Journal: bioRxiv

Article Title: B1a B cells require autophagy for metabolic homeostasis and self-renewal

doi: 10.1101/240523

Figure Lengend Snippet: A . Experimental schematic and example histograms. 6-week-old C57BL/6 mice were injected intravenously with BODIPY FL C 16 then analysed after 1 hour. Example distributions of fluorescence from peritoneal B1a and B2 B cells analysed by flow cytometry, compared with an uninjected control. B . Geometric mean fluorescence intensity of BODIPY FL C 16 from peritoneal B cells following injection. n=3 biological replicates representative of or pooled from two independent experiments. Mean ±SEM is depicted. Unpaired t -test used. C . Geometric mean fluorescence intensity of CD36 on peritoneal B1a and splenic follicular B2 B cells from 6-week-old C57BL/6 mice. Each point represents a single mouse, and data are pooled from two independent experiments. Mean ±SEM is depicted. Unpaired t -test used. D . Peritoneal B1a and splenic follicular B2 B cells were sorted by flow cytometry from 6-week-old C57BL/6 mice and qRT-PCR was performed for the indicated genes, relative to B2m . Each point represents a single mouse, and data are pooled from two independent experiments. Mean ±SEM is depicted. Unpaired t -test p -value is adjusted for multiple testing using FDR method (5% threshold). E . Geometric mean fluorescence intensity of CellROX from splenic and peritoneal B cells of 6-week-old C57BL/6 mice. n=5 biological replicates. Mean ±SEM is depicted. One-way ANOVA with Dunnett correction for multiple comparison used. n=5 biological replicates. Representative of two independent experiments. F . Geometric mean fluorescence intensity of Alexa Fluor 488-ClickIT lipid peroxidation assay for peritoneal B1a and splenic Fo B2 B cells of 6-week-old C57BL/6 mice. n=5 biological replicates. Mean ±SEM is depicted. Unpaired t -test used. Representative of two independent experiments. G . Expression of Plin3 relative to B2m in flow sorted peritoneal B1a and splenic follicular B2 B cells from 6-week-old C57BL/6 mice. n=3 biological replicates. Mean ±SEM is depicted. Unpaired t -test used. Representative of two independent experiments. H . Geometric mean fluorescence intensity of BODIPY 493/503 in splenic follicular B2 B cells, and peritoneal B1a and B2 B cells of 6-week-old C57BL/6 mice. n=5 biological replicates, pooled from two independent experiments. Mean ±SEM is depicted. One-way ANOVA with Dunnett correction for multiple comparison used. I . Representative microscope images of BODIPY 493/503 staining in flow sorted peritoneal B1a and splenic follicular B2 B cells (x60 magnification) 6-week-old C57BL/6 mice. Scale bar is 20μm. Representative of two independent experiments. J-M . 6-week-old C57BL/6 mice received i.p . injections of C75 (15mg.kg −1 ) every 2 days for 3 doses, before sacrifice. Shown are representative flow cytometry plots of peritoneal CD19 + B cells from C75 injected and control mice (H). Shown is the percentage of the indicated population of total cells in the analysed compartment (I) or the geometric mean fluorescence intensity of BODIPY 493/503 measured by flow cytometry (J-K). Each point represents one mouse. Mean ± SEM is depicted. Unpaired t -test used in (I), two-way ANOVA with Sidak correction for multiple testing used in (J) and (K). Representative of 3 independent experiments.

Article Snippet: Mice were injected intravenously with 50μg BODIPY FL C 16 (Life Technologies) in 100µl PBS, then sacrificed after 1 hour.

