Journal: bioRxiv

Article Title: Mycobacterium tuberculosis SecA2-dependent activation of host Rig-I/MAVs signaling is not conserved in Mycobacterium marinum

doi: 10.1101/2023.01.27.525853

Figure Lengend Snippet: M. marinum strains were grown to exponential phase in 7H9 supplemented with 10% OADC and 0.2% tyloxapol before being subcultured into fresh media at an optical density (OD 600 ) of 0.05. Bacterial growth was monitored by measuring the OD 600 every 24-hours for 7 days.

Article Snippet: Mycobacterium marinum M bacterial strains were grown on Middlebrook’s 7H11 (Sigma-Aldrich) agar plates supplemented with 10% OADC [oleic acid (Fischer Scientific), dextrose (Sigma and VWR), albumin (Sigma Aldrich), and catalase(Fischer)] and 0.5% glycerol (VWR, Radnor, PA) at 30°C or in Middlebrook’s 7H9 media (Sigma-Aldrich, St. Louis, MO) supplemented with 10% OADC and 0.2% tyloxapol (Chem-Impex Int’l Inc., Wood Dale, IL) at 30°C.

Techniques:

Journal: bioRxiv

Article Title: Mycobacterium tuberculosis SecA2-dependent activation of host Rig-I/MAVs signaling is not conserved in Mycobacterium marinum

doi: 10.1101/2023.01.27.525853

Figure Lengend Snippet: M. marinum strains were grown to exponential phase in 7H9 supplemented with 10% OADC and 0.2% tyloxapol before being subcultured into fresh media at an optical density (OD 600 ) of 0.05. Bacterial growth was monitored by measuring the OD 600 every 24-hours for 7 days and plotted on a log transformed scale (A). The same strains were examined for growth in sodium dodecyl-sulfate (SDS) by subculturing exponential phase bacteria into 7H9 supplemented with 10% OADC and 0.2% tyloxapol with and without 0.2% SDS at an OD600 of 0.8. Twenty-four hours later, these cultures were serially diluted and plated onto 7H11 agar plates supplemented with 10% OADC in technical triplicate. Images are representative of three biological replicates each plated in technical triplicate (B). Colony forming units were quantified from agars plates following incubation at 32°C with 5% CO 2 for one week (C). Statistical significance was determined using a one-way ANOVA followed by a Dunnett’s test for multiple comparison relative to WT, with p-values ≤ 0.05 considered significant (C only).

Article Snippet: Mycobacterium marinum M bacterial strains were grown on Middlebrook’s 7H11 (Sigma-Aldrich) agar plates supplemented with 10% OADC [oleic acid (Fischer Scientific), dextrose (Sigma and VWR), albumin (Sigma Aldrich), and catalase(Fischer)] and 0.5% glycerol (VWR, Radnor, PA) at 30°C or in Middlebrook’s 7H9 media (Sigma-Aldrich, St. Louis, MO) supplemented with 10% OADC and 0.2% tyloxapol (Chem-Impex Int’l Inc., Wood Dale, IL) at 30°C.

Techniques: Transformation Assay, Subculturing Assay, Bacteria, Incubation, Comparison

Journal: bioRxiv

Article Title: Mycobacterium tuberculosis SecA2-dependent activation of host Rig-I/MAVs signaling is not conserved in Mycobacterium marinum

doi: 10.1101/2023.01.27.525853

Figure Lengend Snippet: M. marinum strains were grown to exponential phase in 7H9 supplemented with 10% OADC and 0.2% tyloxapol and used to infect triplicate wells of BMDMs, at an MOI of 0.2, for 2 hours. At 2, 48, and 96 hours post infection (hpi), macrophage monolayers were lysed and plated onto 7H11 agar supplemented with 10% OADC in technical triplicate. One week after plating, colony forming units were enumerated for each timepoint (A). Bacterial numbers present at 2hpi were plotted separately (B) to better visualize differences in bacterial counts. Uninfected wells were included as a control for across well contamination (not shown, no contamination observed) and the Δ esxBA strain as a control for attenuated growth. Statistical significance was determined using a Kruskal-Wallis test followed by a Wilcoxon Rank Sum test for pairwise comparison relative to WT, with p-values ≤ 0.05 considered significant (*** p<0.001).

