Journal: bioRxiv

Article Title: Far From Home: Evolution of haploid and diploid populations reveals common, strong, and variable pleiotropic effects in non-home environments

doi: 10.1101/2023.02.28.530341

Figure Lengend Snippet: (A) Lineage tracking data of haploid Bulk Fitness Assay, hBFA, for lineages evolved in fluconazole, clotrimazole, and glycerol/ethanol. Each column represents a replicate. The columns are grouped together by the evolution environment: fluconazole, clotrimazole, glycerol/ethanol. Each row represents the “Test Environment”. Each line represents one lineage evolved in the home environments that corresponds with its column group. The color of the line indicates the test environment in which that lineage was remeasured. (B) Lineage tracking data of diploid Bulk Fitness Assay, dBFA, for lineages evolved in fluconazole, clotrimazole, and glycerol/ethanol. (C) Lineage tracking data of combined Bulk Fitness Assay, cBFA, for lineages evolved in fluconazole, clotrimazole, and glycerol/ethanol.

Article Snippet: Conditions with 0.2M NaCl (Fisher Scientific - BP358-212), 0.8M NaCl, 21°C, 2% Glycerol (Fisher - G33-4) + 2 % Ethanol (Goldshield - 412804), Fluconazole 13 µM (4 mg/L, Acros Organics #455480050), Clotrimazole 6 µM (or 2 mg/L, Chem-Impex International #01311) were performed with 48 hour transfers at Stanford.

Techniques:

Journal: bioRxiv

Article Title: Far From Home: Evolution of haploid and diploid populations reveals common, strong, and variable pleiotropic effects in non-home environments

doi: 10.1101/2023.02.28.530341

Figure Lengend Snippet: These heatmaps show the fitnesses of all lineages including lineages that had the same mutation and were collapsed into a single row using the median fitnesses in . The number at the end of the name represents numerical barcode identification number. (A) Mutations identified in clotrimazole 1N measured in hBFA, (B) Mutations identified in clotrimazole 2N measured in dBFA, (C) Mutations identified in fluconazole 2N measured in cBFA, (D) Mutations identified in fluconazole 2N measured in dBFA, (E) Mutations identified in glycerol/ethanol 1N measured in hBFA, (F) Mutations identified in glycerol/ethanol 2N measured in cBFA.

Article Snippet: Conditions with 0.2M NaCl (Fisher Scientific - BP358-212), 0.8M NaCl, 21°C, 2% Glycerol (Fisher - G33-4) + 2 % Ethanol (Goldshield - 412804), Fluconazole 13 µM (4 mg/L, Acros Organics #455480050), Clotrimazole 6 µM (or 2 mg/L, Chem-Impex International #01311) were performed with 48 hour transfers at Stanford.

Techniques: Mutagenesis

Journal: bioRxiv

Article Title: Far From Home: Evolution of haploid and diploid populations reveals common, strong, and variable pleiotropic effects in non-home environments

doi: 10.1101/2023.02.28.530341

Figure Lengend Snippet: The columns are the home environments that lineages evolved in and the rows are the test environments in which their fitnesses were remeasured. X axis is the fitness of the lineages remeasured in the BFA in their home environment. Y axis is the fitness of the lineages in a non-home environment. No fitness remeasurements are available from BFAs grown in clotrimazole.

Article Snippet: Conditions with 0.2M NaCl (Fisher Scientific - BP358-212), 0.8M NaCl, 21°C, 2% Glycerol (Fisher - G33-4) + 2 % Ethanol (Goldshield - 412804), Fluconazole 13 µM (4 mg/L, Acros Organics #455480050), Clotrimazole 6 µM (or 2 mg/L, Chem-Impex International #01311) were performed with 48 hour transfers at Stanford.

Techniques:

Journal: bioRxiv

Article Title: Far From Home: Evolution of haploid and diploid populations reveals common, strong, and variable pleiotropic effects in non-home environments

doi: 10.1101/2023.02.28.530341

Figure Lengend Snippet: Distribution of Fitness Effects for Fitness Estimates of All Adaptive Diploid Lineages remeasured in Clotrimazole, Fluconazole, and Glycerol/Ethanol: The distribution of fitness effects for all adaptive diploid lineages remeasured in clotrimazole, fluconazole, and glycerol/ethanol. The gray vertical line delineates the fitness threshold of the top 10% of mutants that were evolved in the labeled environment. The red line delineated the top fitness of all mutants that were evolved in the labeled environment. The y axis delineates the percentage of mutants that have a specific fitness (x axis).

