Journal:

Article Title: Brain Lipid Binding Protein in Axon-Schwann Cell Interactions and Peripheral Nerve Tumorigenesis

doi: 10.1128/MCB.23.6.2213-2224.2003

Figure Lengend Snippet: mRNA expression analysis in Nf1 mutant mouse Schwann cells. (A) Microarray analysis was used to compare genome-wide expression levels between normal mouse Schwann cells and Nf1 mutant Schwann cells. The control for each comparison was Cy3-labeled cDNA generated from normal mouse Schwann cell mRNA. For each of four Nf1 mutant Schwann cell samples (Nf1+/−, Nf1−/−, Nf1−/− TXF, and Nf1−/− TXF treated with FTI), mRNA was used as a template to synthesize Cy5-labeled cDNA. Cy3- and Cy5-labeled cDNA probes were hybridized simultaneously to the Incyte Genomics MouseGEM 1.0 cDNA microarray. Relative intensities of Cy3 versus Cy5 fluorescent signals for each cDNA target sequence were analyzed with GeneSpring software. The most changes were observed in the Nf1−/− TXF cells (genes upregulated in Nf1−/− TXF are red; genes downregulated in Nf1−/− TXF are green). Expression of one target cDNA, BLBP (black line), was 26-fold above normal in the Nf1−/− TXF cells and not normalized by FTI treatment. (B) RT-PCR analysis confirmedthe microarray result of elevated BLBP expression in Nf1−/− TXF cells. Reverse transcriptase (RT) was omitted from duplicate samples to control for DNA contamination. Primers for BLBP (∼200-bp amplicon) and actin control primers (∼500-bp amplicon) were included in the mixture for each 40-cycle reaction. The plasmid positive control for BLBP amplification is the UniGEM clone (Incyte Genomics) containing the BLBP cDNA insert spotted on the microarray. (C) Quantitative real-time PCR of BLBP normalized to GAPDH resulted in a 145-fold change over expression in Nf1−/− TXF cells compared to wild-type mouse Schwann cells. Rn, fluorescent signal intensity; horizontal starred line, chosen threshold at geometric phase of amplification.

Article Snippet: In DRGN cocultures, neurofilament was visualized by incubation with anti-NF-15g1 antibodies ( 38 ) diluted 1:5, followed by donkey anti-mouse Cy3-labeled secondary antibodies (Jackson Immunoresearch).

Techniques: Expressing, Mutagenesis, Microarray, Genome Wide, Labeling, Generated, Sequencing, Software, Reverse Transcription Polymerase Chain Reaction, Amplification, Plasmid Preparation, Positive Control, Real-time Polymerase Chain Reaction, Over Expression

Journal:

Article Title: Brain Lipid Binding Protein in Axon-Schwann Cell Interactions and Peripheral Nerve Tumorigenesis

doi: 10.1128/MCB.23.6.2213-2224.2003

Figure Lengend Snippet: Mouse neuron-Schwann cell coculture. Anti-BLBP promotes extension of Nf1−/− TXF cell processes along axons. Wild-type (A and B) or Nf1−/− TXF (C and D) mouse Schwann cells labeled with Cell Tracker green were preincubated with rabbit IgG (A and C) or anti-BLBP antibodies (B and D) and seeded onto DRGN cultures stripped of endogenous Schwann cells. Two days after seeding, cocultures were fixed and stained with antineurofilament antibodies followed by Cy3 (red)-conjugated secondary antibodies. Confocal images obtained with Zeiss LSM Image Browser software are shown. Single cells are representative of the majority observed with each treatment. Arrowheads indicate Schwann cell processes. The asterisk indicates the region which is magnified fivefold in the inset. The scale bar in panel C equals 5 μm and also applies to panels A, B, and D. (E) Lower magnification (scale bar, 5 μm) of Nf1−/− TXF on DRGN cultures in the presence of anti-BLBP antibodies. Arrowheads indicate processes from two cells extending along neurites; other cells lack processes. (F) Extension of Nf1−/− TXF cell processes in the presence of anti-BLBP antibodies is statistically significant. The percentages of Nf1−/− TXF cells extending processes along axons in the presence of control IgG (gray bar) or anti-BLBP antibodies (black bar) are graphed. Error bars reflect standard deviations in a Student t test (P = 0.003).

Article Snippet: In DRGN cocultures, neurofilament was visualized by incubation with anti-NF-15g1 antibodies ( 38 ) diluted 1:5, followed by donkey anti-mouse Cy3-labeled secondary antibodies (Jackson Immunoresearch).

Techniques: Labeling, Staining, Software

Journal: bioRxiv

Article Title: The emerging fungal pathogen Candida auris induces IFNγ to colonize mammalian hair follicles

