EMBO/soda-nel huggingface dataset:
In situ CRISPR-Cas9 base editing for the development of genetically engineered mouse models of breast cancer (2019)
Figure 2

Figure 2

In situ base editing creates allelic series of oncogenic driver mutations.

(A) Sanger-sequencing chromatograms showing the target regions of sgPik3caE542K, sgPik3caE545K and sgPik3caE453K in wild-type (WT) and base edited (BE) cells. Arrowheads highlight cytosines of the protospacers that show base editing 5 days after transduction of BE3-expressing NIH3T3 cells with Lenti-sgPik3caE542K, Lenti-sgPik3caE545K and Lenti-sgPik3caE453K.

(B) EditR was used to calculate the frequency (%) of C-to-T conversion at the indicated target cytosines of the protospacers in BE3-expressing NIH3T3 cells 5 days after transduction with the indicated sgRNA vectors.

(C) Overview of the intraductal injections performed in WB1P-BE3 females with high-titer lentiviruses encoding Myc and either a non-targeting sgRNA (Lenti-sgNT-Myc) or the different sgRNAs targeting Pik3ca (Lenti-sgPik3ca-Myc).

(D) Kaplan-Meier curves showing mammary tumor-specific survival for the different models. WB1P-BE3 females injected with Lenti-sgPik3caE542K-Myc (n=10), Lenti-sgPik3caE545K-Myc (n=10) and Lenti-sgPik3caE453K-Myc (n=10) showed a reduced mammary tumor-specific survival compared to WB1P-BE3 female mice injected with Lenti-sgNT-Myc (n=11) vectors (respectively 47, 44 and 49 days after injection vs 72 days after injection, P < 0.0001 by Mantel-Cox test).

(E) Sanger-sequencing chromatograms showing the target region of sgPik3caE542K, sgPik3caE545K and sgPik3caE453K in 3 independent tumors from WB1P-BE3 females injected with the corresponding Lenti-sgPik3ca-Myc vectors. Arrowheads highlight cytosines of the protospacer that show base editing.

(F) EditR was used to calculate the average frequency (%) of C-to-T conversion at the indicated target cytosines of the protospacers in tumors from WB1P-BE3 females injected with Lenti-sgNT-Myc or Lenti-sgPik3caE542K-Myc, Lenti-sgPik3caE545K-Myc and Lenti-sgPik3caE453K-Myc. Data are plotted as mean + standard deviation (n=7).
In situ CRISPR-Cas9 base editing for the development of genetically engineered mouse models of breast cancer (2019)
Figure 3

Figure 3

In vivo nonsense editing of Pten.

(A) Sanger-sequencing chromatograms showing the target region of sgPtenQ245* in wild-type (WT) and base edited (BE) cells. Arrowheads highlight cytosines of the protospacer that show base editing 5 days after transduction of BE3-expressing NIH3T3 cells with Lenti-sgPtenQ245*.

(B) EditR was used to calculate the frequency (%) of C-to-T conversion at C4 of the protospacer targeted by sgPtenQ245* in BE3-expressing NIH3T3 cells 5 days after transduction with the indicated sgRNA vectors.

(C) Overview of the intraductal injections performed in WB1P-BE3 females with high-titer lentiviruses encoding Myc and either a non-targeting sgRNA (Lenti-sgNT-Myc) or the sgRNA targeting Pten (Lenti-sgPtenQ245*-Myc).

(D) Kaplan-Meier curves showing mammary tumor-specific survival for the different models. WB1P-BE3 females injected with Lenti-sgPtenQ245*-Myc (n=11) showed a reduced mammary tumor-specific survival compared to WB1P-BE3 female mice injected with Lenti-sgNT-Myc (n=11) vectors (37 days after injection vs 72 days after injection, P < 0.0001 by Mantel-Cox test).

(E) BE Analyzer (Hwang et al., 2018) was used to assess from next-generation sequencing data the fraction of wild-type Pten alleles, base edited alleles or alleles with insertions/deletions (indels) in tumors from WB1P-BE3 animals injected with Lenti-sgNT-Myc or Lenti-sgPtenQ245*-Myc.

