Journal: Frontiers in Molecular Neuroscience

Article Title: Chaperone-Mediated Regulation of Choline Acetyltransferase Protein Stability and Activity by HSC/HSP70, HSP90, and p97/VCP

doi: 10.3389/fnmol.2017.00415

Figure Lengend Snippet: Identification of the heat shock proteins HSP70 and HSP90 as putative ChAT protein-interactors by proximity-dependent biotin identification (BioID). (A) Optimization of BioID in HEK293 cells expressing wild-type or P17A/P19A-ChAT fused to the HA-tagged promiscuous biotin ligase BirA-R118G (BirA * ). Control cells were transfected with empty vector or plasmids encoding either untagged ChAT or BirA * . Cells were treated for 24 h with either 50 μM biotin to facilitate proximity-dependent biotinylation of ChAT-interacting cellular proteins or with vehicle-control (water). Biotinylated proteins were isolated from cell lysates by streptavidin pull-downs (PD: Strep) and immunoblotted as indicated ( n = 2). (B) Identification of HSP70 and HSP90 as ChAT proximally-interacting proteins. Streptavidin PD samples prepared from biotin-treated HEK293 cells expressing wild-type-ChAT-BirA * or P17A/P19A-ChAT-BirA * fusion proteins were resolved and visualized on a silver-stained SDS-PAGE gels. Two proteins (~70 and ~90 kDa) that were enriched in samples expressing P17A/P19A-ChAT-BirA * were identified by MALDI-TOF-MS or LC-ESI-MS/MS as HSP70 and HSP90, respectively. Control cells were transfected to express untagged wild-type ChAT ( n = 1). (C) Confirmation of endogenous HSP70 and HSP90 as putative ChAT-interacting proteins by immunoblotting of streptavidin PD samples prepared from biotin-treated HEK293 cells expressing HA-tagged wild-type-ChAT-BirA * or P17A/P19A-ChAT-BirA * . Control cells were transfected with empty vector or vector encoding untagged ChAT ( n = 4).

Article Snippet: The impact of inhibition of HSC/HSP70, HSP90 or p97/VCP function on ChAT steady-state protein levels was determined in SN56 cells expressing either wild-type or mutant ChAT proteins treated with varying concentrations of the HSC/HSP70 inhibitor VER-155008 (5–50 μM; Sigma), HSP90 inhibitor 17-AAG (0.5–2 μM; StressMarq Biosciences) or p97/VCP inhibitor Eeyarestatin-I (5–10 μM; Sigma) for 18–24 h at 37°C.

Techniques: Expressing, Transfection, Plasmid Preparation, Isolation, Staining, SDS Page, Tandem Mass Spectroscopy, Western Blot

Journal: Frontiers in Molecular Neuroscience

Article Title: Chaperone-Mediated Regulation of Choline Acetyltransferase Protein Stability and Activity by HSC/HSP70, HSP90, and p97/VCP