Techniques: Injection, Fluorescence, Flow Cytometry, Quantitative RT-PCR, Peroxidation Assay, Expressing, Microscopy, Staining

Journal: bioRxiv

Article Title: B1a B cells require autophagy for metabolic homeostasis and self-renewal

doi: 10.1101/240523

Figure Lengend Snippet: A . Heatmap of Fluidigm Biomark qRT-PCR gene expression data for peritoneal B1a B cells and follicular splenic B2 B cells (CD19 + CD23 + ) from control and B- Atg7 −/− mice. Data are relative to β-actin, and colouring is based on row Z-score. Each data point is the mean of three technical replicates. Hierarchical clustering is unsupervised. B . Example distributions of fluorescence of BODIPY FL C 16 from peritoneal B1a B cells from control and B- Atg7 −/− mice, measured by flow cytometry, compared with an uninjected wild-type control. C . Geometric mean fluorescence of BODIPY FL C 16 in B1a and B2 B cells from peritoneum and spleen, in control and B- Atg7 −/− mice, measured by flow cytometry. Each point represents one mouse. Mean ±SEM is depicted. Data pooled from two independent experiments. Two-way ANOVA with Sidak correction for multiple testing used. D . Geometric mean fluorescence of BODIPY 493/503 in B1a and B2 B cells from peritoneum and spleen, in control and B- Atg7 −/− mice, measured by flow cytometry. Each point represents one mouse. Mean ±SEM is depicted. Data pooled from two independent experiments. Two-way ANOVA with Sidak correction for multiple testing used. E . Representative immunofluorescent confocal images of the mitochondria of sorted wild type follicular B2 and peritoneal B1a B cells. Cells are stained for TOM20 (red) and nuclei are visualised with DAPI (blue). 60× magnification, scale bar is 5μm. F . Geometric mean fluorescence of TMRM and Mitotracker Deep Red in B cells from the spleen and peritoneum, in control and B- Atg7 −/− mice, measured by flow cytometry. Each point represents one mouse. Mean ±SEM is depicted. Data representative of two independent experiments. Two-way ANOVA with Sidak correction for multiple testing used. G . Geometric mean fluorescence of 2-NBDG in B1a and B2 B cells from peritoneum and spleen, in control and B- Atg7 −/− mice, measured by flow cytometry. Each point represents one mouse. Mean ±SEM is depicted. Data pooled from two independent experiments. Two-way ANOVA with Sidak correction for multiple testing used. H . Flow cytometry gating definition of high and low levels of TMRM fluorescence. The highest and lowest quartiles of the distribution were used to define TMRM hi and TMRM lo populations of B1a B cells. I . Experimental schematic. 10 3 B1a B cells from the lowest and highest quartiles of TMRM fluorescence were sorted and adoptively transferred into the peritonea of B6. Rag7 −/− hosts. These mice were then analysed after 1 month. J . Quantification of peritoneal B1a B cells from I . Each point represents one mouse. Mean ±SEM is depicted. Unpaired t -test used. Data representative of two independent experiments. K . Quantification of serum IgM levels from I . Each point represents one mouse. Mean ±SEM is depicted. Unpaired t -test used. Data representative of two independent experiments.

Article Snippet: Mice were injected intravenously with 50μg BODIPY FL C 16 (Life Technologies) in 100µl PBS, then sacrificed after 1 hour.

Techniques: Quantitative RT-PCR, Expressing, Fluorescence, Flow Cytometry, Staining

Journal: eLife

Article Title: Interplay between acetylation and ubiquitination of imitation switch chromatin remodeler Isw1 confers multidrug resistance in Cryptococcus neoformans

