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 Journal of Clinical Investigation

Article Title: Engineered hydrogel reveals contribution of matrix mechanics to esophageal adenocarcinoma and identifies matrix-activated therapeutic targets

doi: 10.1172/JCI168146

Figure Lengend Snippet: ( A ) Transcriptional expression and ( B ) representative fluorescence images of YAP and SOX9 in organoids within NorHA hydrogels of different stiffness at 14 days after encapsulation. Data are represented as mean ± SEM. n = 3 technical replicates, representative of 3 independent experiments. ( A ) One-way ANOVA with Tukey’s multiple comparisons test showed significant differences between 100 Pa and 1000 Pa, 350 Pa and 1000 Pa, and 1000 Pa and Matrigel. * P < 0.05; ** P < 0.01; *** P < 0.001. RNA levels normalized to 100 Pa. Scale bars: 100 μm. Original magnification, ×5 (insets, 100 Pa and 350 Pa); ×3 (insets, 1,000 Pa). ( C ) Quantification and representative fluorescence microscopy images of CD44 expression in EAC PDOs cultured in NorHA hydrogels of different stiffness at 14 days after encapsulation. Data are represented as mean ± SEM. n = at least 20 organoids analyzed per group. Kruskal-Wallis test with Dunn’s multiple-comparisons test showed significant differences between 100 Pa and 1000 Pa, and 350 Pa and 1000 Pa. * P < 0.05; **** P < 0.0001. Scale bar: 100 μm. ( D ) Schematic of in vitro experiment of EAC PDOs within NorHA hydrogels being treated with YAP inhibitor verteporfin. Created with BioRender.com. ( E ) Representative immunohistochemistry microscopy images and quantification of Yap expression in EAC PDOs cultured in NorHA hydrogels of different stiffnesses at 7 days after encapsulation and treated with 5 nM verteporfin or DMSO. Data are represented as mean ± SEM. n = 4 hydrogels per group. One-way ANOVA with Tukey’s multiple-comparisons test showed significant differences between 1000 Pa+DMSO and every other group (** P < 0.01), and no significant differences among other groups ( P > 0.05). Scale bar: 100 μm. ( A – E ) Three independent experiments were performed, and data are presented for 1 of the experiments. Every independent experiment was performed with 4 gel samples per experimental group. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: For drug treatment experiments, 1 week after transplantation, verteporfin (Selleck Chemicals, 100 mg/kg in DMSO; maximum of 150 μL per injection) or DMSO treatment (150 μL per injection) was given every other day for 3 weeks.

Techniques: Expressing, Fluorescence, Encapsulation, Microscopy, Cell Culture, In Vitro, Immunohistochemistry

Journal: The Journal of Clinical Investigation

Article Title: Engineered hydrogel reveals contribution of matrix mechanics to esophageal adenocarcinoma and identifies matrix-activated therapeutic targets

doi: 10.1172/JCI168146

Figure Lengend Snippet: ( A ) Schematic of in vivo transplantation experiment of EAC PDOs within NorHA hydrogels into mouse subcutaneous pockets and treatment with intraperitoneal injections of verteporfin or DMSO. Created with BioRender.com. ( B ) Histological (H&E) microcopy images and quantification of PDO ( C ) size (area) and ( D ) density within NorHA hydrogels at 28 days after encapsulation, in vivo transplantation, and treatment with verteporfin or DMSO. Data are represented as mean ± SEM. ( C ) n = at least 17 organoids analyzed across 10 samples per group. ( D ) n = at least 55 organoids analyzed across 10 samples per group. Kruskal-Wallis test with Dunn’s multiple-comparisons test showed significant differences between 1000 Pa+DMSO and 1000 Pa +verteporfin, 1000 Pa+DMSO and 100 Pa+DMSO and no significant differences between 100 Pa+DMSO and 100 Pa+verteporfin ( P > 0.05). ** P < 0.01; *** P < 0.001; **** P < 0.0001. Scale bar: 100 μm. ( E ) Fluorescence microcopy images of PDOs within NorHA hydrogels at 28 days after encapsulation, in vivo transplantation, and treatment with verteporfin or DMSO stained for Sox9, Yap, Ki67, and CD44. Scale bar: 100 μm. Original magnification, ×5 (insets). ( F – I ) Quantification of ( F ) percentage of nuclear Yap + cells (%Yap + ), ( G ) percentage of Sox9 + cells (%Sox9 + ), ( H ) percentage of Ki67 + cells (%Ki67 + ), and ( I ) CD44 fluorescence intensity per EAC PDO within NorHA hydrogels at 28 days after encapsulation, in vivo transplantation, and treatment with verteporfin or DMSO. Data are represented as mean ± SEM. ( F – I ) n = at least 6 organoids analyzed per group. Kruskal-Wallis test with Dunn’s multiple-comparisons test showed significant differences between 1000 Pa+DMSO and 1000 Pa+verteporfin, 1000 Pa+DMSO and 100 Pa+DMSO and no significant differences between 100 Pa+DMSO and 100 Pa+verteporfin ( P > 0.05). * P < 0.05; *** P < 0.001; **** P < 0.0001. ( A – G ) Two independent experiments were performed, and data are presented for 1 of the experiments. Every independent experiment was performed with 2 gels per mouse and 5 mice per experimental group.

Article Snippet: For drug treatment experiments, 1 week after transplantation, verteporfin (Selleck Chemicals, 100 mg/kg in DMSO; maximum of 150 μL per injection) or DMSO treatment (150 μL per injection) was given every other day for 3 weeks.

Techniques: In Vivo, Transplantation Assay, Encapsulation, Fluorescence, Staining