the ALDEFLUOR staining. DEAB is an ALDH inhibitor used as negative control. E. Pearson correlation analysis of the variation of the ALDHbr cells proportion upon drug treatment and gene silencing. F. Heat map for the variation of the ALDHbr cells proportion under drug treatment, in five different cell lines. Red and blue indicate the K-score in treated conditions, respectively, above and below the K-score in the untreated condition. G-H. Primary (n=4) (G) and secondary (n=4) (H) tumorsphere-forming efficiency (SFE) of SUM159 cells under treated and untreated conditions. Grey dashed-line corresponds to the level of SFE in the untreated conditions. Statistical test used is Pairwise chi-square test. Data represent mean ±SD.
A genome-wide RNAi screen reveals essential therapeutic targets of breast cancer stem cells (2019)
Figure 4

Figure 4. Drug combination screen identifies salinomycin as acting synergistically with JQ1 to reduce the bCSC proportion. A. Interaction surface built using the estimated combination index (CI) for the Salinomycin and JQ1 drug combination, in four different breast cancer cell lines (top panels). The CI is represented in 2D color code interaction surface. The color code spans from strong synergism (dark blue, CI<1) to strong antagonism (red, CI>1). The concentrations of each particular drug in the combinations are denoted on each axis. On the bottom panels, neighborhood Z-core matrix estimating the significant synergistic interaction. The color code spans from a statistically significant synergism (dark blue, CI<1, p<0.01) to a statistically significant antagonism (dark red, CI>1, p<0.01). B. Representative examples of flow chart for the ALDEFLUOR staining following different JQ1/Salinomycin drug combinations. DEAB is an ALDH inhibitor used as negative control. Data represent mean ±SD. C-D. Primary (n=4) (C) and secondary (n=4) (D) tumorsphere-forming efficiency (SFE) of SUM159 cells under mono- and combined-therapy compared to untreated conditions. Statistical test used is Pairwise chi-square test.. E. Bar plots representing the dose-dependent changes in the proportion of apoptotic cells (YO-PRO-3+) in each cell subpopulations, in response to drug treatments (right panel). Cisplatin (CisPt) is used as a positive control. On the right panel, representative fluorescence images obtained in different treatment conditions (n=6). Nucleus are in blue (Hoechst), bCSCs are in green (ALDH), and apoptotic cells in red (YO-PRO-3). F. Schematic representation of the lineage tracing system protocol. G. Representation of the BFP+/RFP+ cells proportions for increasing drug concentrations of either JQ1 or Salinomycin alone, or in combination. Blue bars: BFP+ cells proportion, Red bars: RFP+ cells proportion. H-I. Representation of the bCSC proportion in the BFP+ (H) and RFP+ (I) progenies in the control cells compared to the JQ1- and Salinomycin-treated cells alone or in combination (n=4). Statistical test used is a student's t-test. JQ1 Vs CTRL and Sal Vs JQ1 p=0.03; JQ1/Sal Vs CTRL p=0.01. Error bars represent mean ± SD.
A genome-wide RNAi screen reveals essential therapeutic targets of breast cancer stem cells (2019)
Figure 5

Figure 5. JQ1/Salinomycin combination decreases the bCSC population in patient-derived xenografts. A. Schematic representation of the in vivo experimental design. B. Effect of JQ1 and Salinomycin treatment on the tumor growth of CRCM434 (n=10) (see Figure EV4 A and E for CRCM404 and CRCM494). The grey area corresponds to the period of drug treatments. C-E. Quantification of the proportion of residual ALDHbr cells in treated PDX normalized to the proportion of ALDHbr cells in the vehicle-treated tumors, measured by flow cytometry. Statistical test used is a student t-test. Data represent mean ±SD (n=10). F-I. Reimplantation assay (CRCM434). Two-week treated PDXs were reimplanted, in serial dilutions, into new recipient mice and tumor growth was monitored (F, see Figure EV4 for CRCM404 and CRCM494). Each curve represents the growth kinetic from one individual injection. G-I. Bar plots displays bCSC frequency calculated using an extreme limiting dilution analysis (ELDA). Results are expressed as the estimated number of bCSCs for 10,000 tumor cells. Statistical test used is Pairwise chi-square test. J-L. Effect of JQ1 and Salinomycin treatments on the metastasis formation of CRCM404 and CRCM434. Metastasis formation was monitored using bioluminescence imaging in the treated mice livers and lungs (J). Quantification of the normalized photon flux revealed a statistically significant decrease in metastasis formation in Sal/JQ1 treated mice compared to the control mice (K, CRCM404, p=0.04; L, CRCM434, p=0.02). Statistical test used is a Wilcoxon test. Error bars represent mean ± SD (n=10).
Co-translational insertion and topogenesis of bacterial membrane proteins monitored in real time (2020)
Figure 1

