c jun jun  (Cell Signaling Technology Inc)

Journal: bioRxiv

Article Title: Attenuation of IFITM proteins’ antiviral activity through sequestration into intraluminal vesicles of late endosomes

doi: 10.1101/2025.05.27.656272

Figure Lengend Snippet: (A) Antibodies recognizing extracellular or intracellular domains of EGFR or IFITM1-FLAG proteins were used to probe the accessibility of these domains in cells treated with the EGFR ligand or CsA. Not drawn to scale. (B) A549 cells were incubated in the presence of CHX for one hour, placed on ice, treated with EGF (EGFR samples) or CsA (IFITM1 samples) for 30 minutes, and returned to 37 °C for indicated times. Cells were fixed, permeabilized with digitonin, and stained for extracellular and intracellular domains of a respective target protein, EGFR (C) or IFITM1/FLAG (D). The integrated intensity for each respective antibody targeting domains of EGFR (E) or IFITM1 (F) was calculated and plotted as a function of time of incubation. (G) The ratios between integrated intensities of intracellular and extracellular domains of EGFR and IFITM1 at indicated times are plotted. Data are means and S.D. of two independent experiments, each acquiring three fields of view. ***, p < 0.001; ns, not significant.

Article Snippet: Antibodies used were rabbit IgG against the N-terminus of IFITM3 (Abgent, San Diego, CA, USA), mouse anti-IFITM2/3 (Proteintech, San Diego, CA, USA), rabbit anti-IFITM1 (Sigma), mouse anti-GM130 (BD Bioscience, Franklin Lakes, NJ, USA), sheep anti-TGN46 (Bio-Rad AbD Serotec Limited, Luxembourg), mouse anti-Rab6A, clone 5B10 (a gift from Prof. Angelika Barnekow, Münster University, Germany), mouse anti-flag® M2 (Sigma), mouse anti-Human CD63 (BD Biosciences), Influenza A NP recombinant rabbit monoclonal antibody (Fisher) and antibody to the EGFR N-terminus (Calbiochem), antibody to the EGFR C-terminus (Cell Signaling, Danvers, MA, USA), AlexaFluor 568 Goat anti-Mouse IgG (H+L) (Invitrogen, Waltham, MA, USA), Goat anti-rabbit IgG (H+L) conjugated with AlexaFluor 647 (ThermoFisher, Waltham, MA, USA), and Donkey anti-sheep IgG (H+L) conjugated with AlexaFluor 568 (Abcam).

Techniques: Incubation, Staining

Journal: bioRxiv

Article Title: Attenuation of IFITM proteins’ antiviral activity through sequestration into intraluminal vesicles of late endosomes

doi: 10.1101/2025.05.27.656272

Figure Lengend Snippet: A549.IFITM1-C-FLAG cells were treated with CHX, EGF and either DMSO (A) or CsA (B), as in . Cells were fixed, permeabilized with TX-100, incubated with anti-IFITM1 and anti-EGFR antibodies, and stained with secondary antibodies conjugated to STED-compatible fluorophores, STAR RED and STAR 580. Representative images of n>20 analyzed endosomes are shown. Line histograms for selected endosomes are shown. Scale bar is 0.5 µm.

Article Snippet: Antibodies used were rabbit IgG against the N-terminus of IFITM3 (Abgent, San Diego, CA, USA), mouse anti-IFITM2/3 (Proteintech, San Diego, CA, USA), rabbit anti-IFITM1 (Sigma), mouse anti-GM130 (BD Bioscience, Franklin Lakes, NJ, USA), sheep anti-TGN46 (Bio-Rad AbD Serotec Limited, Luxembourg), mouse anti-Rab6A, clone 5B10 (a gift from Prof. Angelika Barnekow, Münster University, Germany), mouse anti-flag® M2 (Sigma), mouse anti-Human CD63 (BD Biosciences), Influenza A NP recombinant rabbit monoclonal antibody (Fisher) and antibody to the EGFR N-terminus (Calbiochem), antibody to the EGFR C-terminus (Cell Signaling, Danvers, MA, USA), AlexaFluor 568 Goat anti-Mouse IgG (H+L) (Invitrogen, Waltham, MA, USA), Goat anti-rabbit IgG (H+L) conjugated with AlexaFluor 647 (ThermoFisher, Waltham, MA, USA), and Donkey anti-sheep IgG (H+L) conjugated with AlexaFluor 568 (Abcam).

