EMBO/SourceData huggingface dataset:
Figure 2Sanger-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.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.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).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. 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.
Figure 3Sanger-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*. 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.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).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. 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. 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.
Figure 4Sanger-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 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.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.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. 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. 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.
Figure 2Myc-tagged ATG2A-wild type (WT) and ATG2A-mLIR (orange) were co-expressed with GFP-alone, GFP-LC3B or GFP-GABARAP in HEK293T cells, lysed and anti-GFP nanobodies coupled to agarose were used to immunoprecipitate GFP-tagged proteins. Samples were subjected to western blotting and probed for the presence of Myc-ATG2A in immunoprecipitated samples. Anti-p62/SQSTM1 was used as an internal control for the immunoprecipitated samples.   As in (B) but using Myc-tagged ATG2B-WT or ATG2B-mLIR (orange) co-expressed with GFP-alone, GFP-LC3B or GFP-GABARAP. Samples were subjected to western blotting and probed for the presence of Myc-ATG2B in immunoprecipitated samples. Anti-p62/SQSTM1 was used as an internal control for the immunoprecipitated samples.   HA-tagged ATG2A-WT, -mLIR (orange) and -mYFS (YFS/AAA; purple) were stably expressed in U2OS ATG2A/B double knock-out cells using retrovirus transduction. Cells were transfected with GFP-GABARAP and 24 h later grown in complete medium (CM) or starved for 2 h (EBSS). Cells were lysed and anti-HA beads were used to immunoprecipitate HA-tagged ATG2A and processed for western blot. Blots were then probed with antibodies against HA-tag (ATG2A), anti-WIPI4, and anti-GFP for the presence/absence in immunoprecipitated samples. All blots are representative of at least n=3 independent experiments.   Quantification of WIPI4 and GFP-GABARAP-ii co-precipitation with HA-ATG2A from (D). bands were normalized against HA-ATG2A immunoprecipitate and expressed as a fold change compared to HA-ATG2A-WT (complete media; CM). Each symbol represents an independent experiment (n=3) and error bars mean ± SD.   ATG2A/B do8uble knock-out cells (DKO) stably expressing HA-tagged ATG2A-WT, -mLIR (orange) or -mYFS (YFS/AAA; purple) were transfected with GFP-GABARAP and 24 h later grown in complete medium (CM) or starved for 2 h (EBSS). Cells were lysed and GFP-GABARAP was immunoprecipitated using anti-GFP trap beads. Samples were processed for western blot and probed with anti-HA-tag (ATG2A), anti-WIPI4, and anti-GFP for their presence/absence in immunoprecipitated samples. All blots are representative of at least n=3 independent experiments.   Quantification of HA-ATG2A and WIPI4 co-precipitation with GFP-GABARAP from (F). Results were expressed as a fold change compared to HA-ATG2A-WT (complete media; CM). Each symbol represents an independent experiment (n=3) with error bars mean ± SD.   GFP alone, GFP-GABARAP or GFP-GABARAP with increasing concentrations of mCherry-WIPI4 were expressed in HEK293T cells for 24 h, lysed and GFP-trap beads used to immunoprecipitate GFP alone or GFP-GABARAP. Samples were then probed with antibodies to detect endogenous ATG2A or ATG2B in immunoprecipitated samples. Blots are representative of n=3 independent experiments.   Quantification of ATG2A (green line, round symbols) and ATG2B (grey line and squares) co-precipitation with GFP-GABARAP in the presence of increasing concentrations of mCherry-WIPI4 from (H). Co-precipitation was normalised to GFP-GABARAP alone. Line and error bars are mean ±SD of n=3 independent experiments.
Figure 3U2OS ATG2A/B double knock-out (DKO) CRISPR/Cas9 cells stably expressing Tandem tagged LC3B (mCherry-GFP-LC3B) were retrovirally transduced to express vector, or HA-tagged ATG2A-WT, -mLIR (FCIL/AAAA) or -mYFS (YFS/AAA). Cells were grown in complete medium (CM) or starved for 2 h (EBSS) or treated with CM plus BafilomycinA1 (200nM, 4 h), fixed and analysed by confocal microscopy. Merged images of GFP (green) and mCherry (red) channels show the presence of autophagosomes/phagophores (GFP and mCherry positive, yellow puncta) or autolysosomes (mCherry only, red puncta) Scale bar 10µm. Images are representative of n=3 independent experiments.Quantification using flow cytometry of measuring GFP and mCherry fluorescence. Cells were gated based on GFP and mCherry fluorescence and % mCherry positive cells gated used as an indication of autolysosome formation due to GFP quenching. Each symbol represents 1 independent experiment with 10,000 cells analysed per condition. A total of n=3 independent experiments were performed, and horizontal bar indicates mean ± SD.U2OS ATG2A/B DKO cells reconstituted with vector only, HA-ATG2A-WT, -mLIR and -mYFS were stimulated with complete medium, (CM), 2 h starvation (EBSS) or 4 h BafilomycinA1 (BafA1, 200nM), lysed in total cell lysis buffer and subjected to western blot analysis. Blots were probed for the presence of HA-tag (ATG2A), p62/SQSTM1, LC3B, pan-GABARAP (GABARAP, GABARAP-L1 and GABARAP-L2) and vinculin (loading control).D, E p62/SQSTM1 (D) and LC3B-II (E) levels were normalized to loading control and quantified as fold change of DKO proteins levels. Each symbol represents an independent experiment. Quantification of at least n=3 independent experiments is shown. Horizontal bar represents mean ± SD.
