EMBO/sd-character-level-ner huggingface dataset:
R3hdml KO mice-derived satellite cells show reduced proliferation and differentiation compared with those from wild type controls.Figure 4A Satellite cells were isolated by enzymatic digestion from R3hdml KO (n = 6) and wild type control (n = 6) mice and placed in culture medium. Then, cell proliferation was evaluated by Ki67 staining. Cell nuclei were stained with DAPI. B The ratio of Ki67 positive cells to total cells (DAPI-positive) was evaluated. The number of cells was counted under approximately 60 fields of view for each well. Closed columns, wild type controls; open columns, R3hdml KO mice. * p < 0.05; Student's t-test. Thirty freshly isolated myofibers from 8-12-week-old R3hdml KO (n = 7) and wild type mice (n = 7) were cultured for 72 h. Then, the isolated myofibers were fixed with 4% PFA followed by immunostaining using anti Pax-7, anti MyoD, and anti Myog antibodies. (C, D) Co-immunostaining parallel cultures for Pax7 and MyoD showed that there were fewer cells with the self-renewing Pax7+/MyoD− phenotype in muscles from R3hdml KO mice compared with control; Pax7+/MyoD− cells (arrowheads), Pax7−/MyoD+ cells (yellow arrows) and Pax7+/MyoD+ cells (white arrows). * p < 0.05; Student's t-test. (E, F) Co-immunostaining parallel cultures for MyoD and Myog showed that there were fewer cells with the differentiating MyoD−/Myog+ phenotype in muscles from R3hdml KO mice compared to control; MyoD−/Myog+ cells (arrowheads), MyoD-/Myog+ cells (yellow arrows), and MyoD+/Myog+ cells (white arrows). * p < 0.05; Student's t-test.
Identification of ER8 elements in 1,25‐dihydroxyvitamin D3 and RA target genes.Figure 1(A) Chromatin immunoprecipitation (ChIP) analysis of receptor binding to the ER8 element (region 1) of the nrf2 promoter. Immunoprecipitation with nonspecific IgG and PCR amplification of an adjacent region (2) served as controls. ChIP assays were also carried out to analyse binding to ER8s in the il10, ddit3 and tyrobp genes. RAR, retinoic acid receptor; VDR, vitamin D receptor. (B) Reverse transcription-PCR analysis of 1,25‐dihydroxyvitamin D3 (1,25D3)‐ or retinoic acid (RA)‐regulated expression of ddit3, il10, and tyrobp in U937, Calu‐3 and MCF‐7 cells.
Figure 6A. Immunoblot of AXL expression in PANC-1 and A549 cancer cells upon transfection of control siRNA (CNT) or hMENA(t) siRNA (sihMENA(t)), indicating that the knock‐down of hMENA isoforms, resulted in a reduction of AXL protein expression. B. Real time qRT-PCR analysis of the relative AXL mRNA expression in PANC-1 and A549 cancer cells transfected with control siRNA (siCNT) or hMENA(t) siRNA (sihMENA(t)), indicating that the knock‐down of hMENA isoforms, resulted in a significant reduction of mRNA AXL expression. Data are presented as the mean ± SD of three replicates. P values were calculated by two‐sided Student's t‐test. **P<0.01, ***P<0.001. C. Real time qRT-PCR analysis of the relative levels of mature AXL mRNA or AXL pre-mRNA in PANC-1 (left) and A549 (right) cell lines, transfected with control siRNA (siCNT) or hMENA(t) siRNA, sihMENA(t). Data represent percent of AXL mRNA or pre-mRNA levels in hMENA(t) silenced cells relative to siCNT control cells (set at 100). Data are presented as the mean ± SD of two biological replicates. P values were calculated by two‐sided Student's t‐test. *P< 0.05, ***P<0.001. D. Immunoblot analysis with the indicated antibodies of PANC-1 cells transfected with control siRNA (CNT) or hMENA(t) siRNA, showing that the knock‐down of total hMENA isoforms, (hMENA(t)) inhibits GAS6-mediated pAXL and pAKT expression. Cells were serum starved overnight and subsequently stimulated with DMSO (0.02%) in control culture medium (-) or rGAS6 (200 ng/mL), for 30 and 60 min. The fold change of pAXL or pAKT expression respect to siCNT untreated cells is reported. E. Quantification of in vitro matrigel invasion assay of PANC-1 siCNT cells (siCNT) and hMENA/hMENA∆v6 silenced cells (sihMENA(t)) toward rGAS6 as chemo-attractant (siCNT + rGAS6 and sihMENA(t) + rGAS6), showing that the knock‐down of hMENA(t) reduced cancer cell invasion toward GAS6. The number of invading cells were counted in 6 random fields. Data are presented as the mean ± SD of three biological replicates. Statistical analysis was performed with one-way ANOVA P<0.0001, followed by Bonferroni's multiple comparison test. **P<0.01. F. Quantification of in vitro matrigel invasion assay of PANC-1 siCNT cells (siCNT) and hMENA/hMENA∆v6 silenced cells (sihMENA(t)), untreated (-) or treated with conditioned media derived from CAFs (P-CAF#36-CM) for 48 h. The number of invaded cells were counted in 6 random fields. Data are presented as the mean ± SD of three biological replicates. Statistical analysis was performed with one-way ANOVA P<0.0001, followed by Bonferroni's multiple comparison test. *** P< 0.001.