Figure 1 - Monitoring co-translational membrane protein insertion in real time by FRET.

Expected FRET changes during co-translational insertion of an N-out membrane protein. The FRET acceptor (Atto655, red star) was placed at the N-terminus of the nascent chain, the FRET donor (Atto488, yellow star) at one of two positions in SecY (see panel B).

Positions on SecY (cyan) used for site-specific Atto488 donor labeling at the cytoplasmic face (position 111; red sphere) or periplasmic face (position 212; black sphere). (SecE, orange; SecG, green). The SecYEG translocon is embedded into phospholipids contained in a nanodisc held together by membrane scaffold proteins. Model based on PDB ID: 3J45 .

Time-dependent acceptor fluorescence changes due to FRET during co-translational insertion of acceptor-labeled LepB75 into donor-labeled translocon.
Co-translational insertion and topogenesis of bacterial membrane proteins monitored in real time (2020)
Figure 2

Figure 2 - Translation and topogenesis of LepB.

Analysis of LepB94 translation products on SDS-PAGE. Translation products are visualized by the fluorescence of N-terminal Atto655. Pausing intermediates are indicated P1 and P2.

Fitting of time courses (filled circles) of the translation of LepB mRNA constructs of increasing length (Appendix Fig S3) by delay-exponential functions (red lines).

Kinetic model used for fitting of both translation and FRET time courses. Ribosome pausing at intermediates P1 and P2 is modeled as off-pathway states.

Stopped-flow time courses of co-translational LepB insertion into SecYEG (Atto488 donor-in at position 111 of SecY), as monitored by the fluorescence of the Atto655 acceptor placed at the N-terminus of the nascent peptide. Fits (red lines) were obtained by global fitting (Materials and Methods).

Intrinsic fluorescence intensities (IFI) derived from global fitting are shown as black lines and filled circles along with amplitudes obtained from exponential fitting (red squares). Confidence contours (shaded areas) were calculated using the Fitspace algorithm (Materials and Methods).
Co-translational insertion and topogenesis of bacterial membrane proteins monitored in real time (2020)
Figure 3

Figure 3 - Co-translational insertion of N-in membrane protein EmrD.

Time course of EmrD135 translation. Translation was monitored by SDS-PAGE as in Fig 2A. For translation time courses of shorter mRNA constructs see Appendix Fig S5.

Time course of EmrD(-)135 translation.

Kinetic model of EmrD synthesis. Ribosome stalling (peptide P1) is modeled at EmrD48.

Kinetic model of EmrD(-) synthesis. Stalling (peptide P1) is modeled at EmrD(-)48.

Stopped-flow time courses for translocon insertion of TM1 of EmrD (black) and EmrD(-) (blue) with mRNA constructs of varying chain length (50 to 135 aa).The FRET acceptor (Atto655) was placed at the N-terminus of the nascent peptide, the donor (Atto488) at position 111 (donor-in) of SecY. The results of global fitting are indicated (red lines).

Intrinsic fluorescence intensities (IFIs) calculated for EmrD (black) and EmrD(-) (blue). Error margins (shaded areas) are calculated using the Fitspace algorithm (Materials and Methods).

Stopped-flow traces for co-translational insertion of LepB94 (red, from Fig 2), EmrD135 (black, from Fig 4E), and EmrD(-)135 (blue, from Fig 4E), rescaled according to the translation efficiency.
Co-translational insertion and topogenesis of bacterial membrane proteins monitored in real time (2020)
Figure 4

Figure 4. Protection of nascent chains against PK digestion.