Techniques: Incubation, Staining

Journal: bioRxiv

Article Title: Attenuation of IFITM proteins’ antiviral activity through sequestration into intraluminal vesicles of late endosomes

doi: 10.1101/2025.05.27.656272

Figure Lengend Snippet: A549.IFITM3 (IFITM3+) or A549.vector (IFITM3-) cells were incubated in the presence of CHX for one hour, placed on ice, treated with EGF and either DMSO (A, C) or CsA (B, D) for 30 minutes, and returned to 37 °C for indicated times. Cells were fixed, permeabilized with TX-100, and stained using anti-EGFR (targeting N-terminus) and anti-IFITM3 primary antibodies and secondary antibodies conjugated to STED-compatible fluorophores, STAR RED and STAR 580. Normalized linear intensity profiles across endosomes are shown for each channel. To measure the endosome diameter, local maxima of EGFR signals were used. Endosomes with low EGFR signal, excessive background noise, or indistinguishable features were excluded. Representative linear histograms for IFITM3 positive or negative cells in the presence or absence of CsA are shown. (E) Endosome diameters based on EGFR signal are plotted. Endosomes from two independent experiments (n>20 endosomes, n>15 cells) were analyzed per condition and per cell line (A549.IFITM3 and A549.vector). (F) The endosome diameters based on IFITM3’s signal for IFITM3+ cells were calculated based upon the distance between the normalized linear profile intensities corresponding to 25% of signal. Endosomes with a high background were omitted. Lines and bars are medians and interquartile range. Scale bar is 0.5 µm. ***, p < 0.001; ns, not significant.

Article Snippet: Antibodies used were rabbit IgG against the N-terminus of IFITM3 (Abgent, San Diego, CA, USA), mouse anti-IFITM2/3 (Proteintech, San Diego, CA, USA), rabbit anti-IFITM1 (Sigma), mouse anti-GM130 (BD Bioscience, Franklin Lakes, NJ, USA), sheep anti-TGN46 (Bio-Rad AbD Serotec Limited, Luxembourg), mouse anti-Rab6A, clone 5B10 (a gift from Prof. Angelika Barnekow, Münster University, Germany), mouse anti-flag® M2 (Sigma), mouse anti-Human CD63 (BD Biosciences), Influenza A NP recombinant rabbit monoclonal antibody (Fisher) and antibody to the EGFR N-terminus (Calbiochem), antibody to the EGFR C-terminus (Cell Signaling, Danvers, MA, USA), AlexaFluor 568 Goat anti-Mouse IgG (H+L) (Invitrogen, Waltham, MA, USA), Goat anti-rabbit IgG (H+L) conjugated with AlexaFluor 647 (ThermoFisher, Waltham, MA, USA), and Donkey anti-sheep IgG (H+L) conjugated with AlexaFluor 568 (Abcam).

Techniques: Plasmid Preparation, Incubation, Staining

Journal: Nature Communications

Article Title: Mechanisms of multidrug resistance caused by an Ipi1 mutation in the fungal pathogen Candida glabrata

doi: 10.1038/s41467-025-56269-z

Figure Lengend Snippet: a A schema representing sequential exposure of C. glabrata wild-type (CBS138) cells to increasing concentrations of micafungin. C. glabrata cells were spread on YPD plates containing micafungin at the indicated concentrations, incubated at 30 °C for 2 days, and subjected to antifungal susceptibility tests. b , c Spot dilution assay. C. glabrata strain Cg50 was obtained from a plate containing micafungin at the concentration of 0.03 µg/mL as described in ( a ). Strain Cg51 was obtained after the repeated subculture of Cg50 in YPD broth without micafungin for 20 days. An ipi1 R70H mutant was constructed as described in the methods. Serial 10-fold dilutions of C. glabrata cells were spotted onto an SC plate containing an antifungal agent at the indicated concentrations. Plates were incubated at 30 °C or the indicated temperatures for 2 days. All susceptibility tests were performed on at least three separate occasions. d Doxycycline-mediated transcriptional repression of IPI1 resulted in a growth defect in C. glabrata . Logarithmic-phase cells of the C. glabrata wild-type (ACG22) and tet- IPI1 strain, in which the native IPI1 promoter was replaced with the tetracycline regulatable promoter, were adjusted to ~ 1 × 10 5 cells/mL in SD broth and incubated at 30 °C for 24 h with or without 20 µg/mL of the tetracycline analog doxycycline (Dox). Data are expressed as mean ± standard deviation for biological triplicates (**** P < 0.0001; ns, not significant; one-way ANOVA with Dunnett’s multiple comparison test). The experiment was repeated twice with similar results. Source data are provided as a Source Data file. e Northern blotting. Logarithmic - phase C. glabrata cells grown at 30 °C were subsequently incubated at 42 °C, a non-permissive temperature for the ipi1 R70H mutant. Total RNA was extracted from each strain at the indicated time points. Top , northern hybridization was performed using an ITS2 probe. Bottom , total RNA was analyzed using 1.0% agarose gel with ethidium bromide, and 25S and 18S rRNA are indicated. The experiment was repeated twice with similar results. Source data including uncropped and unprocessed scans with molecular weight markers are provided as a Source Data file.