(F) TIDE analysis showing the spectrum of indels of the targeted Pten alleles in two independent representative tumors from WB1P-BE3 mice injected with Lenti-sgPtenQ245*-Myc.

(G) For the two tumors shown in (F), Sanger-sequencing chromatograms showing the target region of sgPtenQ245* (PCR products were subcloned for clarity). Arrowheads highlight cytosines of the protospacer that show base editing. In the lower example the gene was inactivated by indels at both alleles, while in the upper one by Q245* base editing in one allele and a deletion at the second copy of the gene.
In situ CRISPR-Cas9 base editing for the development of genetically engineered mouse models of breast cancer (2019)
Figure 4

Figure 4

Multiplexed in vivo base editing.

(A) Sanger-sequencing chromatograms showing the target region of sgTrp53Q97* in wild-type (WT) and base edited (BE) cells. Arrowheads highlight cytosines of the protospacer that show base editing 5 days after transduction of BE3-expressing NIH3T3 cells with Lenti-sgTrp53Q97*.

(B) EditR was used to calculate the frequency (%) of C-to-T conversion at C8 of the protospacer targeted by sgTrp53Q97* in BE3-expressing NIH3T3 cells 5 days after transduction with the indicated sgRNA vectors.

(C) Overview of the intraductal injections performed in WapCre;Brca1F/F;Trp53F/+;Col1a1invCAG-BE3/+ (Trp53F-het WB1P-BE3) females with high-titer lentiviruses encoding Myc and either a non-targeting sgRNA (Lenti-sgNT-Myc), the sgRNA targeting Trp53 (Lenti-sgTrp53Q97*-Myc) or two arrayed sgRNA cassettes encoding sgPik3caE545K and sgTrp53Q97* (Lenti-sgPik3caE545K/sgTrp53Q97*-Myc).

(D) Kaplan-Meier curves showing mammary tumor-specific survival for the different models. WapCre;Brca1F/F;Trp53F/+;Col1a1invCAG-BE3/+ females injected with Lenti-sgPik3caE545K/sgTrp53Q97*-Myc (n=6) showed a reduced mammary tumor-specific survival compared to animals injected with Lenti-sgTrp53Q97*-Myc (n=5) vectors (76 days after injection vs 101 days after injection, P < 0.05 by Mantel-Cox test). Females injected with Lenti-sgNT-Myc (n=11) did not develop any palpable tumors during the 150 days observation period.

(E) BE Analyzer was used to assess from next-generation sequencing data the fraction of wild-type Trp53 alleles, base edited alleles or alleles with indels in tumors from WapCre;Brca1F/F;Trp53F/+;Col1a1invCAG-BE3/+ animals injected with Lenti-sgTrp53Q97*-Myc. Tumors from WB1P-BE3 animals injected with Lenti-sgNT-Myc mice were used as control.

(F) TIDE analysis showing the spectrum of indels of the targeted Trp53 alleles in two independent representative tumors from WapCre;Brca1F/F;Trp53F/+;Col1a1invCAG-BE3/+ mice injected with Lenti-sgTrp53Q97*-Myc.

(G) For the two tumors shown in (F), Sanger-sequencing chromatograms showing the target region of sgTrp53Q97*. Arrowheads highlight cytosines of the protospacer that show base editing. In the lower example the gene was inactivated by a deletion, while in the upper one by Q97* base editing. Of note, the allele with the indel also displays base editing at C6 of the protospacer.

Expanded View figure legends
Key phosphorylation sites in GPCRs orchestrate the contribution of β-Arrestin 1 in ERK1/2 activation (2020)
Figure 1

Figure 1. βarr1 has opposite contributions in agonist-induced ERK1/2 activation for V2R and B2R.

(A) G-protein-coupling preference and phospho-site patterns in the carboxyl-terminus of V2R and B2R, deduced based on a previous study (Zhou et al., 2017). The phospho-site patterns in the form of PXPXXP and PXXPXXP are underlined and color-coded. Both, V2R and B2R recruit βarrs in "class B" pattern (Oakley et al., 2000) as reflected by stable interaction and endosomal trafficking of the receptor-βarr complexes.