Article Snippet: Mycobacterium marinum M bacterial strains were grown on Middlebrook’s 7H11 (Sigma-Aldrich) agar plates supplemented with 10% OADC [oleic acid (Fischer Scientific), dextrose (Sigma and VWR), albumin (Sigma Aldrich), and catalase(Fischer)] and 0.5% glycerol (VWR, Radnor, PA) at 30°C or in Middlebrook’s 7H9 media (Sigma-Aldrich, St. Louis, MO) supplemented with 10% OADC and 0.2% tyloxapol (Chem-Impex Int’l Inc., Wood Dale, IL) at 30°C.

Techniques: Infection, Control, Comparison

Journal: Microbiology Spectrum

Article Title: Fatty acid synthase may facilitate the trafficking of bovine alpha herpesvirus 1 out of the Golgi apparatus, potentially promoting viral infection

doi: 10.1128/spectrum.01388-25

Figure Lengend Snippet: Detection of FASN protein in TG neurons during viral acute infection and latency by IHC. ( A ) TG tissues were collected from calves subjected to mock infection, acute infection (at 4 days post-infection), or latent infection (at 60 days post-infection). Thin sections of 10 µm were cut from formalin-fixed, paraffin-embedded TG tissues and subjected to FASN protein detection by IHC using FASN-specific polyclonal antibody (Proteintech, cat#10624-2-AP, 1:500). Biotinylated goat anti-rabbit IgG (Vector Laboratories) was used as the secondary antibody. ( B ) The percentage of FASN-positive neurons was calculated from 468 neurons of uninfected calves, 467 neurons from acutely infected calves, and 413 TG neurons from latency calves. Data shown are representative of two independent experiments. Scale bars = 100 µm.

Article Snippet: FASN polyclonal antibody (pAb) (cat#10624-2-AP) and GP73 mouse monoclonal antibody (mAb) (cat# 66331-1-lg) were provided by Proteintech (Rosemont, IL, USA).

Techniques: Infection, Formalin-fixed Paraffin-Embedded, Plasmid Preparation

Journal: Microbiology Spectrum

Article Title: Fatty acid synthase may facilitate the trafficking of bovine alpha herpesvirus 1 out of the Golgi apparatus, potentially promoting viral infection

doi: 10.1128/spectrum.01388-25

Figure Lengend Snippet: BoAHV-1 infection decreases the expression of FASN protein across various cell cultures. ( A ) MDBK cells that were confluent in 60 mm dishes were either mock infected or infected with BoAHV-1 at an MOI of 1. After 4, 8, 16, and 24 h of infection, cell lysates were prepared and analyzed by Western blotting using antibodies against FASN (Proteintech, cat# 10624-2-AP, 1:10,000) and virion-associated proteins (VMRD, cat# P170703-001, 1:5,000). ( C ) MDBK cells were either mock-infected or infected with BoAHV-1 at MOIs of 0.1, 1, and 10, respectively. At 24 hpi, cell lysates were prepared for Western blotting to assess the protein expression of FASN and virion-associated proteins. ( E ) Neuro-2A cells were either mock-infected or infected with BoAHV-1 at an MOI of 10. After 12, 24, 36, and 48 hours of infection, cell lysates were prepared and analyzed by Western blotting to detect FASN protein and virion-associated proteins. Tubulin was probed and served as a protein loading control and for subsequent quantitative analysis. ( B, D, and F ) Quantitative analysis of FASN band intensities was performed using the freeware software Image J. Changes in FASN protein levels after infection were calculated relative to mock-infected controls, which were set at 100%. ( G ) Neuro-2A cells in 24-well plates were either mock-infected or infected with BoAHV-1 at an MOI of 10. At 36 and 48 hpi, cell survival was assessed using the CCK-8 assay kit (Beyotime, Shanghai, China, cat# C0038), following the manufacturer’s instructions. ( H ) MDBK and Neuro-2A cells in six-well plates were either mock infected or infected with the virus (MOI of 1 for MDBK cells and MOI of 10 for Neuro-2A cells) for 24 h. Total RNA was then extracted from the cells for the detection of FASN mRNA using RT-qPCR. The data shown are means of three independent experiments with error bars indicating standard deviations. Significance was assessed by standard t -test (ns, not significant; * P < 0.05; ** P < 0.01; *** P < 0.001).

Article Snippet: FASN polyclonal antibody (pAb) (cat#10624-2-AP) and GP73 mouse monoclonal antibody (mAb) (cat# 66331-1-lg) were provided by Proteintech (Rosemont, IL, USA).