Article Snippet: Logarithmic-phase C. glabrata cells were lysed using the Minute Total Protein Extraction Kit for Microbes with Thick Cell Walls (Invent Biotechnologies, Plymouth, MN) according to the manufacturer’s instructions.

Techniques: Incubation, Dilution Assay, Concentration Assay, Mutagenesis, Construct, Standard Deviation, Comparison, Northern Blot, Hybridization, Agarose Gel Electrophoresis, Molecular Weight

Journal: Nature Communications

Article Title: Mechanisms of multidrug resistance caused by an Ipi1 mutation in the fungal pathogen Candida glabrata

doi: 10.1038/s41467-025-56269-z

Figure Lengend Snippet: a Sterol analysis. C. glabrata wild-type (WT) and ipi1 R70H strains were grown in synthetic defined (SD) broth or SD broth with 8 μg/mL fluconazole ( + FLC). Sterol contents were analyzed by reverse-phase HPLC as described in the methods. The means and standard errors for three independent experiments are shown (ns, not significant; ordinary one-way ANOVA with Sidak’s multiple comparison test). b qRT-PCR. C. glabrata wild-type (WT) and the ipi1 R70H mutant were grown as described in ( a ) and total RNA was extracted. mRNA abundance of ERG11 was measured by qRT-PCR and normalized using ACT1 as an internal control. Data are expressed as the means ± standard deviations (ns, not significant; ordinary one-way ANOVA with Sidak’s multiple comparison test). qRT-PCR was repeated on three independent occasions with similar results. c qRT-PCR. Total RNA was extracted from C. glabrata wild-type, Cg50, and two different clones of ipi1 R70H mutant strains. mRNA abundance was measured by qRT-PCR and normalized using ACT1 as an internal control. Expression of wild-type strain was defined as 1 in each assay, and relative mRNA abundances in other strains were calculated. Data are expressed as the means ± standard deviations (ordinary one-way ANOVA with Dunnett’s multiple comparison test). qRT-PCR was repeated on three independent occasions with similar results. Source data for panels ( a – c ) are provided as a Source Data file. d Rhodamine 6 G (R6G) accumulation assay. Intracellular concentration of the fluorescent dye R6G, a substrate of azole efflux pumps, was measured by flow cytometry in three different wild-type strains, two different clones of the ipi1 R70H mutant, and a mutant lacking Cdr1 efflux pump ( cdr1 Δ). Fluorescence intensity of R6G is shown on the X-axis. Left panel: Cells were incubated with R6G under normal growth conditions to examine the activity of R6G efflux. Right panel: Cells were exposed to R6G under the de-energized condition to examine the levels of R6G infused passively into the cells. These experiments were repeated twice with similar results.

Article Snippet: Logarithmic-phase C. glabrata cells were lysed using the Minute Total Protein Extraction Kit for Microbes with Thick Cell Walls (Invent Biotechnologies, Plymouth, MN) according to the manufacturer’s instructions.

Techniques: Comparison, Quantitative RT-PCR, Mutagenesis, Control, Clone Assay, Expressing, Concentration Assay, Flow Cytometry, Fluorescence, Incubation, Activity Assay

Journal: Nature Communications

Article Title: Mechanisms of multidrug resistance caused by an Ipi1 mutation in the fungal pathogen Candida glabrata

doi: 10.1038/s41467-025-56269-z

Figure Lengend Snippet: a Effects of CDR1 and PDR1 deletion on azole susceptibility were examined using a spot dilution assay in the wild-type and ipi1 R70H backgrounds. Logarithmic-phase cells of the C. glabrata strains were serially diluted and spotted on SC plates containing fluconazole or voriconazole at the indicated concentrations. Plates were incubated at 30 °C for 2 days. b Pdr1 P927S, which is known as a gain-of-function mutation in C. glabrata , was introduced in the wild-type and ipi1 R70H backgrounds. A spot dilution assay was performed as described above except that plates were incubated at 37 °C. All susceptibility tests were performed on at least three separate occasions.