(B) Confocal microscopy reveals typical "class B" pattern of βarr1 recruitment for V2R and B2R as reflected by first the localization at the plasma membrane and subsequently, internalization in endosomal vesicles upon agonist-stimulation. HEK-293 cells expressing V2R/B2R and βarr1-mCherry were stimulated with agonist (AVP; 100nM and Bradykinin; 100nM) and the localization of βarr1 was visualized using confocal microscopy. Representative images from three independent experiments are shown here, and the scale bar is 10μm. Visual scoring of images from three independent experiments revealed agonist-induced βarr1 recruitment (i.e. membrane and endosomal localization) in approximately 77% of the cells for V2R (221 cells) and 75% of the cells for B2R (662 cells).

(C,D) Agonist-induced phosphorylation of ERK1/2 in HEK-293 cells expressing either V2R or B2R in the absence (control; CTL) and presence of βarr1 knock-down (βarr1-KD) are measured using Western blotting. Densitometry-based quantification of data (mean ±SEM) from four independent experiments is presented as bar-graphs in the right panels, normalized with respect to maximal signal under control condition (treated as 100%), and analyzed using Two-way-ANOVA with Bonferroni multiple comparisons test (***p<0.001, **p <0.01).
Key phosphorylation sites in GPCRs orchestrate the contribution of β-Arrestin 1 in ERK1/2 activation (2020)
Figure 2

Figure 2. Spatial distribution of phospho- sites and βarr1 recruitment of B2R mutants.

(A) Phospho-site clusters in the carboxyl-terminus of V2R and B2R are underlined and color-coded to reflect the PXPXXP and PXXPXXP patterns as proposed in a previous study (Zhou et al., 2017).

(B) A number of B2R mutants were generated to mimic the spatial distribution of phospho-site pattern of Ser/Thr as present in the V2R. The first three mutants were designed to target the proximal part of the phospho-site pattern (indicated in red dotted box) while the fourth mutant was designed to target the distal part (indicated in blue dotted box).

(C) Confocal microscopy reveals robust recruitment of βarr1 to B2R mutant constructs upon agonist-stimulation. HEK-293 cells expressing either B2RWT or B2R mutants along with βarr1-mCherry were stimulated with agonist (Bradykinin; 100nM) and the localization of βarr1 was visualized using confocal microscopy. Representative images from three independent experiments are shown here, and the scale bar is 10μm. Visual scoring from three independent experiments revealed agonist-induced βarr1 recruitment (i.e. membrane and endosomal localization) in approximately 75% of the cells for B2RWT (662 cells), 74% of the cells for B2RΔG368 (169 cells), 75% of the cells for B2RL370T (130 cells), 85% of the cells for B2RΔG368+L370T (132 cells) and 77% of the cells for B2RΔI374 (116 cells).

(D) HEK-293 cells expressing B2R constructs with C-terminal SmBiT and βarr1 with N-terminal LgBiT were treated with indicated concentrations of bradykinin, and ligand-induced change in luminescent signal was measured. Concentration-response curves were plotted using GraphPad Prism, and pEC50 (top) and Emax (bottom) were calculated. Data represent mean ± SEM of five independent experiments (three for B2RΔI374), each performed in duplicate.
Key phosphorylation sites in GPCRs orchestrate the contribution of β-Arrestin 1 in ERK1/2 activation (2020)
Figure 3

Figure 3. Contribution of βarr1 in ERK1/2 phosphorylation for B2R mutants.

(A-D) Knock-down of βarr1 leads to an increase in agonist-induced ERK1/2 phosphorylation for B2RΔG368 and B2RΔI374 but a decrease for B2RL370T and B2RΔG368+L370T mutants. HEK-293 cells expressing the indicated B2R mutants in presence and absence of βarr1 knock-down were stimulated with indicated doses of bradykinin (Brady) for 10 min followed by detection of phosphorylated ERK1/2 using Western blotting. Densitometry-based quantification of data (mean±SEM) from three independent experiments (five for B2RΔG368 and B2RΔI374) is presented as bar graphs in the lower panels. Data are normalized with respect to maximal signal under control condition (treated as 100%), and analyzed using Two-way-ANOVA with Bonferroni multiple comparisons test (*p <0.05; **p <0.01; ***p<0.001).
Key phosphorylation sites in GPCRs orchestrate the contribution of β-Arrestin 1 in ERK1/2 activation (2020)
Figure 4

Figure 4. An intrabody sensor suggests distinct conformations of βarr1 for V2R and B2R.