doi: 10.3389/fnmol.2017.00415

Figure Lengend Snippet: Co-immunoprecipitation (co-IP) of ChAT with heat shock proteins HSC70, HSP70, and HSP90 is altered by mutation of N-terminal proline-rich motif in ChAT. (A) Immunoblots showing co-IP of ChAT with endogenous HSC70, HSP70 and HSP90 from HEK293 cells expressing either wild-type or P17A/P19A-ChAT. Control cells were transfected with empty vector. Using HEK293 cells, co-IP of P17A/P19A-ChAT with HSP70 (B) , HSP90 (C) and HSC70 (D) , respectively, is greater than that of wild-type ChAT ( *** p ≤ 0.001, Student's t -test, mean ± SEM, n = 4). (E) Co-IP of ChAT with endogenous HSC70 and HSP90 from mouse cholinergic SN56 cells expressing either wild-type or P17A/P19A-ChAT or CMS-related mutant proteins V18M- or A513T-ChAT. Control cells were transfected with empty vector. (F) Using SN56 cells, Co-IP of P17A/P19A-ChAT ( *** p ≤ 0.001) and V18M-ChAT ( * p ≤ 0.05), but not A531T-ChAT, with HSC70 is greater than that of wild-type ChAT (mean ± SEM, n = 5). (G) While there was a trend toward increased HSP90 interaction with P17A/P19A-ChAT ( p = 0.09), no significant differences were observed for HSP90 interaction with mutant ChAT compared to wild-type ChAT in SN56 cells (mean ± SEM, n = 5). Statistical analysis for (F) and (G) was performed by one-way ANOVA with Dunnett's post-hoc test. (H) Detection of in situ interactions of wild-type ChAT with endogenous HSC70 and HSP90 by proximity ligation assay (PLA) in SN56 cells. Formalin-fixed cells were first co-labeled with goat anti-ChAT together with either mouse anti-HSC70 or mouse anti-HSP90 primary antibodies, then incubated with oligonucleotide-linked secondary antibodies. Following DNA ligation and DNA amplification using the Duolink in Situ Orange Kit (Sigma), in situ ChAT-HSP interactions were imaged by confocal microscopy. Positive in situ ChAT-HSP interactions where visualized as fluorescent red dots while nuclei were stained with DAPI (blue). Control cells were either transfected with empty vector or had primary antibodies omitted from the assay (No 1° antibodies). Images are representative of 3 independent experiments; scale bars are 10 μm.

Article Snippet: The impact of inhibition of HSC/HSP70, HSP90 or p97/VCP function on ChAT steady-state protein levels was determined in SN56 cells expressing either wild-type or mutant ChAT proteins treated with varying concentrations of the HSC/HSP70 inhibitor VER-155008 (5–50 μM; Sigma), HSP90 inhibitor 17-AAG (0.5–2 μM; StressMarq Biosciences) or p97/VCP inhibitor Eeyarestatin-I (5–10 μM; Sigma) for 18–24 h at 37°C.

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Mutagenesis, Western Blot, Expressing, Transfection, Plasmid Preparation, In Situ, Proximity Ligation Assay, Labeling, Incubation, DNA Ligation, Amplification, Confocal Microscopy, Staining

Journal: Frontiers in Molecular Neuroscience

Article Title: Chaperone-Mediated Regulation of Choline Acetyltransferase Protein Stability and Activity by HSC/HSP70, HSP90, and p97/VCP

doi: 10.3389/fnmol.2017.00415

Figure Lengend Snippet: Inhibition of HSP90 ATPase activity specifically reduces steady-state levels of mutant ChAT protein. Immunoblots from SN56 cells expressing wild-type (A) , P17A/P19A- (B) , V18M- (C) , or A513T-ChAT (D) that were treated for 24 h with 0.5–2 μM with 17-AAG, an inhibitor of HSP90 activity, or with DMSO-vehicle. (E) Treatment of cells with 17-AAG at concentrations up to 2 μM has no effect on the steady-state levels of wild-type ChAT protein. Conversely, steady-state protein levels of P17A/P19A- (F) , V18M- (G) , and A513T-ChAT (H) are reduced following treatment of cells with 17-AAG compared to vehicle-control ( *** p ≤ 0.001; one-way ANOVA with Dunnett's post-hoc test, mean ± SEM, n = 4). (I) Proteasome inhibition by co-treatment with 5 μM MG132 for 18 h attenuates the effects of inhibition of HSP90 (1 μM 17-AAG, 24 h) on ChAT steady-state protein levels in ChAT-expressing SN56 cells. Control cells were treated with DMSO-vehicle. Proteasome inhibition was validated by immunoblotting for the accumulation of ubiquitinated cellular proteins ( n = 4). (J) Lysosomal inhibition by co-treatment with 50 μM chloroquine (CQ) for 18 h did not prevent the effects of 17-AAG treatment (1 μM, 24 h) on steady-state ChAT protein levels in SN56 cells. Lysosomal inhibition was validated by immunoblotting for the accumulation of the lysosome-associated protein LC3B-II ( n = 3).