A, B, C LepB (A), EmrD (B), and EmrD(-) (C) are translated for the times indicated and subjected to PK treatment (Materials and Methods). Sensitivity to PK in the absence of SecYEG (black circles) or in the presence of SecYEG (red squares), and co-translational translocon insertion monitored by FRET (blue trace) are depicted. Error bars represent standard deviations (n≥3) and the corresponding lines represent fits to delay-exponential functions.
Atoh1+ secretory progenitors possess renewal capacity independent of Lgr5+ cells during colonic regeneration (2018)
Figure 1

Figure 1. Lgr5+ stem cells are sensitive to colonic injury and dispensable for epithelial regeneration in colitis.

(a) Illustration of experimental protocol outlining DSS induced colitis in Lgr5GFP-CreERT2;ROSA26tdTomato mice.

(b) RNAscope analysis of Lgr5 mRNA in DSS colitis demonstrating loss of Lgr5 transcript (N=3 per condition).

(c) Hematoxylin and Eosin staining of the colonic epithelium following DSS induced damaged. Crypts are dramatically injured by d5. At d8 through d12, regenerating crypts are observed.

(d-e) Compared to untreated controls, DSS treatment ablates Lgr5-GFP+ stem cells and results in functional loss of tdTomato+ lineage tracing as early as d5. Lgr5-GFP+ stem cells reappear by d12 (N=3-4 per condition).

(f) Effects of DSS colitis injury on RNA expression levels of stem (Lgr5 and Krt19) and progenitor (Atoh1 and Notch1) cell markers. Lgr5 mRNA expression is significantly decreased after exposure to DSS (N=6-7 per condition).

(g) DSS and DT treatment of Lgr5DTR-GFP mice.

(h-i) Mice body weight (h) and survival (i) are comparable between three treatments: DSS+DT (N=12), only DSS (N=8) or only DT (N=3).

(j) Hematoxylin and Eosin staining of the colonic epithelium shows no morphological difference between DSS alone and DSS+DT treated mice.

(k) The extent of epithelial damage is similar in mice treated with DSS alone versus DSS+DT (to ablate Lgr5+ stem cells) (N=3 DSS; N=6 DSS+DT).

(l) RNAscope for Lgr5 in Lgr5DTR-GFP mice showing loss of Lgr5 transcripts after DT treatment (N=3-4 per condition).

Data information: Nuclei are counterstained with Dapi (blue); white dashed lines indicate basement membrane. Scale bars (b, c, d, l) =50µm; scale bar (j) =500µm. Data are represented as mean ± SD (e) and mean ± SEM (f-k) analyzed using Student's t-test. *P≤0.05, ** P≤0.01.
Atoh1+ secretory progenitors possess renewal capacity independent of Lgr5+ cells during colonic regeneration (2018)
Figure 2

Figure 2. Lgr5-negative/Krt19+ cells contribute to epithelial renewal during homeostasis and following colitis.

(a) Illustration of experimental protocol outlining DSS induced colitis in Krt19BAC-CreERT2;ROSA26tdTomato mice.

(b-c) Krt19+ cells lineage trace entire colonic crypts during both normal homeostasis and injury (N=3-4 per condition).

(d) DSS and DT treatment of Krt19CreERT2;ROSA26tdTomato/DTR mice.

(e-f) DT ablation of Krt19+ cells significantly reduced the number of lineage labeled crypts (N≥3 per condition).

(g) Mice body weight and survival remain unchanged between DSS and DSS+DT treated groups (N=6 control; N=7 DT).

(h-i) Hematoxylin and Eosin staining of the colonic epithelium after DSS versus DSS+DT (h) and quantification of damaged epithelial area (i), showing more damage is seen in mice treated with both DSS+DT treatment (N=6 control; N=7 DT).