Techniques: Infection, Expressing, Western Blot, Control, Software, CCK-8 Assay, Virus, Quantitative RT-PCR

Journal: Microbiology Spectrum

Article Title: Fatty acid synthase may facilitate the trafficking of bovine alpha herpesvirus 1 out of the Golgi apparatus, potentially promoting viral infection

doi: 10.1128/spectrum.01388-25

Figure Lengend Snippet: Examination of FASN localization in BoAHV-1-infected MDBK cells using IFA assay. MDBK cells were seeded into 24-well plates containing coverslips and cultured until they reached 90% confluence. The cells were then either mock-infected or infected with BoAHV-1 at an MOI of 1 for 4, 16, and 24 h, respectively. The cells were immunostained with antibodies against FASN (Green; Abclonal, cat# A21182 , 1:200) and the viral protein gD (Red; VMRD, cat# 1B8-F11, 1:1,000). Nuclei were counterstained with DAPI (4′,6-diamidino-2-phenylindole; Blue). Immunofluorescence was visualized, and images were captured using confocal microscopy (Zeiss). Zoomed-in images framed in red highlight the typical co-localization of FASN with the viral protein gD. These images are representative of results from three independent experiments. Scale bars = 20 µm. ND, not done.

Article Snippet: FASN polyclonal antibody (pAb) (cat#10624-2-AP) and GP73 mouse monoclonal antibody (mAb) (cat# 66331-1-lg) were provided by Proteintech (Rosemont, IL, USA).

Techniques: Infection, Cell Culture, Immunofluorescence, Confocal Microscopy

Journal: Microbiology Spectrum

Article Title: Fatty acid synthase may facilitate the trafficking of bovine alpha herpesvirus 1 out of the Golgi apparatus, potentially promoting viral infection

doi: 10.1128/spectrum.01388-25

Figure Lengend Snippet: Examination of FASN localization in BoAHV-1-infected Neuro-2A cells using IFA assay. Neuro-2A cells were seeded into 24-well plates containing coverslips and cultured until they reached 90% confluence. The cells were then either mock-infected or infected with BoAHV-1 at an MOI of 1 for 48 and 72 h, respectively. They were immunostained with antibodies against the FASN protein (Red; Proteintech, cat# 10624-2-AP, 1:600) and the virion-associated protein (Green; VMRD, cat# P170703-001, 1:2,000). Nuclei were counterstained with DAPI (Blue). Immunofluorescence was visualized, and images were captured using confocal microscopy (Zeiss). These images are representative of results from three independent experiments. Scale bars = 20 µm.

Article Snippet: FASN polyclonal antibody (pAb) (cat#10624-2-AP) and GP73 mouse monoclonal antibody (mAb) (cat# 66331-1-lg) were provided by Proteintech (Rosemont, IL, USA).

Techniques: Infection, Cell Culture, Immunofluorescence, Confocal Microscopy

Journal: Microbiology Spectrum

Article Title: Fatty acid synthase may facilitate the trafficking of bovine alpha herpesvirus 1 out of the Golgi apparatus, potentially promoting viral infection

doi: 10.1128/spectrum.01388-25

Figure Lengend Snippet: Examination of FASN protein in the Golgi apparatus in MDBK cells. ( A ) MDBK cells were seeded into 24-well plates containing coverslips and cultured until they reached 90% confluence. The cells were then either mock-infected or infected with BoAHV-1 at an MOI of 1 for 24 h. They were immunostained with antibodies against the FASN protein (Green; Proteintech, cat# 10624-2-AP, 1:600) and GP73 (Red; Proteintech, cat# 66331-1-1g, 1:200). Nuclei were counterstained with DAPI (Blue). Immunofluorescence was visualized, and images were captured using confocal microscopy (Zeiss). These images are representative of results from three independent experiments. Scale bars = 20 µm. ( B ) MDBK cells in 100 mm dishes were either mock-infected or infected with BoAHV-1 (MOI = 1) for 24 h. The cells were then collected to isolate the Golgi fraction using a commercial kit (Beijing Biolabo Technology, cat# HR0247-50T), following the manufacturer’s protocol. The lysates were subjected to Western blot analysis using antibodies against FASN (Proteintech, cat# 10624-2-AP, 1:10,000) and GOLGA1 (Abclonal, cat# A14688, 1:1,000). GOLGA1 served as a marker for the Golgi apparatus and as a protein loading control. The data presented are representative of three independent experiments. ( C ) Quantitative analysis of FASN band intensities was performed using the freeware software Image J. Changes in FASN protein levels after infection were calculated relative to mock-infected controls, which were set at 100%. The data shown are means of three independent experiments with error bars indicating standard deviations. Significance was assessed by a standard t -test (** P < 0.01).