Article Snippet: The impact of inhibition of HSC/HSP70, HSP90 or p97/VCP function on ChAT steady-state protein levels was determined in SN56 cells expressing either wild-type or mutant ChAT proteins treated with varying concentrations of the HSC/HSP70 inhibitor VER-155008 (5–50 μM; Sigma), HSP90 inhibitor 17-AAG (0.5–2 μM; StressMarq Biosciences) or p97/VCP inhibitor Eeyarestatin-I (5–10 μM; Sigma) for 18–24 h at 37°C.

Techniques: Inhibition, Activity Assay, Mutagenesis, Western Blot, Expressing

Journal: Frontiers in Molecular Neuroscience

Article Title: Chaperone-Mediated Regulation of Choline Acetyltransferase Protein Stability and Activity by HSC/HSP70, HSP90, and p97/VCP

doi: 10.3389/fnmol.2017.00415

Figure Lengend Snippet: Inhibition of HSC/HSP70 and HSP90 activity enhances ChAT ubiquitination and reduces cellular ChAT enzymatic activity. (A) Immunoblots following anti-ChAT IP from ChAT-expressing SN56 cells that were co-treated for 24 h with 40 μM VER-155008 and for the final 6 h with 20 μM MG132 to inhibit degradation of ubiquitinated ChAT. Control cells were transfected with empty vector and/or treated with DMSO-vehicle. Inhibition of HSC/HSP70 by VER-155008 treatment enhanced ChAT ubiquitination, where levels of ubiquitinated mutant ChAT, particularly P17A/P19A-ChAT, are greater than that of wild-type ChAT ( n = 3). (B) Immunoblots following anti-ChAT IP from ChAT-expressing SN56 cells that were co-treated for either 8 or 24 h with 1 μM 17-AAG and for the final 6 h with 20 μM MG132. Inhibition of HSP90 by treatment with 1 μM 17-AAG for 8 h, but not for 24 h, enhanced ChAT ubiquitination where levels of ubiquitinated mutant ChAT, particularly P17A/P19A-ChAT, are greater than that of wild-type ChAT ( n = 3). (C) Cellular activity of wild-type ChAT is reduced following treatment of SN56 cells for 24 h with either 40 μM VER-155008 or 1 μM 17-AAG compared to vehicle-treated (DMSO) cells ( ** p ≤ 0.01). Treatment with VER-155008 trended toward a greater reduction in ChAT activity compared to 17-AAG ( p = 0.074; one-way ANOVA with Tukey's post-hoc test, mean ± SEM, n = 3).

Article Snippet: The impact of inhibition of HSC/HSP70, HSP90 or p97/VCP function on ChAT steady-state protein levels was determined in SN56 cells expressing either wild-type or mutant ChAT proteins treated with varying concentrations of the HSC/HSP70 inhibitor VER-155008 (5–50 μM; Sigma), HSP90 inhibitor 17-AAG (0.5–2 μM; StressMarq Biosciences) or p97/VCP inhibitor Eeyarestatin-I (5–10 μM; Sigma) for 18–24 h at 37°C.

Techniques: Inhibition, Activity Assay, Western Blot, Expressing, Transfection, Plasmid Preparation, Mutagenesis

Journal: Pharmaceutical Biology

Article Title: Berberine regulates the Notch1/PTEN/PI3K/AKT/mTOR pathway and acts synergistically with 17-AAG and SAHA in SW480 colon cancer cells

doi: 10.1080/13880209.2020.1865407

Figure Lengend Snippet: BBR did not inhibit Hsp90 and HDACs in SW480 cells. (A) The binding effect between BBR (100 μM) and Hsp90 was detected by the thermal stabilization-based CESTA method at 48–76 °C after 30 min of incubation at room temperature. (B) Hsp90 protein expression was quantified and the data are presented as means ± S.D. of three independent experiments. (C) Western blot analysis of Hsp70, IKKβ and EGFR in BBR treated SW480 cells after 24 h incubation. The Hsp90 inhibitor 17-AAG was included as positive control. (D) Western blot analysis of HDAC proteins in BBR treated SW480 cells after 24 h incubation.