doi: 10.1101/2025.01.15.632653

Figure Lengend Snippet: (A) Mice were colonized on days 0,2,4,6 with C. albicans and back skin was harvested for CFU on day 14 (8 days after the final application). Mice that lack IL-17 receptor on keratinocytes (Ker14 Cre ;IL17Ra F/F , i.e. Ker14 ΔIL17Ra ) were colonized alongside littermate controls (IL17Ra F/F lacking Cre recombinase) and IL-17-/- mice, which globally lack IL-17a and IL-17f. (B) Representative photographs of back skin from Ker14 ΔIL17Ra and littermate controls at day 14. (C) Schematic cartoon showing keratinocyte subsets. (D) From scRNAseq, keratinocytes were pre-selected and further analyzed to reveal subset clusters: hair follicle bulge, isthmus, infundibulum, basal layer, spinous layer, sebaceous gland (SG), and proliferating. (E,F) scRNAseq dot plot by (E) treatment condition or (F) cell type, showing modules upregulated following C. albicans colonization associated with epithelial stem cells, differentiation, migration and cornification associated modules and anti-Candida defenses. (G) Schematic showing experimental design where Lrig1 CreERT ; R26 Tdt mice were injected with tamoxifen 1 day prior to mock, C. auris or C. albicans colonization. Mice were harvested for confocal microscopy on day 5 post colonization. (H) Representative image from slices of 3D image showing lineage traced Lrig1+ cells 1-day post tamoxifen (I) Top panel showing representative 3D image of lineage traced Lrig1+ cells 5 days post-colonization with mock, C. auris, or C. albicans . Bottom panel showing overlapping Lrig1 and CD49f epithelial surface, using Imaris imaging software. (J) Graphical representation of overlapping Lrig1 + surface with the CD49f + epithelia surface subdivided into the hair follicle and interfollicular epidermis, using Imaris imaging software. (K) Schematic showing proposed C. albicans induced changes to keratinocytes. *P<0.05, **P<0.01, ****P<0.0001 for one-way analysis of variance (ANOVA) and Tukey’s multiple comparison test (A,I).

Article Snippet: To generate keratinocyte specific knock out of IL-17 and IFNγ signaling, we crossed Ker14 Cre mice (B6N.Cg-Tg(KRT14-cre)1Amc/J) from Jackson with Il17Ra floxed mice (B6.Cg-Il17ratm2.1Koll/J; MGI:J:237978) and IFNγR1 floxed mice (C57BL/6N-Ifngr1tm1.1Rds/J, MGI:J:206257) from Jackson Labs. To track type 1 lymphocytes we used Tbet (Tbx21)-zsGreen transgenic mice (generously provided by Jinfang Zhu, Lab of Immune System Biology, NIH).

Techniques: Migration, Injection, Confocal Microscopy, Imaging, Software, Comparison

Journal: bioRxiv

Article Title: The emerging fungal pathogen Candida auris induces IFNγ to colonize mammalian hair follicles

doi: 10.1101/2025.01.15.632653

Figure Lengend Snippet: (A-C) scRNAseq dot plot of epithelial cells separated by (A) fungal colonization type or (B) epithelial cell type or (C) IFNγ -/- , WT, IL-17 -/- , and IFNγ OE genotype. Mice were treated with Candida spp indicated on days 0,2,4,6 and back skin was harvested on day 14. (D) Representative 3D confocal microscopy of Stat1 expression in the upper hair follicle in WT mice treated with either mock colonization or C. auris colonization on day 14. (E) Correlation between Stat1 expression, assessed via mean fluorescence index (MFI), and CD8 T cells per hair follicle. (F) Quantitation of Stat1 mean fluorescence intensity (MFI) per hair follicle in mice indicated. WT C. auris colonized mice were subdivided based on having less than or greater than 15 CD8 T-cells per hair follicle. (G) Stat1 MFI in the epithelial surface (CD49f) compared to all other skin structures (non-CD49f). (H) Stat1 MFI between the interfollicular epithelium (IFE), upper hair follicle, and lower hair follicle in mock colonized (white) and C. auris colonized (red) mice. (I,J) Schematics showing models used to test colonization and epicutaneous infection. For epicutaneous infection, intact back skin was pre-colonized with C. auris on day 0,2,4,6; on day 14, hair was removed, and the skin was abraded with sandpaper. C. auris was reapplied on day 15 and back skin was harvested 2 days later for fungal burden. For ‘Re-Colonization’, intact back skin was pre-colonized with C. auris on days 0,2,4,6 and intact back skin was re-colonized on day 14. Back skin was harvested for fungal CFU 5 days after final colonization. Graph showing fungal burden (CFU) after C. auris (I) epicutaneous infection and (J) recolonization of B6 wild type (WT), IFNγ -/- , IL-17 -/- mice, IFNγ OE mice (Yeti/+), and Ker14 ΔIFNγR1 (Ker14 Cre ;IFNγR1 Flox ). (K) IFNγ overexpressing mice ( IFNγ OE ) colonized with C. auris , at day 14 with numerous yeast forms at the hair follicle orifice. *P<0.05, ****P<0.0001 for one-way analysis of variance (ANOVA) and Tukey’s multiple comparison test (F) . In (E), statistics shown comparing C. auris <15 CD8 per hair follicle to all other groups. **P<0.01, ***P<0.001 for Kruskal-Wallis test with Dunn’s multiple comparison test (I,J) . *P<0.05, **P<0.01 student T-Test (G,H). *P<0.05, **P<0.01 Mann-Whitney Test (I,J) .

Article Snippet: To generate keratinocyte specific knock out of IL-17 and IFNγ signaling, we crossed Ker14 Cre mice (B6N.Cg-Tg(KRT14-cre)1Amc/J) from Jackson with Il17Ra floxed mice (B6.Cg-Il17ratm2.1Koll/J; MGI:J:237978) and IFNγR1 floxed mice (C57BL/6N-Ifngr1tm1.1Rds/J, MGI:J:206257) from Jackson Labs. To track type 1 lymphocytes we used Tbet (Tbx21)-zsGreen transgenic mice (generously provided by Jinfang Zhu, Lab of Immune System Biology, NIH).

Techniques: Confocal Microscopy, Expressing, Fluorescence, Quantitation Assay, Infection, Comparison, MANN-WHITNEY