Data information: Nuclei are counterstained with Dapi (blue); white dashed lines outline the basement membrane. Scale bars (b) =45µm; scale bar (e) =30µm; scale bar (h) =1mm. Data are represented as mean ± SEM (c, f, g, i) using Student's t-test (f-i). *P≤0.05, **P≤0.01.
Atoh1+ secretory progenitors possess renewal capacity independent of Lgr5+ cells during colonic regeneration (2018)
Figure 3

Figure 3. Colonic Notch1+ absorptive progenitors aid in homeostatic renewal but do not show renewal capacity in injury.

(a) Illustration of experimental protocol outlining DSS induced colitis in Notch1CreERT2; ROSA26tdTomato;Lgr5DTR-GFP mice.

(b-c) Notch1+ progenitors are able to generate Lgr5+ stem cells and contribute to epithelial renewal in homeostasis; however, do not contribute to regeneration after colitis (N=4 per condition).

(d) Illustration of experimental protocol outlining DT ablation of Lgr5+ cells in the context of Notch1+ cell lineage tracing using Notch1CreERT2; ROSA26tdTomato;Lgr5DTR-GFP mice.

(e-f) Images of whole mount (e) and tissue sections Notch1CreERT2; ROSA26tdTomato;Lgr5DTR-GFP mice (f) showing Notch1 lineage labeled crypts. No partially labeled crypts were found after Lgr5+ stem cell ablation.

Data information: Nuclei are counterstained with Dapi (blue); white dashed lines indicate basement membrane. Scale bars=50μm. Data are represented as mean ± SD analyzed using two-way ANOVA with Sidak's multiple comparisons test. ****P≤0.0001.
Atoh1+ secretory progenitors possess renewal capacity independent of Lgr5+ cells during colonic regeneration (2018)
Figure 4

Figure 4. Atoh1+ secretory progenitors display plasticity and contribute to colonic regeneration in homeostasis and injury.

(a-b) Atoh1+ progenitors show stem cell capacity during normal homeostasis and regeneration. At early stages of recovery, d10-12, Atoh1+ cells lineage trace the colonic crypt (N=4 per condition).

(c-d) By d30 after DSS, Atoh1 derived stem cells continue to lineage trace complete colonic crypts (N=4 per condition).

(e) Illustration of experimental protocol outlining DT treatment of Atoh1CreERT2;ROSA26tdTomato;Lgr5DTR-GFP mice.

(f-g) Whole mount (f) and section (g) images of Atoh1 lineage labeled crypts. Majority of crypts are labeled after Lgr5+ stem cell ablation and remain label with the appearance of Lgr5+ stem cells (inset, arrowheads). Atoh1 lineage labeled crypts remain by d30.

(h) Significantly increased labeling of crypt base was observed at d8 in DT treated mice (N=4 per condition).

(i) DSS and DT treatment of Atoh1CreERT2;ROSA26tdTomato;Lgr5DTR-GFP mice.

(j-k) Scattered labeling of Atoh1+ cells in control conditions, while DSS injury lead to Atoh1 lineage labeled crypts lacking Lgr5+ stem cells.

Data information: Nuclei are counterstained with Dapi (blue); white dashed lines indicate basement membrane. Scale bars (a, first two panels of c, f, j) =50μm; scale bars (last panel of c, g) =100μm. Data are represented as mean ± SD analyzed using two-way ANOVA with Sidak's multiple comparisons test (b, d) and Student's t-test with Welch's correction (h, k). *P≤0.05, ***P≤0.001.
Atoh1+ secretory progenitors possess renewal capacity independent of Lgr5+ cells during colonic regeneration (2018)
Figure 5

Figure 5. Atoh1+ colonic progenitor cells show renewal capacity in vitro upon injury.

(a) Illustration of experimental protocol outlining 4-OHT induction and irradiation of Atoh1CreERT2;ROSA26tdTomato colonic organoids.

(b-c) Sporadic labeling of Atoh1+ cells after 4-OHT induction. Following irradiation, entire colonic organoids exhibit full lineage labeling of Atoh1+ cells, characteristic of multipotent stem cells (N=3 per condition).

(d-e) Flow cytometry for tdTomato+ cells in crypt epithelium from DT treated Atoh1CreERT2;Lgr5DTR-GFP;ROSA26tdTomato mice.