Article Snippet: Conditions with 0.2M NaCl (Fisher Scientific - BP358-212), 0.8M NaCl, 21°C, 2% Glycerol (Fisher - G33-4) + 2 % Ethanol (Goldshield - 412804), Fluconazole 13 µM (4 mg/L, Acros Organics #455480050), Clotrimazole 6 µM (or 2 mg/L, Chem-Impex International #01311) were performed with 48 hour transfers at Stanford.

Techniques: Labeling

Journal: bioRxiv

Article Title: Far From Home: Evolution of haploid and diploid populations reveals common, strong, and variable pleiotropic effects in non-home environments

doi: 10.1101/2023.02.28.530341

Figure Lengend Snippet: Heatmaps Representing Pleiotropic Profiles of Adaptive Mutant Lineages from populations evolved in clotrimazole. Each heatmap shows the lineages evolved in a particular condition and their fitness remeasurements in a specific bulk fitness assay. Each square on the heatmap shows the average fitness of the lineage measured in each environment (columns) for approximately 40 generations, specifically for mutant lineages we identified in (rows). The “+” indicates that in that lineage there are other background mutations, the “++” indicates that this specific mutation was observed in multiple lineages and what is shown in the row is the median fitness of all the lineages that have that mutation. (A) shows the haploids and (B) shows the diploids from the fluconazole evolutions.

Article Snippet: Conditions with 0.2M NaCl (Fisher Scientific - BP358-212), 0.8M NaCl, 21°C, 2% Glycerol (Fisher - G33-4) + 2 % Ethanol (Goldshield - 412804), Fluconazole 13 µM (4 mg/L, Acros Organics #455480050), Clotrimazole 6 µM (or 2 mg/L, Chem-Impex International #01311) were performed with 48 hour transfers at Stanford.

Techniques: Mutagenesis

Journal: The EMBO Journal

Article Title: The histone methyltransferase Setd8 acts in concert with c-Myc and is required to maintain skin

doi: 10.1038/emboj.2011.421

Figure Lengend Snippet: Long-lived progenitors of IFE and sebaceous glands are irreversibly lost in K14CreERSetd8 Δ/Δ mice and recovered skin derives from hair follicles (HFs). ( A ) Schematic overview of the 4-OHT-treatment regime. ( B , C ) Haematoxylin & Eosin (H&E) staining of skin section from control ( B ) and K14CreERSetd8 Δ/Δ ( C ) mice at the end of treatment with 4-OHT and after 7, 14 and 21 days of recovery. Arrows point to IFE and arrowheads to sebaceous glands. ( D – G ) Co-labelling of control ( D , F ) and K14CreERSetd8 Δ/Δ epidermis ( E , G ) for K14 (green) and GFP (red) ( D , E ) or GFP (red) only ( F , G ) after treatment with 4-OHT (end treatment) or after 7 and 21 days of recovery. ( H ) BrdU incorporation (BrdU; green) and labelling for K14 (red) in the IFE, sebaceous glands (SGs) and HF bulges/bulbs (Bu/Bb) in control (upper panels) and K14CreERSetd8 Δ/Δ (Setd8 Δ/Δ ; lower panels) epidermis. ( I ) Quantification of BrdU-positive cells from ( H ). ( J ) Detection of quiescent label-retaining cells (LRCs) (BrdU; green) in bulges of control and K14CreERSetd8 Δ/Δ (Setd8 Δ/Δ ) mice. ( K ) Quantification of ( J ). KO: K14CreERSetd8 Δ/Δ . Nuclei are counterstained with DAPI (blue) in ( D – H , J ). Scale bars: 250 μm ( A – G ); 50 μm ( H ) and 75 μm ( J ).

Article Snippet: To activate K14CreER, ∼5-week-old mice were treated topically with 1.4 mg 4-OHT (Sigma) dissolved in acetone or acetone alone as a control every other day.

Techniques: Staining, BrdU Incorporation Assay

Journal: The EMBO Journal

Article Title: The histone methyltransferase Setd8 acts in concert with c-Myc and is required to maintain skin