Article Snippet: Tanespimycin (17-AAG) and Vorinostat (SAHA) were from Medchem Express (Monmouth Junction, NJ).

Techniques: Binding Assay, Incubation, Expressing, Western Blot, Positive Control

Journal: Pharmaceutical Biology

Article Title: Berberine regulates the Notch1/PTEN/PI3K/AKT/mTOR pathway and acts synergistically with 17-AAG and SAHA in SW480 colon cancer cells

doi: 10.1080/13880209.2020.1865407

Figure Lengend Snippet: BBR enhanced the anti-proliferative activity of 17-AAG and SAHA, and the combination of the three has more potent synergistic effect on SW480 cells. The incubation time of SW480 cells and the test drugs was 24 h. (A) SW480 cells were exposed to specified concentrations of 17-AAG for 24 h in the presence of 0–2 µM of BBR. (B) The CI values of BBR and 17-AAG combinations. (C) SW480 cells were exposed to specified concentrations of SAHA for 24 h in the of 0–2 µM of BBR. (D) The CI values of BBR and SAHA combinations. (E) SW480 cells were treated with specified concentrations of 17-AAG and SAHA in the presence of 0–2 µM of BBR. (F) The CI values for BBR, 17-AAG and SAHA combinations. The above data are all presented as the means ± S.D. of three independent experiments. * p < 0.05, ** p < 0.01 when comparing with 17-AAG, SAHA and both 17-AAG and SAHA treatment in the absence of BBR.

Article Snippet: Tanespimycin (17-AAG) and Vorinostat (SAHA) were from Medchem Express (Monmouth Junction, NJ).

Techniques: Activity Assay, Incubation

Journal: Oncotarget

Article Title: TNF induced cleavage of HSP90 by cathepsin D potentiates apoptotic cell death

doi: 10.18632/oncotarget.12411

Figure Lengend Snippet: a. Apoptosis was measured after 20 h by quantifying nuclear fragmentation by ImageStream analysis: U937 cells were left untreated (~2%) or TNF treated (~30%) and in combination with Pepstatin A (PepA) (~20%) or 17AAG (~70%). PepA and 17AAG showed no cytotoxicity alone. b. HSP90-V5 was either mutated at F437 to L or at Y465 to W or then overexpressed in U937 (left) and Jurkat (right) cells. The expression was analysed by Western blot via a V5-tag, tubulin served as loading control. c. Overexpression of mutated HSP90 resulted in reduced apoptosis compared to mock transfected cells (ctrl). The Y465W mutant showed higher protective efficiency than the F437L mutant. d. Scheme of the molecular mass matched (compared to the Western blots from Figure ) V5-tagged N- and C-terminal cleavage products. e. The expression of the two V5-tagged fragments in U937 (left panels) and Jurkat cells (right panels) is shown and indicated by asterisks. The lower panels show the endogenous HSP90 and actin as loading control. f. The normalised apoptotic response towards TNF after transfecting the cells with the V5 tagged constructs or mock plasmid (ctrl) is shown. For quantification, the nuclear fragmentation measured by ImageStream was analysed after 20h incubation with the apoptosis inducing agent.

Article Snippet: 17AAG was purchased from Tocris.