(f) tdTomato+ colonic organoids from single Atoh1-tdTomato+/Lgr5-negative cells (N=1; n=3 technical replicates per condition).

(g) Post-passage colonic organoids from RFP+ cells of control or DT treated mice continue growing. tdTomato-negative cells of control or DT treated mice also form colonic organoids after passaging.

(h) tdTomato+ colonic organoids established from mice in which Lgr5+ stem cells have been ablated show higher growth efficiency compared to their colonic organoid counterparts (N=1; n=3 technical replicates per condition).

Data information: Scale bars (b, f) =100μm; scale bars (g) =50μm. Data are represented as mean ± SD analyzed using Student's t-test (c), and mean ± SD analyzed using two-way ANOVA with Sidak's multiple comparisons test (h). *P≤0.05, **P≤0.01, ***P≤0.001.
Atoh1+ secretory progenitors possess renewal capacity independent of Lgr5+ cells during colonic regeneration (2018)
Figure 6

Figure 6. Atoh1+ colonic progenitor cells are essential for epithelial regeneration post-injury. (a) Illustration of experimental protocol outlining DSS-induced colitis in Atoh1CreERT2;ROSA26tdTomat/DTR mice.

(b) Atoh1+ lineage labelling post-DSS and DSS+DT.

(c) DSS+DT treated-mice exhibit significant weight loss and lower survival compared to DSS alone (N=4-7 control; N=3-4 DSS).

(d-e) Hematoxylin and Eosin staining of colonic epithelium after DSS versus DSS+DT (d). Extensive epithelial damage was observed (d, arrow) and quantified of in DSS+DT treated mice (N=7 control; N=4 DSS).

(f) Illustration of experimental protocol outlining 4-OHT induction and irradiation in Atoh1CreERT2;ROSA26tdTomato/DTR colonic organoids.

(g-h) DT treatment effectively ablates Atoh1+ cells shown by reduced number of tdTomato+ cells per organoid.

(i-j) Concurrent irradiation injury and Atoh1+ cell ablation results in significantly reduced organoid survival (N=3; n=3 technical replicates per condition).

Data information: Scale bars (b)=200μm; scale bars (d)=400μm; scale bars (g, i)=100μm. Data are represented as mean ± SEM analyzed using Student's t-test. *P≤0.05.

Expanded View Figure Legends
Preperoxisomal vesicles can form in the absence of Pex3. (2014)
Figure 1.

pex3 atg1 cells harbor Pex14-containing structures. (A) EM analysis of KMnO4-fixed pex3 atg1 and WT cells grown for 16 h on MM-M/G. The inset shows a cluster of vesicles (enlarged from the boxed region). (B) iEM analysis of pex3 atg1 cells using α-Pex14 antibodies. (C and D) FM images of pex3 atg1 cells producing Pex14-mCherry and the ER marker BiPN30-eGFP-HDEL (C), or Pex14-mGFP complemented with Mitotracker orange staining (D). (E) Electron tomography analysis of a serial-sectioned pex3 atg1 cell (a) containing a perinuclear membrane cluster (b, arrows). (d–f) 10-nm-thin digital slices through the tomogram reconstruction (viewing direction indicated in c) revealed vesicles (black arrows) and reticular structures (red arrows). The surface-rendered reconstruction in g shows the reticulovesicular structures in 3D viewed at right angles from d. CW, cell wall; M, mitochondrion; N, nucleus; P, peroxisome; V, vacuole. Bars: (A) 500 nm; (A, inset; and B) 100 nm; (C and D) 2.5 µm; (E) 250 nm.
Preperoxisomal vesicles can form in the absence of Pex3. (2014)
Figure 2.

pex3 atg1 cells contain enhanced numbers of Pex14-containing structures. (A) iEM analysis of pex3 cells using α-Pex14 antibodies, identifying structures (arrows) in the vicinity of mitochondria. CW, cell wall; M, mitochondrion. (B) Quantification of Pex14-mGFP spots in pex3 and pex3 atg1 cells. (C) FM images of pex3 atg1 or pex3 cells, producing Pex14-mGFP complemented with FM4-64 vacuolar staining. The inset (enlarged from the boxed region) shows optimized intensities for pex3 cells, highlighting the Pex14-mGFP spot and vacuolar mGFP. (D) WB analysis of cells grown for 16 h on MM-M/G using α-Pex11 or α-Pex14 antibodies. Pyruvate carboxylase 1 (Pyc1) was used as a loading control. Error bars indicate SEM. Bars: (A) 100 nm; (C) 1 µm.
Preperoxisomal vesicles can form in the absence of Pex3. (2014)
Figure 3.