STAMP2 increases intracellular ROS by decreasing intracellular NADPH levels.Figure 9A LNCaP cells stably expressing an empty vector (LN-Vec) or a vector expressing STAMP2 (LN-ST2) were established by lentivirus delivery. Cell lysates were prepared and subjected to Western blot analysis with the indicated antisera. B Relative ferrireductase activities were measured in the cells described in (A). *P = 0.0003 C NBT staining was performed in LNCaP cells stably expressing either an empty vector (LN-Vec) or a vector expressing wild-type STAMP2 (LN-ST2/WT) or a STAMP2 mutant (LN-ST2/dGSR) as indicated. The stained cells were then photographed and quantified. *P = 0.004. D LNCaP cells were transfected with either scrambled siRNA (siCtrl) or siRNA against STAMP2 (siST2) and were cultured in the presence of 10 nM R1881 or vehicle for 2 days. Intracellular ROS levels were then measured by CellROX reagent staining. *P = 0.002. ns, not significant. E LNCaP cells stably expressing an empty vector (Vec), a vector expressing wild-type STAMP2 (WT), or a STAMP2 mutant (dGSR) were cultured, and the NADPH/NADP+ ratio was determined as described in Materials and Methods. *P = 0.015. F LNCaP cells were transfected with either control siRNA (siCtrl) or STAMP2 siRNA (siST2). Cells were then cultured for 2 days and harvested, and the NADPH/NADP+ ratio was determined as above. *P = 0.026. G LNCaP cells stably expressing wild-type STAMP2 were treated with or without DPI (1 μM) for 4 h, harvested, and used in the NADPH/NADP+ assay. *P = 0.018.
a) GFP-LC3 puncta were visualized by fluorescent microscopy of tissue from each type of mouse in fed state, and the numbers of GFP-LC3 puncta counted.f4(a) GFP-LC3 puncta were visualized by fluorescent microscopy of tissue from each type of mouse in fed state, and the numbers of GFP-LC3 puncta counted. Scale bars, 20 μm. *P0.05, **P0.01; Student's t-test. (b) Tissues of ob/w and ob/ob mice were subjected to EM, and the number of autophagosomes was counted. ***P0.001; Student's t-test. (c) Immunoblotting using anti-GFP or -p62 antibody. (d) Tissue lysates were prepared from the liver of fed ob/w or ob/ob mice 4 h after leupeptin administration, and immunoblotting was performed. Numbers below immunoblot bands indicate the fold changes normalized to control bands. (e) After labelling SK-Hep1 cells with C14-leucine, cells were treated with FFAs (600 μM PA or 1,200 μM OA). Percent release of radioactivity was measured for 3-6 h as described in the Methods. The difference between the percent proteolysis without E64d/pepstatin A/NH4Cl and that with E64d/pepstatin A/NH4Cl was regarded as lysosomal proteolysis. *P0.05, **P0.01; one-way ANOVA, n=3. (f) Atg7+/+ and Atg7+/− primary MEFs were loaded with a mixture of the indicated concentrations of PA and OA for 48 h, and TG content was estimated by ORO staining. **P0.01, ***P0.001; two-way ANOVA, n=4.