Article Snippet: Logarithmic-phase C. glabrata cells were lysed using the Minute Total Protein Extraction Kit for Microbes with Thick Cell Walls (Invent Biotechnologies, Plymouth, MN) according to the manufacturer’s instructions.

Techniques: Dilution Assay, Incubation, Mutagenesis

Journal: Nature Communications

Article Title: Mechanisms of multidrug resistance caused by an Ipi1 mutation in the fungal pathogen Candida glabrata

doi: 10.1038/s41467-025-56269-z

Figure Lengend Snippet: a –d Logarithmic-phase C. glabrata cells were serially 10-fold diluted and spotted onto SC plates containing micafungin, fluconazole, or voriconazole at the indicated concentrations. Plates were incubated at 30 °C for 96 h. e , f Expression levels of CDR1, CDR2 , and PDR1 are elevated in the ssb ∆ and ssz1 ∆ strains. qRT-PCR was performed as described in the methods section and in Fig. . Data are expressed as the means ± standard deviations (* P < 0.05; *** P < 0.001; **** P < 0.0001, ordinary one-way ANOVA with Dunnett’s multiple comparison test). qRT-PCR was repeated on three independent occasions with similar results. Source data for panels (e) and (f) are provided as a Source Data file.

Article Snippet: Logarithmic-phase C. glabrata cells were lysed using the Minute Total Protein Extraction Kit for Microbes with Thick Cell Walls (Invent Biotechnologies, Plymouth, MN) according to the manufacturer’s instructions.

Techniques: Incubation, Expressing, Quantitative RT-PCR, Comparison

Journal: Nature Communications

Article Title: Mechanisms of multidrug resistance caused by an Ipi1 mutation in the fungal pathogen Candida glabrata

doi: 10.1038/s41467-025-56269-z

Figure Lengend Snippet: a Growth curves of C. glabrata wild-type and various mutant strains. C. glabrata cells were grown in SC broth at 37 °C with agitation at 250 rpm. Based on the echinocandin susceptibility, C. glabrata strains were classified into two groups: group S (echinocandin-susceptible [wild-type level susceptibility to echinocandins]) and group R (echinocandin-resistant compared to the wild-type strain), shown as blue and red lines, respectively. The growth curves represent the mean of biological duplicates. b Optical densities at 600 nm (OD 600 nm) were compared at the indicated time points between the groups S and R ( n = 7, each). Data are expressed as the means ± standard deviations. Individual dots represent the mean of biological duplicates for each strain as indicated in Fig. 6a. Statistical analysis was performed using Mann–Whitney U test. ns, not significant. Source data for all panels are provided as a Source Data file.

Article Snippet: Logarithmic-phase C. glabrata cells were lysed using the Minute Total Protein Extraction Kit for Microbes with Thick Cell Walls (Invent Biotechnologies, Plymouth, MN) according to the manufacturer’s instructions.

Techniques: Mutagenesis, MANN-WHITNEY

Journal: Nature Communications

Article Title: Mechanisms of multidrug resistance caused by an Ipi1 mutation in the fungal pathogen Candida glabrata

doi: 10.1038/s41467-025-56269-z

Figure Lengend Snippet: C. glabrata cells were exposed to 1 µg/mL of micafungin in SC broth at 37 °C for 1 h. RNA-seq analysis was performed as described in the materials and methods. Venn diagrams show the number of differentially expressed genes in cells treated with micafungin relative to untreated controls (>2-fold change in expression, adjusted P -value of <0.05, DESeq2 analysis applying a two-sided test for each gene with multiple comparison adjustments using the Benjamini-Hochberg method). Representative Gene Ontology (GO) terms are shown ( P < 0.05, ~5 terms in order from the term with the lowest P -value, one-sided binomial test with multiple comparison adjustments using the Bonferroni method). Note that no statistically significant term was found for genes upregulated only in the ipi1 R70H mutant. Source data are provided in Supplementary Data .

Article Snippet: Logarithmic-phase C. glabrata cells were lysed using the Minute Total Protein Extraction Kit for Microbes with Thick Cell Walls (Invent Biotechnologies, Plymouth, MN) according to the manufacturer’s instructions.