doi: 10.7554/eLife.85728

Figure Lengend Snippet: ( a ) Spotting assays of ISW1 mutant strains. Wild-type H99, isw1Δ , and ISW1 complementation strains were spotted onto YPD agar supplemented with indicated concentrations of antifungal agents. Plates were incubated at 30°C for 3 days. ( b ) Drug inhibitory tests. The H99 and isw1Δ ( n = 3 each) strains were tested to determine the drug inhibition of several antifungal agents. Twofold dilutions of fluconazole (FLC) from 0 to 64 µg/ml, ketoconazole (KTC) from 0 to 2 µg/ml, or 5-fluorocytosine (5-FC) from 0 to 400 µg/ml were added to YPD medium. After 24 hr at 30°C, absorbance at 600 nm was used to measure growth. The optical densities of duplicate measurements were averaged and normalized relative to the control without FLC. Quantitative data were depicted in color (see color bar) and bar plots. Two-tailed unpaired t -tests were used. Data are expressed as mean ± standard deviation (SD). ( c ) Transcriptome analysis of isw1Δ . Samples of RNA were isolated from H99 and isw1Δ cells ( n = 3 each) supplemented with or without 10 μg/ml FLC. Transcriptome analysis was performed, and a heat map of the expressions of drug-resistance genes was generated. Statistical significant genes are labeled with asterisks. ( d ) Intracellular concentration of FLC. H99 and isw1Δ cells ( n = 3 each) were incubated in the presence of 40 μg/ml FLC at 30°C for 5 hr. Cells were then washed and weighed, and the intracellular FLC was quantified using high-performance liquid chromatography. A two-tailed unpaired t -test was used. Data are expressed as mean ± SD. ( e ) Scheme of the mechanism for 5-FC and 5-fluorouracil (5-FU) resistance. The red arrow indicates the entry of 5-FC via Fcy2. Green arrows indicate the downregulation of gene expression in response to 5-FC in the isw1Δ strain. ( f ) Analyses of 5-FC-resistance genes using quantitative reverse transcription PCR(qRT-PCR). Indicated strains ( n = 3 each) were grown with or without 400 μg/ml 5-FC, then qRT-PCR was performed to determine gene expressions of FCY1 , FUR1 , UGD1 , UXS1 , and FCY2 . Two-tailed unpaired t- tests were used. Data are expressed as mean ± SD. ( g ) Chromatin immunoprecipitation (ChIP)-PCR analysis of Isw1-FLAG. ISW1-FLAG cells ( n = 3) were incubated without drug treatment at 30°C for 5 hr. ChIP-PCRs were performed in the ISW1-FLAG strain ( n = 3 each). Isw1 target genes, INO1 and INO80 , were amplified and used as positive controls. CNAG_06338 were used as a negative control. Potential target genes, CNAG_03600 , UXS1 , UGD1 , FCY1 , and FCY2 were tested. Two-tailed unpaired t- tests were used. Data are expressed as mean ± SD. ( h ) Truncation analysis of cryptococcal Isw1. Truncated Isw1s were cloned and transformed into the isw1Δ strain to generate the DNA-binding domain (DBD) truncation ( isw1Δ/ISW1 DBDΔ ), SNF truncation ( isw1Δ/ISW1 SNFΔ ) and helicase C truncation ( isw1Δ/ISW1 Helicase-CΔ ) strains, which were spotted onto YPD agar plates supplemented with indicated antifungal drugs. ( i ) qRT-PCR analysis in truncated strains supplemented with 5-FC or FLC. Indicated strains were treated with 5-FC or FLC. RNA samples were isolated. qRT-PCRs were performed using oligos from UGD1 , UXS1 , FCY2 , CNAG_03600 , and CNAG_06338 . Figure 1—source data 1. The source data comprises unprocessed data.

Article Snippet: Drug-resistance tests were performed using 16 μg/ml or 20 μg/ml FLC (MedChemExpress), 0.2 μg/ml or 0.3 μg/ml KTC (MedChemExpress), 100 μg/ml or 200 μg/ml 5-FC (MedChemExpress), 0.5 μg/ml or 1.0 μg/ml Amp B (MedChemExpress) or 50 μg/ml or 100 μg/ml 5-FU (MedChemExpress).

Techniques: Mutagenesis, Incubation, Inhibition, Control, Two Tailed Test, Standard Deviation, Isolation, Generated, Labeling, Concentration Assay, High Performance Liquid Chromatography, Expressing, Reverse Transcription, Quantitative RT-PCR, Chromatin Immunoprecipitation, Amplification, Negative Control, Clone Assay, Transformation Assay, Binding Assay