Techniques: Expressing, Western Blot, Control, Over Expression, Transfection, Mutagenesis, Construct, Plasmid Preparation, Incubation

Journal: Oncotarget

Article Title: TNF induced cleavage of HSP90 by cathepsin D potentiates apoptotic cell death

doi: 10.18632/oncotarget.12411

Figure Lengend Snippet: a. The impact of TNF treatment in combination with HSP90 inhibition on the activation of caspase 9 and Bid was analysed by Western blot. The blots show the decrease in pro-caspase 9 levels and the increase in cleaved caspase 9 levels after 120 min. After the same time, more Bid-cleavage can be observed. Densitometric quantification of the Western blots is shown below (representative result of three experiments). b. Increases in enzymatic activity of caspase 9 after TNF and combined TNF/17AAG treatment compared to untreated cell lysates was analysed by luminescence measurement (n=3).

Article Snippet: 17AAG was purchased from Tocris.

Techniques: Inhibition, Activation Assay, Western Blot, Activity Assay

Journal: Oncotarget

Article Title: TNF induced cleavage of HSP90 by cathepsin D potentiates apoptotic cell death

doi: 10.18632/oncotarget.12411

Figure Lengend Snippet: Binding of TNF to TNF-R1 initially triggers survival signalling via TRADD/RIP1/TRAF2 and NF-kB activation. K63 ubiquitination of TNF-R1 mediates internalization of the receptor and formation of TNF-receptosomes, concomitantly switching off NF-kB signalling and recruiting the DISC proteins FADD and caspase-8. The TNF-receptosomes maturate by fusing with trans-golgi vesicles to form multivesicular body compartments. Cathepsin D is activated and released into the cytosol, where it can cleave Bid and also HSP90. Cleavage of HSP90 or inhibition of HSP90 by 17AAG finally results in enhanced Bid cleavage, Caspase 9 activation and apoptosis.

Article Snippet: 17AAG was purchased from Tocris.

Techniques: Binding Assay, Activation Assay, Ubiquitin Proteomics, Inhibition

Journal: Journal of Virology

Article Title: 9-Butyl-Harmol Exerts Antiviral Activity against Newcastle Disease Virus through Targeting GSK-3β and HSP90β

doi: 10.1128/jvi.01984-22

Figure Lengend Snippet: HSP90 facilitates NDV proliferation, and NDV L protein is a client protein of HSP90β. (A) 9-butyl-harmol suppresses the expression of the F48E9 L protein. DF-1 cells were transfected with plasmids encoding the NP, P, or L protein. At 6 h posttransfection, the medium was changed to fresh medium containing 9-butyl-harmol, and this was followed by 24 h of incubation. The cells were harvested to assess the expression of viral proteins via Western blotting. (B and C) 17-AAG inhibits NDV proliferation. DF-1 cells were infected with F48E9 (0.01 MOI) and incubated with 17-AAG. At 24 h postinfection, the cells were harvested to assess the expression of viral protein via Western blotting (B). The supernatant was harvested to assess the viral titer via plaque assay (C). Mean values ± SDs are shown ( n = 3). Significance was assessed via two-tailed, unpaired Student’s t tests. * * , P < 0.01; ** * , P < 0.001. (D) The knockdown of HSP90α inhibits NDV proliferation. DF-1 cells were transfected with siHSP90AA1-1. At 48 h posttransfection, the cells were infected with F48E9 (0.01 MOI). At 24 h postinfection, the cells were harvested to assess the expression of viral protein via Western blotting (left panel). The supernatant was harvested to assess the viral titer via plaque assay (right panel). Mean values ± SDs are shown ( n = 3). Significance was assessed via two-tailed, unpaired Student’s t tests. * * , P < 0.01. (E) The knockdown of HSP90β inhibits NDV proliferation. DF-1 cells were transfected with siHSP90AB1-2. At 48 h posttransfection, the cells were infected with F48E9 (0.01 MOI). At 24 h postinfection, the cells were harvested to assess the expression of viral protein via Western blotting (E). The supernatant was harvested to assess the viral titer via plaque assay (F). Mean values ± SDs are shown ( n = 3). Significance was assessed via two-tailed, unpaired Student’s t tests. ** * , P < 0.001. (F) NDV viral proteins do not interact with HSP90α. DF-1 cells were transfected with plasmids of viral proteins (NP, P, or L) together with pCAGGS-Flag-HSP90AA1. At 48 h posttransfection, the interaction between viral proteins and HSP90α was analyzed via coimmunoprecipitation assay. (G) NDV L protein interacts with HSP90β. DF-1 cells were transfected with plasmids of viral proteins (NP, P, or L) together with pCAGGS-Flag-HSP90AB1. At 48 h posttransfection, the interaction between viral proteins and HSP90β was analyzed via coimmunoprecipitation assay. (H) Colocalization of HSP90β and the NDV L protein. DF-1 cells were transfected with pCAGGS-myc-L together with pCAGGS-Flag-HSP90AB1. At 48 h posttransfection, the subcellular localization of two proteins was analyzed via immunofluorescence microscopy. (I) HSP90β enhances the expression level of the NDV L protein. DF-1 cells were transfected with pCAGGS-Flag-HSP90AA1, pCAGGS-Flag-HSP90AB1, or both plasmids together with pCAGGS-myc-L. At 48 h posttransfection, the expression of the protein was analyzed via Western blotting.