XIAP‐modulated autophagy is associated with the tumorigenecity.Figure 5(A-C) 1 × 106HCT116XIAP WT and 1 × 106XIAP KO cells (group 1), 1 × 106HCT116XIAP KO cells (KO‐Ctrl) and 1 × 106HCT116XIAP KO cells stably expressing wild‐type XIAP (KO‐XIAP) (group 2), 1 × 106HCT116XIAP KO cells (KO‐Ctrl) and 1 × 106HCT116XIAP KO cells stably expressing XIAP S87A mutant (KO‐XIAP SA) (group 3), or 1 × 106HCT116XIAP KO cells (KO‐Ctrl) and 1 × 106HCT116XIAP KO cells stably expressing XIAP S87D mutant (KO‐XIAP SD) (group 4) were individually injected to the left flank (Left) and right flank (Right) of nude mice as indicated. Five weeks after injection, the mice were sacrificed and photographed (A). The volumes of all flank tumours excised from six mice in each group were compared (n=6) (B). Tumour weights were represented as means±s.d. from six mice in each group (C). (D-F) 1 × 106HCT116XIAP KO cells or 1 × 106HCT116XIAP KO cells stably expressing XIAP D148A/310A were injected to the right flank of nude mice as indicated (n=3). Five weeks after injection, the mice were sacrificed and photographed (D). The volumes of excised tumours were shown (E). Tumour weights were represented as means±s.d. from three mice in each group (F). (G, H) The human tumour tissues (T) from Oesophagus (G) and Colon (H) and their adjacent normal tissues (N) were homogenized for protein extraction. Protein extracts were analysed by western blotting with the indicated antibodies. The data are representative of three biological replicates. The blots in (G, H) were qualified and the ratio of LC3II/LC3I to actin was then calculated and shown in Supplementary Figure 9A and B. (I) The schematic model of autophagic regulation by XIAP is shown. Under non‐stressed condition, phosphorylated XIAP interacts with Mdm2 and mediates its rapid degradation, thus maintaining the relative high levels of p53 and inhibiting basal levels of autophagy. In response to serum starvation, XIAP undergoes dephosphorylation due to AKT inhibition, thereby promoting the dissociation of XIAP from Mdm2. This in turn accelerates the degradation of p53 and facilitates serum starvation‐induced autophagy.
Figure 5BMDMs were stimulated with an increasing dose of S63845 in the presence of ABT-737 (0.5 μM) and LDH release was quantified at 6 h. BMDMs were stimulated with an increasing dose of S63845 in the presence of ABT-737 (0.5 μM) and LDH release was quantified at 6 h. BMDMs were stimulated with ABT-737 (0.5 μM) and S63845 (0.5 μM) and LDH release was quantified BMDMs were stimulated with ABT-737 (0.5 μM) and S63845 (0.5 μM) mixed supernatant and extracts were analysed by immunoblot at 6 h BMDMs were stimulated with ABT-737 (0.5 μM) and S63845 (0.5 μM) and LDH release was quantified BMDMs were stimulated with ABT-737 (0.5 μM) and S63845 (0.5 μM) and mixed supernatant and extracts were analysed by immunoblot at 6 h BMDMs were primed with ultrapure E. coli K12 LPS (100 ng/ml) for 3 h and further stimulated with ABT-737 (1 μM) and S63845 (1 μM) for 24 h and mixed supernatant and extracts were analysed by immunoblot BMDMs were primed with ultrapure E. coli K12 LPS (100 ng/ml) for 3 h and further stimulated with ABT-737 (1 μM) and S63845 (1 μM) for 24 h and IL-1β in cell-free supernatant were quantified by ELISA
a) Ser 317/Ser 777 is required for Ulk1 to protect cells from glucose starvation.figf7(a) Ser 317/Ser 777 is required for Ulk1 to protect cells from glucose starvation. Viability (24 h, mean ± s.d., n = 4; top) and PARP cleavage (8 h; western blot, middle; quantification, n = 2, bottom) was examined in Ulk1+/+ (WT), Ulk1−/− (KO), Ulk1−/− re-expressing wild-type Ulk1 (KO-WT), and Ulk1−/− re-expressing Ulk1 S317/777A mutant (KO-S317/777A) MEFs. Arrows in western blots indicate non-cleaved and cleaved PARP. (b) The Ulk1 S317/777A mutant is compromised in LC3 lipidation in response