doi: 10.1038/emboj.2011.421

Figure Lengend Snippet: Setd8 is transcriptionally regulated by c-Myc and required to mediate skin-specific functions of c-Myc. ( A ) Relative expression of Setd8 RNA in wild-type (WT) and K14MycER transgenic (TG) mice measured by QPCR. ( B ) Log2 fold-change (FC) of Setd8 RNA expression in K14MycER epidermis relative to wild-type skin in time course of treatment with 4-OHT for the days indicated. Data are obtained from gene expression arrays. ( C ) ChIP for Setd8 and Nucleolin (Ncl1) promoters using a c-Myc antibody in skin of WT and K14MycER TG animals using two different sets of primers (Setd8.1; Setd8.2). Ctr indicates the negative control. The insert is a graphical overview of the Setd8 promoter and the putative c-Myc binding sites. Red lines indicate putative c-Myc binding sites amplified by QPCR. ( D , E ) Measuring proliferation, using Ki67 as a marker ( D ) or BrdU incorporation ( E ) in control, K14CrERSetd8 Δ/Δ , K14MycER animals and K14MycER mice with deleted Setd8 alleles (K14MycER-Setd8 Δ/Δ ). K14 (red) was used as counterstain in ( E ). Lines in ( D , E : K14MycER-Setd8 Δ/Δ ) indicate loss of cycling cells. Areas outside the lines may have not deleted Setd8 due to short treatment with 4-OHT (6 days) . ( F – K ) Labelling for K10 ( F ), p63 ( G ), K8 ( H ), 14-3-3σ ( I ) (red) and p53 ( J ) (brown), or TUNEL (green) ( K ). Arrows in ( F ) mark cells of the basal layer and arrows in ( H ) indicate K8-positive cells in the IFE. Nuclei are counterstained with DAPI (blue) in ( E – I , K ). Scale bars: 100 μm ( D , E ) and 50 μm ( F – K ). ( L ) QPCR for p63 RNA levels in the indicated transgenic lines. ( M ) Quantification of ( J ) as percentage of p53-positive cells in the IFE. ( N ) Colour code for transgenic lines used in ( L – N ) and survival of epidermal cells in culture for the indicated hours. Y axis shows relative survival of the 4-OHT-treated lines to their untreated controls.

Article Snippet: To activate K14CreER, ∼5-week-old mice were treated topically with 1.4 mg 4-OHT (Sigma) dissolved in acetone or acetone alone as a control every other day.

Techniques: Expressing, Transgenic Assay, RNA Expression, Negative Control, Binding Assay, Amplification, Marker, BrdU Incorporation Assay, TUNEL Assay

Journal: The EMBO Journal

Article Title: The histone methyltransferase Setd8 acts in concert with c-Myc and is required to maintain skin

doi: 10.1038/emboj.2011.421

Figure Lengend Snippet: Setd8-induced erosion of the IFE can be rescued by decreasing levels of p53 and survival of Setd8-depleted cells is enhanced by overexpression of ΔNp63. ( A – I ) Staining for Haematoxylin & Eosin ( A – C ), Ki67 ( D – F ), K10 (red), GFP (green) and collagen IV (Coll IV) (blue) ( G – I ; left hand panels), and Ivl ( G – I ; middle panels), and p63 (red) ( G – I ; right hand panels) in epidermis from control ( A , D , G ), K14CreERSetd8 Δ/Δ ( B , E , H ) and K14CreERSetd8 Δ/Δ mice with a deleted p53 allele (Setd8 Δ/Δ p53 Δ/wt ) ( C , F , I ). Arrows in ( D – F ) indicate Ki67-positive nuclei. Dotted lines in ( G – I ) mark the basement membrane. Nuclei are counterstained with DAPI (white, blue) ( G – I ). Scale bars: 100 μm ( A – C ) and 50 μm ( G – I ). ( J , K ) Quantification of Ki67 ( D – F ) and p63 ( G – I ; right hand panels) positive nuclei in control (ctr), K14CreERSetd8 Δ/Δ (Set8 Δ/Δ ) and Setd8 Δ/Δ p53 Δ/wt mice. ( L , M ) Relative expression (rel. expr.) of p53 ( L ) and p63 ( M ) in epidermal cells isolated from the indicated lines. Cells in ( M ) were treated with 4-OHT. ( N , O ) Luciferase assays using a p53 reporter ( N ) or the ΔNp63 promoter ( O ). (+) Indicates presence and (−) absence of the indicated plasmids. (+/+) Indicates double concentration of the plasmid. Empty V.: empty vector control; Neg. Ctr: negative control; Scr: scrambled RNAi construct; p63wt: wild-type p63 promoter construct; p63 mut p53 site: p63 promoter with mutated p53 binding site. ( P ) Survival of cultured epidermal cells isolated from wild-type and K14CreERSetd8 Δ/Δ mice for the indicated hours. Cells were either transfected with the empty vector as control (wild-type, Setd8 Δ/Δ ) or with ΔNp63 construct (Setd8 Δ/Δ deltaNp63). Y axis shows relative survival of the 4-OHT-treated lines to their untreated controls (Ctr).

Article Snippet: To activate K14CreER, ∼5-week-old mice were treated topically with 1.4 mg 4-OHT (Sigma) dissolved in acetone or acetone alone as a control every other day.

Techniques: Over Expression, Staining, Expressing, Isolation, Luciferase, Concentration Assay, Plasmid Preparation, Negative Control, Construct, Binding Assay, Cell Culture, Transfection