Figure 4U2OS ATG2A/B DKO cells reconstituted with vector only, HA-ATG2A-WT, -mLIR and -mYFS were stimulated with complete medium, (CM), 4 h starvation (EBSS) plus BafilomycinA1 (BafA1, 200nM) treatment. Cells were centrifuged and resuspended in PBS digitonin, spun, washed and the membrane fractions incubated with proteinase K with and without 0.1% TritonX-100. Sampels were then subjected to western blotting using anti-p62/SQSTM1 and anti-HA (ATG2A) antibodies. Percentage p62/SQSTM1 remaining was calculated using densitometry analysis. Blots are representative of n=3 independent experiments.U2OS ATG2A/B DKO cells reconstituted with vector only, HA-ATG2A-WT, -mLIR and -mYFS and stably expressing GFP-Syntaxin17 (STX17) were stimulated starvation (EBSS) plus BafilomycinA1 (BafA1, 200nM) for 4 hours to stimulate autophagosome generation and prevent their degradation in the lysosome. Cells were fixed and immune-stained for LC3B (magenta). DAPI was included (blue) to mark the DNA/nucleus. Confocal analysis of LC3B and GFP-STX17 (green) localization was performed. Closed arrows (ATG2A/B DKO and DKO + ATG2A mLIR) highlight aggregate structures. Open arrows (DKO + ATG2A-WT and ATG2A-mYFS) highlight STX17/LC3B positive vesicles. Scale bar 10µm.   Quantification of (C) expressed as a percentage of cells with STX17/LC3B positive vesicles. Each symbol represents a single field of cells with 5-10 cells per field. A total of 300-600 cells were analysed over n=3 independent experiments. Data is shown as mean ± SD.
Figure 5(A-C) U2OS ATG2A/B double knock out (DKO) cells or DKO reconstituted with ATG2A-WT, -mLIR (FCIL/AAAA) or -mYFS (YFS/AAA) were starved for 2 h, fixed and stained for LC3B (green) or WIPI2 (magenta), (B) ATG9A (magenta) or (C) GABARAP (Magenta) and imaged using a Zeiss 880 Airyscan super-resolution confocal microscope. Images are representatives of n=3 independent experiments. Scale bars 10µm.Transmission electron micrographs of ATG2A/B DKO cells (upper left), DKO + ATG2A-WT (upper right), DKO+ ATG2A-mLIR (lower left) and DKO+ATG2A-mYFS( lower right) starved (EBSS) for 2 h. Clustered small vesicles (open arrowheads; DKO and DKO-mLIR) and autophagosomes (closed arrow heads, DKO+ATG2A-WT and -mYFS) indicated. Images are representative of n=3 independent experiments. Scale bars 500nm. ER= endoplasmic reticulum; N = Nucleus; Mt = Mitochondria; G = Golgi; AP = Autophagosome.