Journal: eLife

Article Title: Interplay between acetylation and ubiquitination of imitation switch chromatin remodeler Isw1 confers multidrug resistance in Cryptococcus neoformans

doi: 10.7554/eLife.85728

Figure Lengend Snippet: ( a ) qRT-PCR of ISW1 . Samples of RNA were isolated from H99 cells treated with fluconazole (FLC) or 5-fluorocytosine (5-FC). qRT-PCR was performed on each to confirm the gene expression of ISW1 . Oligos of actin were used as a control. Three independent assays were performed and quantified. Two-tailed unpaired t- tests were used. Data are expressed as mean ± standard deviation (SD). ( b ) Immunoblotting analysis of the ISW1 complementation strain. The complementation strain was constructed, and an immunoblotting assay was performed to confirm the expression of the Isw1-Flag protein. ( c ) Genomic copy number assays. Genomic DNA was isolated from the wild-type and complementation strains, then qRT-PCR was performed on each to confirm the gene copy number of ISW1 . Oligos of actin were used as a control . Three independent assays were performed and quantified. Two-tailed unpaired t- tests were used. Data are expressed as mean ± SD. ( d ) Analyses of the ISW1 complementation strain using qRT-PCR. Samples of RNA were isolated from the wild-type and complementation strains, then qRT-PCR was performed on each to confirm the gene expression of ISW1 . Oligos of actin were used as a control. Three independent assays were performed and quantified. Two-tailed unpaired t- tests were used. Data are expressed as mean ± SD. Figure 1—figure supplement 1—source data 1. The source data comprises unprocessed data.

Article Snippet: Drug-resistance tests were performed using 16 μg/ml or 20 μg/ml FLC (MedChemExpress), 0.2 μg/ml or 0.3 μg/ml KTC (MedChemExpress), 100 μg/ml or 200 μg/ml 5-FC (MedChemExpress), 0.5 μg/ml or 1.0 μg/ml Amp B (MedChemExpress) or 50 μg/ml or 100 μg/ml 5-FU (MedChemExpress).

Techniques: Quantitative RT-PCR, Isolation, Expressing, Control, Two Tailed Test, Standard Deviation, Western Blot, Construct

Journal: eLife

Article Title: Interplay between acetylation and ubiquitination of imitation switch chromatin remodeler Isw1 confers multidrug resistance in Cryptococcus neoformans

doi: 10.7554/eLife.85728

Figure Lengend Snippet: ( a ) Spotting analysis of isw1Δ in stress conditions. H99, is w1Δ, and ISW1 complementation strains were spotted onto YPD agar plates supplemented with indicated chemicals. ( b ) Melanin formation of the isw1Δ strain. ( c ) Capsule formation and quantification of the isw1Δ strain. H99 and isw1 Δ cells were induced for capsule structure. Capsular sizes were quantified. Two-tailed unpaired t- tests were used. Data are expressed as mean ± standard deviation (SD). ( d ) Animal survival analysis and the Kaplan–Meier survival curves of wild-type and isw1Δ . Significance was determined using a log-rank (Mantel–Cox) test. ( e ) Colony-forming unit (CFU) analysis of the isw1Δ strain. Mice ( n = 10 each) were infected with H99 or isw1Δ cells. At 7-day post infection, animals were treated with phosphate-buffered saline (PBS), or 5 or 45 mg/kg of fluconazole (FLC), or 100 or 200 mg/kg 5-fluorocytosine (5-FC). Animals were treated with drugs in a 24-hr interval on a daily basis. At 14-day post infection, lung tissues were removed and homogenized. CFUs were performed. Data are expressed as mean ± standard deviation (SD). Two-tailed unpaired t- tests were used for H99-PBS and isw1Δ -PBS group. Two-way analyses of variance (ANOVAs) were used for drug-treated groups. Figure 3—source data 1. The source data comprises unprocessed data.

Article Snippet: Drug-resistance tests were performed using 16 μg/ml or 20 μg/ml FLC (MedChemExpress), 0.2 μg/ml or 0.3 μg/ml KTC (MedChemExpress), 100 μg/ml or 200 μg/ml 5-FC (MedChemExpress), 0.5 μg/ml or 1.0 μg/ml Amp B (MedChemExpress) or 50 μg/ml or 100 μg/ml 5-FU (MedChemExpress).