to glucose starvation. ULK1MEFs were cultured in MEFs-free medium for the indicated times. LC3-II level was determined by western blotting and the LC3-II accumulation was normalized by α-tubulin and quantified (bottom, n = 3, mean ± s.d.). A representative western blot was shown. The LC3 antibody used in this experiment seemed to preferentially recognise the lipid-modified form of LC3-II, which migrated faster on the gel. (c) The Ulk1 S317/777A mutant is defective in autophagosome formation. The indicated MEFs were starved of glucose (4 h) and the formation of GFP-LC3-positive autophagosomes was examined by confocal microscopy. GFP-LC3; green and DAPI; blue. Scale bar, 20 μm. (d) Autophagy vacuole analysis by electron microscopy. Low-magnification images of Ulk1−/− (KO, upper left panel), Ulk1−/− reconstituted with wild-type Ulk1 (KO-WT, two middle panels with accompanying higher magnification images), and Ulk1−/− reconstituted with Ulk1 S317/777A (KO-S317/777A, lower left panel) are shown. High-magnification images of autophagosomes from KO-WT are shown in upper right and lower right panels. Autophagosome/autolysosome-like structures indicated by arrowheads on the lower-magnification images and arrows in higher-magnification images. Scale bars; lower-magnification, 1 μm; higher-magnification, 200 nm.
Figure 6(A) Mesoscale analysis for the Aβ1-40 protein in the TBS (*p=0.0208), Triton-X (*p=0.0440) and SDS (p=0.0540) fractions of brain homogenates from APP23p40+/+(n=7) and APP23p40-/- (n=8) mice. Total protein concentration of each sample was used as an internal reference. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test. (B) Stereological analysis of cortical area covered by 4G8-positive plaques (left) and representative images of 4G8-staining in APP23p40+/+ (n=8) and APP23p40-/- (n=10) mice (right), scale bar = 500 µm. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test, p=0.1831. (C) Fluorescence intensity based analysis of pFTAA-positive area covered in the cortex of APP23p40+/+ (n=8) and APP23p40-/- (n=10) mice (left) and representative images for each genotype (right), scale bar = 1 mm. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test, p=0.3406. (D) Stereological analysis of cortical area covered by Congo Red-positive plaques in APP23p40+/+ (n=8) and APP23p40-/- (n=10) mice (left) and representative images (right), scale bar = 500 µm. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test, p=0.4542. (E) Native filter assay analysis of TBS (p=0.6823), Triton-X (p=0.1146), SDS (p=0.0508) and formic acid (FA) (p=0.6603) fractions from APP23p40+/+ (n=7) and APP23p40-/- (n=7) mouse brain homogenates. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-tests between the same fractions.
Figure 5A. Nuclear fractionation followed by simple western analysis (Peggy Sue) shows an increase in nuclear actin in AECs exposed to TGFβ1 for 48 h. Quantification (right panel) shows mean + s.d., n = 3 biological replicates , ns = non-significant, *p = 0.014, ANOVA/Tukey's). B. Simple western analysis (Peggy Sue) of EZH2 co-immunoprecipitates shows increased levels of EZH2-bound POL2, ph-EHZ2, and actin in injured AECs. Unspecific IgG binding was used as a negative control. A representative from 3 biological replicates is shown. C, D. ChIP-qPCR shows increased occupancy of (C) POL2-S5p at promoters of profibrotic genes in AECs subjected to TGFβ1 for 24 h, whereas no enrichment of (D) POL2-S2p at the gene bodies of these genes was detected. Negative IgG control is shown in Appendix Figure S5B (mean + s.d., n = 3 biological replicates). E, F. ChIP-qPCR shows increased occupancy of (E) POL2-S5p at promoters and (F) POL2-S2p at the gene bodies of profibrotic genes in AECs subjected to TGFβ1 for 48 h. Negative IgG control is shown in Appendix Figure S5C (mean + s.d., n = 3 biological replicates).