Figure 1D Zygotic grk2i283 mutant at 4dpf compared to wild type (N=80). Arrows indicate the position of swim bladder. Scale bar, 1mm
Figure 2A Western blot analysis of the different forms of Gli2a in MZgrk2 embryos compared to wild type, shh mRNA and dnPKA mRNA injected wild type and cyclopamine (Cyc(A)) treated wild type 20hpf embryos. Gli2a-FL levels are low relative to Gli2aR levels in wild type embryos, but are elevated in response to pathway activation (shh and dnPKA mRNA injected). Gli2aR levels are increased while Gli2a-FL is undetectable in CycA treated and MZgrk2 mutant embryos. Probing the same blot with rabbit anti-GRK3 (which recognizes the zebrafish Grk2 protein) reveals a complete loss of full length Grk2 in MZgrk2 embryos. Probing with anti-γ-tubulin was performed as a loading control. Three biological replicates of this analysis were performed.B Phenotype of wild type, MZgrk2, and grk2-GFP mRNA injected MZgrk2 embryos at 24hpf (n=20 for each sample). The white lines indicate the shape of the somites (middle panels) and the separation of the eyes (right hand panels). Scale bar, 200μmC In situ hybridisation of ptch2, nkx2.2a and olig2 transcripts in wild type, MZgrk2 and grk2-GFP injected MZgrk2 24hpf embryos. Each panel shows a full view of the embryo on the left and a cross sectional view of a somite on the right. (n=30 for each sample). Scale bars, 200μm (whole mounts), 50μm (sections).D Expression of Prox1a and Engrailed proteins (Eng) in somites of wild-type, MZgrk2 and grk2-GFP mRNA injected MZgrk2 embryos at 30hpf. Each panel shows Prox1a in green, Eng in red and the merge images with DAPI staining in blue. (n=10 for each sample). Scale bar, 50μm.
Figure 3A Prox1a and Eng expression in the myotome of 30hpf wild type and MZgrk2 embryos injected with mRNAs encoding Shh, dnPKA or mSmoA1-GFP. Each panel shows Prox1a in green, Eng in red, and the merged images with DAPI staining in blue. The co-labelling for Prox1a and Eng (orange) is indicative of MP differentiation (n=30 for each sample) Scale bar, 50μm.B Western blot showing levels of Gli2a-FL and Gli2a-R forms in 20hpf wild type and MZgrk2 embryos injected with mRNA encoding Shh, dnPKA or mSmoA1-GFP, respectively. γ-tubulin was used as loading control. Three biological replicates of this analysis were performed.
Figure 4A Western blot analysis of GRK2, GLI1, PTCH1, and GLI3 protein levels in Flp-In-3T3 cells with lentiCRISPR of 4 different guide RNAs in presence or absence of SAG. α-tubulin was used as loading control. Guide RNA 2 was found to remove Grk2 efficiently and used for subsequent experiments. GLI3 panel shows both full-length (GLI3FL) and repressor form of GLI3 (GLI3R). (n=3)B, C Hh reporter activity assay for wild type and Grk2-/- cells. Cells were treated with PKA peptide inhibitor (PKI) and mutant form of PKA peptide inhibitor (PKI-M) respectively, in presence or absence of SHH (B) Cells were transfected with mSmoA1-GFP expressing constructs in the presence of SHH or SAG (C). Data represent the mean and ± SD (n=3). Unpaired Students t-test was used for analysis. ***P<0.001; **P<0.01; *P<0.05 and n.s. (not significant).
Figure 5A Expression of Prox1a (green) and the eng2a:GFP reporter (red) in otherwise wild-type 30hpf embryos injected with mRNA encoding wild type and mutant forms of mouse Smo. Ectopic MPs are indicated by fibres co-labelled with Prox1a and GFP; ectopic MFFs are labeled only with GFP. Images are representative of embryos in the following proportions of each sample: 28/28 (mSmo; mSmoA1; mSmoSD; mSmoKRA); 9/28 (mSmoA1SA); 12/28 (mSmoA1SD). Scale bar, 50μm.B Average number of Prox1a+ve slow fibres in wild type embryos injected with mRNA encoding different forms of mSmo. Prox1 positive cells were quantified in 4 somites in each of 4 embryos for each sample. The error bars indicate SD. Unpaired Student's t-test was used to determine the statistical significance between uninjected embryos and the various Smo mutants (black asterisk); and between mSmoA1 and mSmoA1SA or mSmoA1SD (red asterisk). ***P<0.001; **P<0.01; *P<0.05 and n.s. (not significant).C Prox1a and Eng expression in smohi1640 mutant embryos injected with mRNA encoding mSmo (n=10), mSmoSA (n=9) and mSmo14SA (n=6). Note the full recovery of SSFs and MPs compared to the un-injected controls. Scale bar, 50μm.D In situ hybridisation for transcripts of ptch2, olig2 and nkx2.2 in 24hpf smohi1640 mutant embryos injected with mRNA encoding mSmo and mSmoSA (n=6 for each sample). Scale bars, are 100μm (lateral view), 50μm (sections).E Hh reporter assay of the activity of wild type and mutant forms of mSmo in Smo-/- MEFs in response to Shh or SAG stimulation. Note that mSmoA1 shows constitutive activity in the absence of either Shh or SAG whereas mSmoKRA dose not; all mutants affecting phosphorylation failed to restore the response to Shh or SAG. Data represent the mean and ± SD (n=3). Unpaired Students t-test was used for analysis. ****P<0.0001; **P<0.01; and n.s. (not significant).