Techniques: Two Tailed Test, Standard Deviation, Infection, Saline

Journal: eLife

Article Title: Interplay between acetylation and ubiquitination of imitation switch chromatin remodeler Isw1 confers multidrug resistance in Cryptococcus neoformans

doi: 10.7554/eLife.85728

Figure Lengend Snippet: ( a ) Immunoblotting analysis. The ISW1-FLAG strain was preincubated with 200 μM cycloheximide (CHX) for 1 hr followed by exposure to various concentrations of fluconazole (FLC) for 0.5 hr. Anti-Flag and anti-histone 3 antibodies were used. Three biological replicates were performed, and results were used for quantification. Two-tailed unpaired t -tests were used. Data are expressed as mean ± standard deviation (SD). ( b ) Immunoblotting analysis. The ISW1-FLAG strain was preincubated with 200 μM cycloheximide (CHX) for 1 hr followed by exposure to 40 μg/ml FLC for 5, 15, or 30 min. Cells not exposed to FLC but held for 30 min were used as a negative control. Anti-Flag and anti-histone 3 antibodies were used. Three biological replicates were performed, and results were used for quantification. Two-tailed unpaired t -tests were used. Data are expressed as mean ± SD. ( c ) Immunoblotting analysis. Testing and data treatment were exactly as described for with the exception that 5-fluorocytosine (5-FC) was used as the antifungal agent. ( d ) Immunoblotting analysis. Testing and data treatment were exactly as described in , except that 5-FC was used as the antifungal agent. ( e ) Ubiquitin analysis of Isw1 via mass spectrometry. The Isw1-Flag proteins were pulled down and analyzed for ubiquitination. Results for Isw1 K441Ub are shown. ( f ) Schematic of Isw1 showing acetylation and ubiquitination sites. Figure 4—source data 1. The source data comprises unprocessed data.

Article Snippet: Drug-resistance tests were performed using 16 μg/ml or 20 μg/ml FLC (MedChemExpress), 0.2 μg/ml or 0.3 μg/ml KTC (MedChemExpress), 100 μg/ml or 200 μg/ml 5-FC (MedChemExpress), 0.5 μg/ml or 1.0 μg/ml Amp B (MedChemExpress) or 50 μg/ml or 100 μg/ml 5-FU (MedChemExpress).

Techniques: Western Blot, Two Tailed Test, Standard Deviation, Negative Control, Mass Spectrometry

Journal: eLife

Article Title: Interplay between acetylation and ubiquitination of imitation switch chromatin remodeler Isw1 confers multidrug resistance in Cryptococcus neoformans

doi: 10.7554/eLife.85728

Figure Lengend Snippet: ( a ) Acetylation analysis of Isw1. Cells were treated with 3 μM trichostatin A (TSA), 20 mM nicotinamide (NAM), and fluconazole (FLC). The Isw1-Flag proteins were pulled down, and immunoblotting assays were performed using anti-Kac and anti-Flag antibodies. ( b ) Acetylation analysis of Isw1. Cells were treated with TSA, NAM, and 5-fluorocytosine (5-FC). The Isw1-Flag proteins were pulled down, and immunoblotting assays were performed using anti-Kac and anti-Flag antibodies. ( c ) Spotting assays of ISW1 mutants. The ISW1 K89R, K97R, K113R and ISW1 K89Q, K97Q, K113Q strains were tested for drug resistance. ( d ) Spotting assays of ISW1 mutants. The ISW1 K89R , ISW1 K97R , and ISW1 K113R strains were tested for drug resistance. ( e ) Immunoblotting assays of ISW1 mutants. The wild-type, ISW1 K89R, K97R, K113R , and ISW1 K89Q, K97Q, K113Q strains were tested for Isw1 levels. Three biological replicates were performed, and results were used for quantification. Two-tailed unpaired t -tests were used. Data are expressed as mean ± standard deviation (SD). ( f ) Immunoblotting assays of ISW1 mutants. The wild-type, ISW1 K89R , ISW1 K97R , and ISW1 K113R strains were tested for Isw1 levels. Three biological replicates were performed, and results were used for quantification. Two-tailed unpaired t -tests were used. Data are expressed as mean ± SD. ( g ) Immunoblotting assay of Isw1. The wild-type strain was preincubated with 200 μM cycloheximide for 1 hr. Proteins were isolated at indicated time points. Three biological replicates of immunoblotting were performed, and results were used for quantification. One-way analysis of variance (ANOVA) was used. ( h ) Immunoblotting assay of Isw1 K97Q . The analysis was performed as described in . ( i ) Immunoblotting assay of Isw1 K97R . The analysis was performed as described in . ( j ) Comparisons of assay results to determine Isw1 stability. The relative intensities from the results shown in were plotted. Two-way ANOVA was used. Data are expressed as mean ± SD. ( k ) Analyses of drug-resistance genes using qRT-PCR. Samples of RNA ( n = 3) were isolated from ISW1 K97Q and ISW1 K97R treated with FLC or 5-FC. Representative drug-resistance genes were quantified using qRT-PCR. Two-tailed unpaired t -tests were used. Data are expressed as mean ± SD. Figure 5—source data 1. The source data comprises unprocessed data.