Article Snippet: FASN polyclonal antibody (pAb) (cat#10624-2-AP) and GP73 mouse monoclonal antibody (mAb) (cat# 66331-1-lg) were provided by Proteintech (Rosemont, IL, USA).

Techniques: Cell Culture, Infection, Immunofluorescence, Confocal Microscopy, Western Blot, Marker, Control, Software

Journal: Microbiology Spectrum

Article Title: Fatty acid synthase may facilitate the trafficking of bovine alpha herpesvirus 1 out of the Golgi apparatus, potentially promoting viral infection

doi: 10.1128/spectrum.01388-25

Figure Lengend Snippet: Determine the roles of FASN in BoAHV-1 productive infection in MDBK cells using transfection of siRNAs. ( A ) MDBK cells in six-well plates were transfected with either scrambled siRNA (150 pmol) or individual siRNAs targeting FASN (150 pmol), referred to as siRNA1, siRNA2, siRNA3, and siRNA4, respectively. At 48 h after transfection, cell lysates were prepared and subjected to Western blot analysis to detect FASN protein levels using an antibody against FASN (Proteintech, cat# 10624-2-AP, 1:10,000). ( C and E ) MDBK cells in six-well plates were transfected with either scrambled siRNA (150 pmol) or the indicated siRNAs (150 pmol). At 36 hpi, the cells were infected with BoAHV-1 at an MOI of 1. After 24 h of infection, the cells were collected and subjected to Western blot analysis using antibodies against FASN (Proteintech, cat# 10624-2-AP, 1:10,000) ( C ) and virus-associated proteins (VMRD, cat# P170703-001, 1:5,000) ( E ). ( B, D, and F ) The band intensities were quantified using the free software Image J. The intensity of each band was first normalized to that of the respective loading control Tubulin, and then normalized to that of the control transfected with scramble siRNA, which was arbitrarily set to 100%. ( G ) MDBK cells in six-well plates were transfected with either scrambled siRNA (150 pmol) or the indicated siRNAs (150 pmol). At 36 h post-transfection, the cells were infected with the virus at an MOI of 1 for 24 h. Subsequently, total RNA was extracted from the cells for the detection of viral mRNA using RT-qPCR. Primers specific for bICP4, bICP27, viral DNA polymerase, and viral protein gC were used, respectively. The data shown are means of three independent experiments, with error bars representing standard deviations. Significance was assessed using a Student’s t -test (ns, not significant; * P < 0.05; ** P < 0.01; *** P < 0.001).

Article Snippet: FASN polyclonal antibody (pAb) (cat#10624-2-AP) and GP73 mouse monoclonal antibody (mAb) (cat# 66331-1-lg) were provided by Proteintech (Rosemont, IL, USA).

Techniques: Infection, Transfection, Western Blot, Virus, Software, Control, Quantitative RT-PCR

Journal: Microbiology Spectrum

Article Title: Fatty acid synthase may facilitate the trafficking of bovine alpha herpesvirus 1 out of the Golgi apparatus, potentially promoting viral infection

doi: 10.1128/spectrum.01388-25

Figure Lengend Snippet: Determine the roles of FASN in BoAHV-1 productive infection in MDBK cells using a chemical inhibitor. ( A and C ) MDBK cells in six-well plates were infected with BoAHV-1 (MOI = 1) and treated either with DMSO control or with the FASN-specific inhibitor Cerulenin at the indicated concentrations. At 24 hpi, the cells were collected either to prepare cell lysates for Western blotting using an antibody against virus-associated proteins (VMRD, cat# P170703-001, 1:5,000) ( A ) or to extract DNA for subsequent qPCR analysis using gB-specific primers ( C ). ( B ) The intensity of bands for virus-associated proteins was quantified using the freeware software Image J. Changes in the levels of individual bands upon treatment with the inhibitor were calculated relative to those of the DMSO control, which was set at 100%. ( D ) Virus titers in the supernatants were determined and expressed as TCID 50 /mL. ( E ) MDBK cells in six-well plates were infected with the virus at an MOI of 1 for 24 h and treated with either DMSO control or 10 µM Cerulenin. Total RNA was then extracted from the cells for the detection of viral mRNA using RT-qPCR. Primers specific for bICP27, viral DNA polymerase, and viral protein gC were used, respectively. The data shown are means of three independent experiments, with error bars representing standard deviations. Significance was assessed by a standard t -test (ns, not significant; * P < 0.05; ** P < 0.01; *** P < 0.001).