(A-D) HEK-293 cells expressing V2R, B2R, B2RΔG/L370Tor B2RL370T along with βarr1-mCherry and YFP-tagged intrabody30 (Ib30-YFP) were stimulated with their respective agonists (AVP, 100nM for V2R) and (bradykinin, 1μM for B2R) and the localization of βarr1 and Ib30 were visualized. Although βarr1 gets recruited for all four constructs, Ib30-YFP does not colocalize with βarr1 either at the surface or upon internalization in case of B2RWT. Representative data from 3-5 independent experiments are shown and the scale bar is 10μm. Visual scoring from 3-5 independent experiments revealed agonist-induced βarr1 recruitment (i.e. membrane and endosomal localization) in approximately 82% of the cells for V2R (691 cells), 10% for B2RWT (254 cells), 63% for B2RL370T (273 cells) and 83% for B2RΔG368+L370T (158 cells). Pearson's correlation coefficients (PCC) were measured to assess the colocalization of βarr1 and Ib30 using JACoP plugin in ImageJ, and the mean±sem for the basal, surface and internalized panels, respectively, are presented here. B2RWT - 0.32±0.02 from 16 cells, 0.18±0.01 from 32 cells, 0.25±0.02 from 31 cells; B2RΔG/L370T - 0.32±0.03 from 13 cells, 0.92±0.01 from 18 cells, 0.90±0.01 from 16 cells; B2RL370T - 0.26±0.01 from 10 cells, 0.85±0.01 from 11 cells, 0.87±0.01 from 25 cells.
Key phosphorylation sites in GPCRs orchestrate the contribution of β-Arrestin 1 in ERK1/2 activation (2020)
Figure 5

Figure 5. βarr1 conformation and G-protein coupling for B2R mutants.

(A) HEK-293 cells expressing the indicated receptor constructs (pcDNA vector control for mock-transfection) together with βarr1-SmBiT and LgBiT-Ib30 were stimulated with the indicated concentrations of respective agonists for 10min. Subsequently, the luminescence signal generated upon the interaction of βarr1-SmBiT and LgBiT-Ib30 was measured, normalized with basal signal (i.e. no agonist stimulation treated as 1), and plotted using GraphPad Prism. pEC50 (top) and Emax (bottom) values are mentioned in the respective graphs and data represent mean±SEM of four independent experiments, each carried out in duplicate.

(B) HEK-293 cells expressing the NanoBiT-Gq protein and indicated B2R constructs were treated different concentrations of bradykinin, and ligand-induced change in luminescent signal was measured. Concentration-response curves were plotted using GraphPad Prism to calculate pEC50 (top) and Emax(bottom) values included in the graph. Data represent mean±SEM of three independent experiments with each performed in duplicate, and normalized with respect to the luminescence signal under basal (no-agonist treatment) condition (treated as 1).
Key phosphorylation sites in GPCRs orchestrate the contribution of β-Arrestin 1 in ERK1/2 activation (2020)
Figure 7

Figure 7. Contribution of βarr1 in ERK1/2 phosphorylation for AT1aR mutants.

(A) Phospho-site clusters in the carboxyl-terminus of V2R and AT1aR are underlined and color-coded to reflect the PXPXXP and PXXPXXP patterns as proposed in a previous study (Zhou et al., 2017).

(B) Two different AT1aR mutants were generated by site-directed mutagenesis to mimic the spatial distribution of Ser/Thr as in V2R with respect to double-Thr cluster. The red dotted box highlights the resulting sequence upon either the delition of Leu337 or Leu337Thr mutation.