Article Snippet: Drug-resistance tests were performed using 16 μg/ml or 20 μg/ml FLC (MedChemExpress), 0.2 μg/ml or 0.3 μg/ml KTC (MedChemExpress), 100 μg/ml or 200 μg/ml 5-FC (MedChemExpress), 0.5 μg/ml or 1.0 μg/ml Amp B (MedChemExpress) or 50 μg/ml or 100 μg/ml 5-FU (MedChemExpress).

Techniques: Western Blot, Two Tailed Test, Standard Deviation, Isolation, Quantitative RT-PCR

Journal: eLife

Article Title: Interplay between acetylation and ubiquitination of imitation switch chromatin remodeler Isw1 confers multidrug resistance in Cryptococcus neoformans

doi: 10.7554/eLife.85728

Figure Lengend Snippet: ( a ) Immunoblotting assays of Isw1-Flag. Proteins Isw1 K97Q , Isw1 K97R , and Isw1 were tested, where the wild-type stain was incubated with 200 μM MG132 and 5 nM rapamycin for 10 hr. Three biological replicates of the assays were performed, and results were used for quantification. Two-tailed unpaired t -tests were used. Data are expressed as mean ± standard deviation (SD). ( b ) Immunoblotting assays of Isw1-Flag. Testing and data treatment were exactly as described in , except that the wild-type sample was treated with 200 μM of MG132 and 40 μg/ml fluconazole (FLC). ( c ) Immunoblotting assays of Isw1-Flag. Testing and data treatment were exactly as described in , except that the wild-type sample was treated with 200 μM MG132 and 400 μg/ml 5-fluorocytosine (5-FC). ( d ) Immunoblotting assays of Isw1 K97Q and Isw1 K97R . Proteins were either treated with MG132 or not before testing. Three biological replicates of the assays were performed, and results were used for quantification. Two-tailed unpaired t- tests were used. Data are expressed as mean ± SD. ( e ) Spotting assays of ISW1 acetylation and ubiquitination mutants. Indicated strains were spotted onto YPD agar either supplemented with an antifungal agent or left blank. ( f ) Immunoblotting assays of ISW1 acetylation and ubiquitination mutants. Protein samples were isolated from the indicated ISW1 mutants. Immunoblotting assays were performed. ( g ) Quantification of immunoblotting results. The immunoblotting assays described for were performed using three independent samples, and the results were used for quantification. Two-tailed unpaired t- tests were used. Data are expressed as mean ± SD. Figure 6—source data 1. The source data comprises unprocessed data.

Article Snippet: Drug-resistance tests were performed using 16 μg/ml or 20 μg/ml FLC (MedChemExpress), 0.2 μg/ml or 0.3 μg/ml KTC (MedChemExpress), 100 μg/ml or 200 μg/ml 5-FC (MedChemExpress), 0.5 μg/ml or 1.0 μg/ml Amp B (MedChemExpress) or 50 μg/ml or 100 μg/ml 5-FU (MedChemExpress).

Techniques: Western Blot, Staining, Incubation, Two Tailed Test, Standard Deviation, Isolation