Figure 4APR-246 content in OVCAR-3 cells (TP53 R248Q) 24h after treatment with APR-246 +/- MK-571, (n = 3). Data information: Indicated values are assessed by LC-MS. Data are represented as mean ± SEM. See also Appendix Figure S4. Glutathione-conjugated MQ (GS-MQ) in OVCAR-3 cells after 24h treatment with APR-246 +/- MK-571 (n = 2). Data information: Indicated values are assessed by LC-MS. Data are represented as mean ± SEM. See also Appendix Figure S4. Amount of GS-MQ at indicated timepoints after incubation of GS-MQ +/- NAC (n = 2). See Fig. EV4 for chemical reaction. Data information: Indicated values are assessed by LC-MS. Data are represented as mean ± SEM. See also Appendix Figure S4. Amount of GS-MQ by LC-MS (green line, left axis) and NAC-MQ (purple line, right axis) over time after incubation of GS-MQ with NAC (n = 2). Values on y-axes are not comparable because GS-MQ and NAC-MQ have different response signals on MS. Data information: Indicated values are assessed by LC-MS. Data are represented as mean ± SEM. See also Appendix Figure S4.
Figure 3(A) Table shows the total number of pups, the number or frequency of human HC-, human LC-and human BCR- KI pups after One-step CRISPR/Cas9 microinjection of CLK09 and CLK19. (B) Binding activity detection of heterozygous CLK09 human BCR KI mice to eOD-GT8.8-week-old mice were detected by FACS. X axis and Y axis represent that BCR were stained with eOD-GT8 tetramer conjugated with Alexa Fluor™ 488 and Alexa Fluor™ 647 respectively. Representative dots were gated as in Figure 2D. (C) Quantification of eOD-GT8 targeting CLK09 KI mice. X-axis represents F0 (n=1) and F2 (n=2) pups, y- axis represents as in Figure 2E. Bars indicate mean ± SD from mice in each group. (D) Binding activity detection of heterozygous and homozygous CLK19 human BCR KI mice to eOD-GT8. 8-week-old mice were detected by FACS. X and Y axes represent as in (B). (E) Quantification of eOD-GT8 binding in HCLK19/WT κCLK19/WT KI mice. X-axis represents F0 (n=3) and F1(n=2) animals, Y-axis represents as in Figure 2E. Bars indicate mean ± SD from mice in each group.
Autoinjector performance: A.Figure 3(Left) Confocal image of a section of mouse telencephalon fixed and stained immediately after microinjection. Dashed lines indicate locations in the slice where microinjections were attempted at different pressures. Microinjections were attempted at pressure of 75 mbar (left area), 100 mbar (center area), and 125 mbar (right area). (Right) Percentage of successful injections achieved by the Autoinjector at 75 mbar (35%, n = 314 total, 110 successful), 100 mbar (35%, n = 185 total, 65 successful), and 125 mbar (8%, n = 287 total, 23 successful). (Left) Confocal images of a section of mouse telencephalon fixed and stained immediately after microinjection. Dashed lines indicate locations in the slice where microinjections were attempted at different depths keeping the pressure constant at 75 mbar. Microinjections were attempted at depths of 10 μm (left area), 15 μm (center area), and 25 μm (right area) into the apical surface. (Right) Percentage of successful injections achieved by the Autoinjector at depths of 10 μm (68%, n = 163 total, 111 successful), 15 μm (22%, n = 170 total, 37 successful), and 25 μm (11%, n = 169 total, 19 successful) into the apical surface. Successful injections represented as a percentage of total for a novice user on the manual microinjection system, an experienced user on the manual microinjection system, and the Autoinjector. Total injection attempts per minute for a novice user on the manual microinjection system, an experienced user on the manual microinjection system, and the Autoinjector. Successful injections per minute for a novice user on the manual microinjection system, an experienced user on the manual microinjection system, and the Autoinjector. Cells targeted at a constant spacing of 30 μm, scale bar is 10 μm.