Techniques: RNA Sequencing Assay, Expressing, Comparison, Mutagenesis

Journal: Nature Communications

Article Title: Mechanisms of multidrug resistance caused by an Ipi1 mutation in the fungal pathogen Candida glabrata

doi: 10.1038/s41467-025-56269-z

Figure Lengend Snippet: a Either calcineurin catalytic subunit A ( CNA1 ) or regulatory subunit B ( CNB1 ) was deleted in the wild-type and ipi1 R70H backgrounds, and the antifungal susceptibility was examined using a spot dilution assay. Logarithmic-phase cells of the C. glabrata strains were serially diluted and spotted on SC plates containing an antifungal agent at the indicated concentrations. Plates were incubated at 37 °C for 2 days. b Expression level of FKS2 in the presence and absence of micafungin was determined by qRT-PCR. Logarithmic-phase cells of the C. glabrata strains were exposed to 1 µg/mL of micafungin in SC broth at 37 °C for 1 h. qRT-PCR was performed as described in the materials and methods and in Fig. . Individual dots are shown for biological replicates ( n = 6, each). The means ± standard deviations are shown (ns, not significant; Kruskal-Wallis test with Dunnett’s multiple comparison test). c C. glabrata cell survival was assessed before and after exposure to micafungin. Logarithmic-phase cells of the C. glabrata strains were exposed to 0.2 µg/mL of micafungin in SC broth at 37 °C. The CFUs were determined at the indicated time points, and percent CFU was calculated relative to the CFU prior to micafungin exposure. Data are presented as mean values ± standard deviations. The experiments shown in panels ( a – c ) were repeated on three independent occasions with similar results. Source data for panels ( b ) and ( c ) are provided as a Source Data file.

Article Snippet: Logarithmic-phase C. glabrata cells were lysed using the Minute Total Protein Extraction Kit for Microbes with Thick Cell Walls (Invent Biotechnologies, Plymouth, MN) according to the manufacturer’s instructions.

Techniques: Dilution Assay, Incubation, Expressing, Quantitative RT-PCR, Comparison

Journal: Nature Communications

Article Title: Mechanisms of multidrug resistance caused by an Ipi1 mutation in the fungal pathogen Candida glabrata

doi: 10.1038/s41467-025-56269-z

Figure Lengend Snippet: a Susceptibility of the C. glabrata ipi1 R70H mutant to macrophage killing. Logarithmic-phase cells of C. glabrata wild-type strain (CBS138) and the ipi1 R70H mutant were cocultured with murine RAW 264 macrophages at 37 °C for 2 h. Percent killing is expressed as the percent reduction of CFU recovered from cocultures compared with the CFU from control cultures ( C. glabrata cells without macrophages). All data obtained from the two independent experiments were compared between the wild-type and ipi1 R70H strain groups ( n = 14, each). The difference was not statistically significant (ns, not significant; two-tailed Mann–Whitney U test). b A mouse model of disseminated candidiasis. Immunocompetent mice were intravenously inoculated with 8 × 10 7 cells of either C. glabrata wild-type or ipi1 R70H strain ( n = 9 for the wild-type strain and n = 10 for the ipi1 R70H mutant). Bilateral kidneys, spleen, and liver were excised 7 days after injection. Appropriate dilutions of organ homogenates were plated, and the numbers of CFU were counted after 2 days of incubation at 30 °C. Numbers of recovered CFU from each organ are indicated for individual mice in the scatter plots. The geometric mean is shown as a bar. ns, not significant (two-tailed Mann–Whitney U test). The representative data of two independent experiments are shown. c , d A silkworm infection model. Larvae were inoculated with 2.0–2.5 × 10 7 cells of each C. glabrata strain and injected with an echinocandin-class antifungal agent (micafungin, caspofungin, or anidulafungin) dissolved in saline or saline alone (50 μL). Twenty-four hours later, hemolymph was collected and spread on YPD plates to calculate CFU. The number of larvae is shown in parenthesis. Data were analyzed and presented as described above (ns, not significant; two-tailed Mann–Whitney U test). Source data for all panels are provided as a Source Data file.

Article Snippet: Logarithmic-phase C. glabrata cells were lysed using the Minute Total Protein Extraction Kit for Microbes with Thick Cell Walls (Invent Biotechnologies, Plymouth, MN) according to the manufacturer’s instructions.

Techniques: Mutagenesis, Control, Two Tailed Test, MANN-WHITNEY, Injection, Incubation, Infection, Saline

Journal: Nature Communications

Article Title: Mechanisms of multidrug resistance caused by an Ipi1 mutation in the fungal pathogen Candida glabrata

doi: 10.1038/s41467-025-56269-z

Figure Lengend Snippet: Survival rates of larvae infected with the C. glabrata wild-type and ipi1 R70H strains

Article Snippet: Logarithmic-phase C. glabrata cells were lysed using the Minute Total Protein Extraction Kit for Microbes with Thick Cell Walls (Invent Biotechnologies, Plymouth, MN) according to the manufacturer’s instructions.

Techniques: Infection, Saline