Article Snippet: Wortmannin (catalog number T6283), iCRT14 (catalog number T4486), 17-AAG (catalog number T6290), protease inhibitor cocktail (catalog number C0001), and phosphatase inhibitor cocktail I (catalog number C0002) were purchased from TargetMol (USA).

Techniques: Expressing, Transfection, Incubation, Western Blot, Infection, Plaque Assay, Two Tailed Test, Knockdown, Co-Immunoprecipitation Assay, Immunofluorescence, Microscopy

Journal: mBio

Article Title: Genetic Analysis of Candida auris Implicates Hsp90 in Morphogenesis and Azole Tolerance and Cdr1 in Azole Resistance

doi: 10.1128/mBio.02529-18

Figure Lengend Snippet: Perturbation of HSP90 results in a loss of viability of C. auris but does not affect azole resistance. (A) Spotting of C. auris wild-type and tetO-HSP90 strains on YPD or YPD agar plus doxycycline (DOX) plates (right panel). C. albicans wild-type and tetO-HSP90 / tetO-HSP90 strains were included for comparison (left panel). A high concentration (50 μg/ml) of DOX was used to ensure strong repression of HSP90 expression. Overnight cultures were diluted 1,000-fold, at which point 5 μl was spotted in 100-fold dilutions. Plates were incubated at 30°C for 48 h. (B) Fluconazole Etest strip in the presence and absence of DOX. A total of 1 × 10 6 cells of wild-type and tetO- repressible HSP90 strains were plated on YPD agar plates with fluconazole Etest strips in the absence and presence of DOX (0.1 μg/ml or 10 μg/ml). The plates were incubated at 30°C for 48 h. (C) Checkerboard assays with geldanamycin and fluconazole. C. albicans clinical isolate CaCi2 and C. auris isolate Ci6684 were inoculated with a 2-fold gradient of geldanamycin in combination with a 2-fold gradient of fluconazole. Cultures were incubated at 30°C for 48 h. Heat maps represent relative growth levels determined from averages of results of technical replicates normalized to the data from a no-drug well.

Article Snippet: Approximately 1 × 10 3 cells were inoculated with a 2-fold gradient matrix of fluconazole (Carbosynth) or of geldanamycin (LC Laboratories) or of a combination of the two, as indicated, in 96-well microtiter plates to reach the final volume of 200 μl in YPD.