Figure 5A, B. GST-pulldowns showing that HUWE1 PIP-box mutants do not interact with PCNA. A. Recombinant GST-tagged HUWE1C-ter, either wild-type or PIP-box mutant variants (QVFF and VFF -the indicated residues, critical for the PIP-box, were mutated to A), were purified from bacteria (see Coomassiestaining) and employed for GST-pulldowns using extracts of 293T cells. PCNA co-precipitated with wild-type, but not PIP-box mutants. B. Reciprocal GST-pulldown using recombinant GST-PCNA and extracts of 293T cells overexpressing Myc-HUWE1C-ter. Wild-type, but not the PIP-box mutant (VFF) HUWE1 fragment bound to GST-PCNA. C, D. Reciprocal co-immunoprecipitationexperiments showing that PIP-box mutation abolishes PCNA interaction. C. 293T cells were transfected with Myc-HUWE1C-ter variants, and subjected to anti-PCNA (C) or anti-Myc (D) immunoprecipitation. Only wild-type, but not PIP-box mutant (VFF) HUWE1 interacted with PCNA in both experimental setups. E. Mutation of the PIP-box in full-length HUWE1 also blocks PCNA interaction. Full-length Myc-tagged HUWE1 (wild-type or PIP-mutant) was transfected in 293T cells. Following Myc-immunoprecipitation, endogenous PCNA was detected in wild-type, but not PIP-box mutant (FF) complexes.
Figure 5Flow cytometric analysis of lymph node γδ T cells in STAT4−/− (−/−) and littermate control mice (+/−). (A, ) Frequency of IL-17A+ (A) γδ T cells before (steady-state) and after (IMQ) IMQ-induced psoriasis. (B) Frequency of IL-17F+ γδ T cells before (steady-state) and after (IMQ) IMQ-induced psoriasis. (C) Quantification of mean fluorescence intensity (MFI) of IL-17A staining in γδ T cells after IMQ-induced psoriasis (D) Quantification of mean fluorescence intensity (MFI) of IL-17F staining in γδ T cells after IMQ-induced psoriasis. Flow cytometric analysis of lymph node γδ T cells in STAT4−/− (−/−) and littermate control mice (+/−). Frequency of IL-22+ γδ T cells before (steady-state) and after (IMQ) IMQ-induced psoriasis. Flow cytometric analysis of lymph node γδ T cells in STAT4−/− (−/−) and littermate control mice (+/−). Frequency of IL-22+ γδ T cells following culture without (−) or with 40ng/ml recombinant IL-23
Overall structure of the HsAtg4B–LC3 complex.Figure 1(A) Ribbon diagram of the HsAtg4B-LC3(1-120) complex. HsAtg4B is coloured salmon red, and LC3 is coloured green. (B) Structural comparison between free and HsAtg4B‐bound LC3. Crystal structure of free LC3(1-120) (PDB code 1UGM) is superimposed on the HsAtg4B‐bound LC3. HsAtg4B‐bound LC3 is coloured green, and free LC3 is coloured grey. (C) Structural comparison between free and LC3‐bound HsAtg4B. Crystal structure of free HsAtg4B (PDB code 2CY7) is superimposed on the LC3‐bound HsAtg4B. LC3‐bound HsAtg4B is coloured salmon red, and free HsAtg4B is coloured grey. The regions with large conformational differences between free and LC3‐bound forms are coloured black (free) and red (LC3 bound). All the figures representing molecular structures were generated with PyMOL ( DeLano, 2002).
Figure 1A. Overview of the lysine metabolism in E. coli. The arrows are color coded according to the different metabolic categories, as defined in the figure legend. Genes targeted in the library are highlighted in green. The insets represent examples of library designs for two targeted proteins, with the targeted residues included inside the gray surface representation B. For each targeted gene, the number of variants (black bars, left y-axis) and the fraction of the single substitution sequence space (green bars, right y-axis) is plotted. The total library size across all genes sums to 16,300 variants C. Description of the two main mechanisms of AEC toxicity. The structural differences between canonical lysine and AEC is shown in the left, with the orange sphere highlighting the sulfur group present in AEC. Lysine binding is shown in the top panels, and AEC in the bottom panels. Mutations described to confer AEC resistance are highlighted in the bottom panels D. Workflow of the strategy to map trajectories of AEC resistance using CREATE. Briefly, designed cassettes were cloned, miniprepped and transformed into strains expressing Cas9 and the lambda red machinery. The library culture was grown for 8 hours in LB media with proper antibiotics, washed with PBS, and inoculated into M9 minimal media containing the AEC selective pressure and antibiotics. An aliquot was stored for initial plasmid barcode sequencing counts. After growth, cells were harvested for deep sequencing of the plasmid barcodes, which were used to map the enrichment scores of the designed mutants