(C-E) HEK-293 cells expressing the indicated AT1aR constructs, in absence (CTL; control) or presence of βarr1 knock-down (βarr1-KD) were stimulated with indicated concentrations of angiotensin II (AngII) for 10 min. Subsequently, ERK1/2 phosphorylation was measured by Western blotting and signal intensities were quantified using densitometry and presented as bar graphs. Data were normalized with respect to the signal at 100nM agonist concentration in control cells (treated as 100%) and represent mean±SEM of five (six for AT1aRΔL337) independent experiments. Data were analyzed using Two-way-ANOVA with Bonferroni multiple comparisons test (***p<0.001, p<0.05).

Expanded view Figure legends
Discovery of Atg5/Atg7-independent alternative macroautophagy (2009)
figf1

a, Etoposide-treated Atg5-/- MEFs were assessed by EM (left) and by Lamp2 immunofluorescence (IF; middle). Right: reverse Lamp2 immunofluorescence image merged with the electron microscopy image. Arrows indicate Lamp2-positive autolysosomes. Magnified photos are provided in Supplementary Fig. 5. b, The size of each autophagic vacuole (AV) (n = 30 cells) in MEFs treated with etoposide. The total number of AVs in 30 cells was 483 in WT cells and 494 in Atg5-/- cells. c, Percentage autophagic area (n ≥ 20 cells) in MEFs treated with etoposide. d, e, Representative macroautophagy in etoposide-treated Atg5-/- MEFs. Conventional (d) and quick freezing and freeze-substitution (e) techniques were used. The autolysosomes (arrows) contain multilamellar bodies (arrowheads). f, The number of autophagosomes (white columns) and autolysosomes (black columns) in MEFs (n = 10 cells) treated with etoposide. g, Reduction of percentage autophagic area by 3-methyladenine (3-MA; n = 22 cells). h, Time-course analysis of long-lived protein degradation (n = 4). i, Inhibition of long-lived protein degradation by bafilomycin A1, protease inhibitor (PI) cocktail and 3-MA (*P  0.05; n = 4). j, k, Similar experiments to those in c and i were performed by starvation. Red and blue lines in b, c, g and j indicate means and s.e.m., respectively. The dashed line in c, g and j indicates the autophagic cell border (6%). Black error bars indicate s.d.
Discovery of Atg5/Atg7-independent alternative macroautophagy (2009)
figf2

a, The production of LC3-II. Gapdh was used as a loading control. b, c, Punctate GFP–LC3 fluorescence in WT MEFs but not in Atg5-/- MEFs. b, Representative photographs. c, Graphical presentation of the percentages of WT MEFs and Atg5-/- MEFs with punctate GFP–LC3 fluorescence. d, The same GFP–LC3-transfected Atg5-/- MEFs were assessed by IF and by EM. The magnified photo is the area indicated by the squares. Arrows indicate AVs. e, Atg5-/- MEFs were incubated without (NT) or with etoposide and immunostained with an anti-Lamp2 antibody (green). Nuclei were counterstained (red). The percentages of cells with punctate Lamp2 immunostaining are shown (means ± s.d., n = 4). f, Microarray analysis of the Atg5-/- MEFs during treatment with etoposide. g, h, Induction of Ulk1 in the etoposide-treated Atg5-/- MEFs was assessed by qPCR (g) and by immunoblotting (h). i–l, Atg5-/- MEFs were transfected with the siRNAs related to the Ulk1 complex (i, j) and the PI(3)K complex (k, l) and a beclin 1 plasmid (k) for 24 h and treated with etoposide for 18 h. Induction of macroautophagy was assessed by Lamp2 IF (i, k) and by EM (j, l). In i and k, *P  0.05 (n = 4); in j and l, *P  0.01 (n = 35 cells each). hBeclin 1, human beclin 1. Red and blue lines in j and l indicate means and s.e.m., respectively. The dashed line in j and l indicates the autophagic cell border. Black error bars indicate s.d. (n = 4). Original magnification (b, e), × 200.
Discovery of Atg5/Atg7-independent alternative macroautophagy (2009)
figf3