Pex10, Pex11, and Pmp47 do not colocalize with Pex14. (A) FM images of pex3 atg1 cells grown for 16 h on MM-M/G. Cells produced Pex14-mCherry and C-terminal mGFP fusions of the indicated proteins. (B) WB analysis of WT (1) and pex3 atg1 cells (2), grown for 6 or 16 h on MM-M/G. (C) FM images showing mGFP-fluorescence in pex3 atg1 cells producing Pex14-mCherry (control) or Pex14-mCherry together with the indicated mGFP fusion protein. Cells were grown for 6 h on MM-M/G. (D and E) iEM of pex3 atg1 cells using α-Pex5 (D) or α-alcohol oxidase antibodies (E). (F) Cell fractionation of the two indicated strains (seperated by the horizontal broken line). Postnuclear supernatants (PNS) were subjected to differential centrifugation resulting in a 30,000 g organelle pellet (P) and supernatant fraction (S). (G) Flotation analysis of the organelle pellet showing the distribution of the indicated proteins in the top (1) to bottom (10) fractions. (H) Colocalization of Pex8-GFP and Pex14-mCherry in pex10 cells. Bars: (A) 2.5 µm; (C) 5 µm; (D and E) 100 nm. AOX, cytosolic alcohol oxidase crystalloid; N, nucleus; V, vacuole.
Preperoxisomal vesicles can form in the absence of Pex3. (2014)
Figure 4.

Pex14-containing vesicular structures mature into peroxisomes upon reintroduction of PEX3. (A) FM images of pex3 cells with Pex14-mCherry upon Pex3-eGFP reintroduction after shifting cells from MM-Glu with ammonium sulfate to MM-M/G with methylamine. (B–E) Live cell FM images of pex3 atg1 cells upon Pex3 reintroduction. Shown are pex3 atg1 cells producing Pex14-mCherry and PAMOPEX3-eGFP (B), PAMOPEX3.PEX10-GFP (C), PAMOPEX3.PMP47-GFP (D), or PAMOPEX3.PTEFGFP-SKL (E). Cells were grown similar to the method in A. The arrows in B–D indicate the first detectable GFP signal. Bars, 2.5 µm.
Preperoxisomal vesicles can form in the absence of Pex3. (2014)
Figure 5.

Pex19 overproduction and PEX19 or PEX25 deletion. (A and B) WB analysis (A) and protein quantification (B) in WT (lane 1; blue), pex3 atg1 (lane 2; red), and pex3 atg1-PAOXPEX19 (lane 3; green) cells grown for 6 h on MM-M/G. The protein levels in WT were set to 100%. (C and D) FM images (C) and quantification (D) of Pmp47-mGFP and Pex10-mGFP in pex3 atg1 (1; red) and pex3 atg1-PAOXPex19 (2; green) cells grown for 6 h on MM-M/G. Control cells in D did not produce mGFP. Significance indications: n.s., P < 0.10; *, 0.10 > P > 0.05; **, 0.05 > P > 0.01; ***, P < 0.01. Error bars indicate SD. (E and F) iEM analysis of pex19 (E) and pex19 atg1 (F) cells using α-Pex14 antibodies. (G) EM analysis of KMnO4-fixed pex3 atg1 pex25 cells grown for 16 h on MM-M/G–showing membrane vesicles (arrows). (H) FM image of pex3 atg1 pex25 cells producing Pex14-mGFP. CW, cell wall; M, mitochondrion; N, nucleus; V, vacuole. Bars: (C) 2 µm; (E and F) 100 nm; (G) 250 nm; (H) 2.5 µm.