Figure 6A Wild type 18hpf embryos injected with mRNA encoding GFP-tagged mSmo (green) showing localisation to the PC of myotomal cells labeled with anti-Acetylated tubulin (AcTub; red), stimulated in response to Shh injection (n = 4). Differences in PC distribution are due to morphological changes in the myotome induced by ectopic expression of Shh as revealed by the distribution of nuclei (DAPI stained; blue). Scale bar, 10μm.B Notochord cells of wild type embryos expressing GFP tagged wild-type and mutant forms of mSmo. Note the localisation to the PC (labeled with anti-AcTub; red) in each case (n=4 for each sample). Scale bar, 10μm.C MZgrk2 18hpf embryos injected with mRNA encoding GFP-tagged wild type mSmo or mSmoSA (green) showing localisation to the PC (labeled with anti- AcTub; red) in myotomal cells. More Smo is localised to the PC in MZgrk2 mutants compared to wild type (panel A). Scale bar, 10μm.
Figure 7B Prox1a (green) and Eng (red) expression in the myotome of 30hpf MZgrk2 embryos that were injected with mRNA encoding: Grk2K220R-GFP; Grk2K220M-GFP; Grk2D335N-GFP; Grk2ΔN7aa-GFP and mGrk2L4AV7AL8A-GFP. Merged panels show nuclear staining with DAPI in blue. (n=20 for each sample). Scale bar, 50μm.C Hh reporter activity assay in wild type and Grk2-/- 3T3 cells transfected with the equivalent wild type and mutant kinase domain forms of mouse Grk2, Grk2K220R, Grk2K220M and Grk2D335N after 24h Shh-N and SAG treatment. Data represent the mean and ± SD (n=3). Unpaired Students t-test was used for analysis. ****P<0.0001; **P<0.01; *P<0.05; and n.s. (not significant).
Figure 1Transgenic 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.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).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.
Figure 2representative SeaHorse time courses of OCR and ECAR respectively; where are indicated the following compounds added: oligomycin (olig.), carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), rotenone plus antimycin A (R/A), glucose (Glu), 2-deoxyglucose (2DG). Each point in the OCR and ECAR time-courses is the average ± S.D. of four technical replicates.OCR/ECAR ratio; (4 technical replicates each); Basal, (resting OCR)/(ECAR +Glu); Max, (OCR+FCCP)/(ECAR+Olig.)Cellular NAD+ and NADH content and NAD+/NADH.Confocal microscopy analysis of the mitochondrial membrane potential DYm. Pictures on the right, representative imaging of cells loaded with tetramethylrhodamine, ethyl ester (TMRE); histogram on the left, averaged fluorescence intensity/cell ± SD of n=3 independent biological replicates; *, P < 0.05.
Figure 3Representative 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).Electron microscopy images of sorted ESZscan4_Emerald and control cells. Scale bar 1μmRepresentative 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.
Figure 4Basal R/A-sensitive OCR measured as in Fig. 2A in the absence and in the presence of a cocktail of inhibitors (BPTES + etomoxir + UK5099); End. Sub., OCR measured with endogenous substrates; Exo. Sub., OCR measured with glutamine (Gln), fatty acids (FA) and glucose (Glc) supplemented in the assay medium. The superimposed bars are means ± SEM of n=3 independent biological replicates (3 technical replicates each);Measurement of dependency and flexibility of the OCR to either of Gln, FA, Glc; the assay was carried out according to the MitoFuel protocol (see Material and Methods); the superimposed bars (whose sum corresponds to the capacity) are means ± SEM of n=3 independent biological replicates (3 technical replicates each);LC-MSMS analysis of the cellular content of free aminoacids; the symbols of aminoacids are indicated in x-axis; the amount expressed as μmol/μg protein is indicated in y-axis. The inset shows the content of ornithine, citrulline and arginine.(D)LC-MSMS analysis of the cellular content of acylcarnitines; the length of the acyl moiety is indicated by the number of carbon atoms in abscissa; bars values are means ± SEM of n=3 independent biological replicates; The inset shows the content of carnitine (C0) and acetylcarnitine (C2).
Figure 1B, Multiplexed activation of Ascl1, Lmx1a, NeuroD1 and of Ascl1, Lmx1a, Nr4a2 in primary astrocytic cultures. n=3, representative experiment from 2-3 independent experiments, additional data in supplement. Activation levels are depicted as fold change between cells transfected with and without sgRNAs. All levels were normalized to β-Actin. Error bars represent mean ± SD between technical replicates.