a–d, Electron micrographs of etoposide-treated Atg5-/- MEFs. a, AVs were observed near the Golgi apparatus (G). Inset, the isolation membrane (I) was extended from the Golgi stack. b, Developing autophagosome. rER, rough endoplasmic reticulum. c, d, Isolation membrane fusing with vesicles containing thick membrane (d) and a complete autophagosome (c) is generated. e, f, Co-localization of Lamp2 with GFP–M6PR, GFP–Stx7 or GFP–Rab9 in etoposide-treated Atg5-/- MEFs. Arrows indicate co-localized dots. Original magnification, × 200. g–i, Atg5-/- MEFs were transfected with the indicated siRNAs for 24 h and treated with etoposide. Induction of macroautophagy was assessed by Lamp2 IF (g) and by EM (h), and the number of isolation membranes was calculated (i). In g, error bars indicate s.d. (n = 4). In h and i, red and blue lines indicate means and s.e.m., respectively; n = 35 cells each. j, No effect of silencing of Rab9 (siRab9) on the production of LC3-II in etoposide-treated WT MEFs.
Discovery of Atg5/Atg7-independent alternative macroautophagy (2009)
figf4

a–e, Typical autophagic structures in the tissues of an Atg5-/- embryo (E14.5): a, midbrain; b–d, liver; c, d, developing autophagosomes; e, heart. Arrows indicate the autophagic vacuoles. f, g, k, l, Electron micrographs of wild-type (WT) and Atg5-/- (KO) reticulocytes (f, g) and erythrocytes (k, l). An isolation membrane (asterisk), autophagosomes (arrows) and autolysosomes (arrowheads) are shown. Inset, engulfed mitochondria. h–j, m–o, The numbers of autophagic vacuoles (h, m) and autophagic vacuoles containing mitochondria (i, n) and persisting mitochondria (j, o) in reticulocytes (h–j) and erythrocytes (m–o). Error bars indicate s.d. (*P  0.05; n = 55 cells).
Reduced proteasome activity in the aging brain results in ribosome stoichiometry loss and aggregation (2020)
Figure 3

Figure 3. Aggregation of ribosomal proteins during brain aging.

(A) Representative Coomassie-stained SDS-PAGE gel showing the isolation of SDS insoluble aggregates from mouse brain lysates. TH = total homogenate, SUP = supernatant, W1 = SDS-soluble fraction, Pellet = formic-acid soluble fraction (see Figure EV4A).

(B) Quantification of the yield of SDS-insoluble aggregates from young and old brain lysates was based on densitometry analysis of Coomassie-stained gel bands obtained from different animals, n=5 per age group (Figure EV4B); ** p < 0.01, unpaired t-test. In boxplots, the horizontal line represents the median, the bottom and top of the box the 25th and 75th percentile, respectively, and the whiskers extend 1.5 folds the interquartile range.

(C) Proteins enriched in aggregates show a predicted higher molecular chaperone requirement for folding (top vs. bottom 20% p=0.0055, Kolmogorov-Smirnov test), and are richer in intrinsically disordered regions (s2D-derived scores, top vs. bottom 20% p=0.0019, Kolmogorov-Smirnov test). Violin plots: the solid line shows the median, the dotted lines the interquartile ranges. The same result was obtained with cleverSuite-derived scores (top vs. bottom 20% p=0.0201, Kolmogorov-Smirnov test) (Figure EV4C). ** p < 0.01, Kolmogorov-Smirnov test.

(D) Volcano plot based on protein quantification by label-free mass spectrometry depicting the enrichment of specific proteins in protein aggregates. The x-axis indicates the log2 ratio between protein abundance in aggregates (Pellet) and starting total homogenate (TH). The horizontal dashed line indicates a p value cut-off of 0.05 and vertical lines a log2 fold change cut off of ± 0.5. Selected proteins are highlighted as colored dots as indicated in the figure legend. Protein quantification was based on samples obtained from 3 independent isolations.

(E) Double-labelling of telencephalic sections of N. furzeri with anti-RPS6 (green) as ribosomal marker and Proteostat as a marker for aggregated proteins (red). Nuclear counterstaining was performed with DAPI (blue). Scale bar = 10 μm.

Data information: Related to Figure EV4 and Dataset EV7.