Techniques: Concentration Assay, Expressing, Incubation, Stripping Membranes

Journal: mBio

Article Title: Genetic Analysis of Candida auris Implicates Hsp90 in Morphogenesis and Azole Tolerance and Cdr1 in Azole Resistance

doi: 10.1128/mBio.02529-18

Figure Lengend Snippet: Hsp90 mediates tolerance of fluconazole in C. auris . (A) MIC assay for fluconazole in a panel of C. auris clinical isolates. Data were analyzed as described for Fig. 1C . (B) Checkerboard assays with geldanamycin and fluconazole. C. auris strains Ci6684, CDC-382, CDC-387, and CDC-388 were inoculated with a 2-fold gradient of geldanamycin in combination with a 2-fold gradient of fluconazole. Data were analyzed as described for Fig. 1C . After measurement of the growth, cultures were spotted onto drug-free YPD agar plates and allowed to grow at 30°C for 24 h to check for cidality.

Article Snippet: Approximately 1 × 10 3 cells were inoculated with a 2-fold gradient matrix of fluconazole (Carbosynth) or of geldanamycin (LC Laboratories) or of a combination of the two, as indicated, in 96-well microtiter plates to reach the final volume of 200 μl in YPD.

Techniques:

Journal: mBio

Article Title: Genetic Analysis of Candida auris Implicates Hsp90 in Morphogenesis and Azole Tolerance and Cdr1 in Azole Resistance

doi: 10.1128/mBio.02529-18

Figure Lengend Snippet: C. auris undergoes filamentous growth under conditions of compromised Hsp90 function. (A) Overnight cultures of C. albicans (strain SN95) and C. auris (strain Ci6684) or of the respective strains with HSP90 under the control of a tetO -repressible promoter were subcultured in YPD without or with doxycycline. Images were captured after 6 h postsubculture. (B) Overnight cultures of wild-type C. albicans and C. auris strains were subcultured in YPD without or with geldanamycin (10 μM for C. albicans and 80 μM for C. auris ) or hydroxyurea (50 mM). Images were captured after 6 h of drug treatment. (C) Overnight cultures of C. albicans and C. auris were subcultured in canonical C. albicans filament-inducing cues. These include 37°C plus 10% serum (Serum), 37°C plus Spider medium (Spider), 37°C RPMI medium, and 42°C. Microscopy images were acquired after 6 h of incubation. The scale bars represent 20 μm in all panels.

Article Snippet: Approximately 1 × 10 3 cells were inoculated with a 2-fold gradient matrix of fluconazole (Carbosynth) or of geldanamycin (LC Laboratories) or of a combination of the two, as indicated, in 96-well microtiter plates to reach the final volume of 200 μl in YPD.

Techniques: Microscopy, Incubation

Journal: mBio

Article Title: Genetic Analysis of Candida auris Implicates Hsp90 in Morphogenesis and Azole Tolerance and Cdr1 in Azole Resistance

doi: 10.1128/mBio.02529-18

Figure Lengend Snippet: CUG clade species undergo filamentous growth under conditions of compromised Hsp90 function. Overnight cultures of C. albicans , C. dubliniensis , C. tropicalis , L. elongisporus , C. lusitaniae , C. auris , S. cerevisiae , and C. glabrata were subcultured in YPD without or with geldanamycin (GdA). (Top left) A phylogenetic tree of the yeast species was constructed using 1,570 single-copy protein-coding genes, maximum likelihood inference based on 1,000 replicates, and RAxML v7.7.8. Scale bar, mean number of nucleotide substitutions per site. (Top right) Microscopy images were acquired after 6 h of incubation. Scale bar, 20 μm. The concentration of geldanamycin used for each species is indicated on the micrograph. (Bottom) Proportions of yeast to filament were quantified.

Article Snippet: Approximately 1 × 10 3 cells were inoculated with a 2-fold gradient matrix of fluconazole (Carbosynth) or of geldanamycin (LC Laboratories) or of a combination of the two, as indicated, in 96-well microtiter plates to reach the final volume of 200 μl in YPD.

Techniques: Construct, Microscopy, Incubation, Concentration Assay