Article Snippet: FASN polyclonal antibody (pAb) (cat#10624-2-AP) and GP73 mouse monoclonal antibody (mAb) (cat# 66331-1-lg) were provided by Proteintech (Rosemont, IL, USA).

Techniques: Infection, Control, Western Blot, Virus, Software, Quantitative RT-PCR

Journal: Microbiology Spectrum

Article Title: Fatty acid synthase may facilitate the trafficking of bovine alpha herpesvirus 1 out of the Golgi apparatus, potentially promoting viral infection

doi: 10.1128/spectrum.01388-25

Figure Lengend Snippet: Determine the roles of FASN in BoAHV-1 productive infection in Neuro-2A cells using both siRNA and chemical inhibitor. ( A ) Neuro-2A cells in six-well plates were transfected with either scrambled siRNA (150 pmol) or individual siRNAs targeting FASN (150 pmol), referred to as siRNA1, siRNA2, siRNA3, and siRNA4, respectively. At 48 h post-transfection, cell lysates were prepared and subjected to Western blot analysis to detect FASN protein levels using an antibody against FASN (Proteintech, cat# 10624-2-AP, 1:10,000). ( C ) Neuro-2A cells in six-well plates were transfected with either scrambled siRNA (150 pmol) or siRNA1 (150 pmol). At 36 h post-transfection, the cells were infected with BoAHV-1 at an MOI of 10. After 24 h of infection, the cells were collected and subjected to Western blot analysis using a monoclonal antibody against viral protein gC (VMRD, cat#F2, 1:2,000). ( E ) Neuro-2A cells in six-well plates were infected with BoAHV-1 (MOI = 10) and treated either with DMSO control or with the FASN-specific inhibitor Cerulenin at the indicated concentrations. At 24 hpi, the cell lysates were prepared and subjected to Western blotting to detect viral protein gC. ( B, D, and F ) The band intensities were quantified using the free software Image J. The intensity of each band was first normalized to that of the respective loading control Tubulin, and then normalized to that of either DMSO-treated or scrambled siRNA-transfected control, which was arbitrarily set to 100%. The data shown are means of three independent experiments, with error bars representing standard deviations. Significance was assessed using a Student’s t -test (ns, not significant; * P < 0.05; ** P < 0.01).

Article Snippet: FASN polyclonal antibody (pAb) (cat#10624-2-AP) and GP73 mouse monoclonal antibody (mAb) (cat# 66331-1-lg) were provided by Proteintech (Rosemont, IL, USA).

Techniques: Infection, Transfection, Western Blot, Control, Software

Journal: Microbiology Spectrum

Article Title: Fatty acid synthase may facilitate the trafficking of bovine alpha herpesvirus 1 out of the Golgi apparatus, potentially promoting viral infection

doi: 10.1128/spectrum.01388-25

Figure Lengend Snippet: Determine the effects of FASN inhibitor, Cerulenin, on the accumulation of gD in the Golgi apparatus. ( A ) Diagram shows the treatment manner of virus-infected cells by both Cerulenin and Monensin. ( B ) MDBK cells of either mock-infected or virus-infected were treated either with vehicle control DMSO or with Cerulenin (10 µM) or Monensin (10 µM) for a duration of 4 h prior to the termination of infection. At 24 hpi, the cells were collected to purify the Golgi apparatus using a commercial purification kit (Beijing Biolabo Technology, cat# HR0247-50T). Subsequently, Western blot analysis was performed to detect the protein levels of gD in the Golgi fractions. GOLGA1, a marker of the Golgi apparatus, was used as a loading control and for subsequent quantitative analysis. ( C ) The band intensity was analyzed with the free software Image J. The intensity of each band was first normalized to that of the respective loading control GOLGA1, and then normalized to that of the control treated with DMSO, which was arbitrarily set to 1. The data shown are means of three independent experiments with error bars indicating standard deviations. Significance was assessed with a Student’s t -test (* P < 0.05, ** P < 0.01).

Article Snippet: FASN polyclonal antibody (pAb) (cat#10624-2-AP) and GP73 mouse monoclonal antibody (mAb) (cat# 66331-1-lg) were provided by Proteintech (Rosemont, IL, USA).

Techniques: Virus, Infection, Control, Purification, Western Blot, Marker, Software