Journal: Nature Communications

Article Title: Increased SOAT2 expression in aged regulatory T cells is associated with altered cholesterol metabolism and reduced anti-tumor immunity

doi: 10.1038/s41467-025-56002-w

Figure Lengend Snippet: A Schematic design for screening the old mice with SOAT2 high or SOAT2 low , and the young mice with SOAT2 low (Created in BioRender. Major, Z. (2023) https://BioRender.com/l79u279 ). B The mRNA expression of SOAT2 in the young and old mice PBMCs was monitored by quantitative RT-PCR using gene-specific primers and probes ( n = 20). C , D The protein expression of SOAT2 in the young and old mice PBMCs was monitored by western blotting analysis ( C ); quantitation of SOAT2 protein concentrations was shown ( n = 20) ( D ). E – G The KLN-205 cells allograft-bearing model ( n = 5). The tumor growth curves ( E ), representative images of subcutaneous tumors ( F ), and tumor weight ( G ) in indicated groups. H Representative immunohistochemical staining of ki67, SOAT2, FOXP3, and CD8 in the tumor nodules in indicated groups. scale bars: 2000 and 50 μm. I , J quantitation of ki67 ( I ); the correlation of SOAT2 with ki67 expression was shown ( n = 5) ( J ). K – M Quantitation of FOXP3 integratedoption density (IntDen) and FOXP3-positive cells ( K ); the correlation of FOXP3-IntD and FOXP3-positive cells with SOAT2 expression were shown ( n = 5) ( L , M ). N , O Quantitation of FOXP3 average optical density (AOD) ( N ); the correlation of FOXP3-AOD with SOAT2 expression was shown ( n = 5) ( O ). P – R Quantitation of CD8-IntDen and FOXP3-positive cells ( P ); the correlation of CD8-IntD and CD8-positive cells with SOAT2 expression were shown ( n = 5) ( Q , R ). S , T Quantitation of CD8 average optical density (AOD) (S); the correlation of FOXP3-AOD with SOAT2 expression were shown ( n = 5) (T). Data represented means ± SD. Statistical difference was evaluated by unpaired two-tailed student’s t -test ( B , D ), and one-way ANOVA test ( E , G , I , K , N , P , S ). Source data are provided as a Source Data file. (** P < 0.01; *** P < 0.001; ns no significance).

Article Snippet: For human samples, single-cell suspensions from human peripheral blood were gathered and extracted with CD4 + CD25 + CD45RA + Regulatory T Cell Isolation Kit (#130-093-631, Miltenyi Biotec) or CD8 + CD45RA + Effector T Cell Isolation Kit (#130-094-485, Miltenyi Biotec).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Quantitation Assay, Immunohistochemical staining, Staining, Two Tailed Test

Journal: Nature Communications

Article Title: Increased SOAT2 expression in aged regulatory T cells is associated with altered cholesterol metabolism and reduced anti-tumor immunity

doi: 10.1038/s41467-025-56002-w

Figure Lengend Snippet: A – C Treg cells were co-cultured with CFSE-labeled CD8 + T lymphocytes at a 0.2:1 ratio in indicated groups, and CD8 + T lymphocytes proliferation expansion was detected by flow cytometry assay ( A ); quantitation of CFSE Low (%) was shown ( n = 3) ( B , C ). D – I Treg cells were co-cultured with CD8 + T lymphocytes at a 0.2:1 ratio in indicated groups, and the release of CD8 + T lymphocytes for granzyme B and perforin were detected by ELISPOT assay ( D , G ); quantitation of granzyme B ( E , H ) and perforin ( F , I ) were shown (biological replicate, n = 3). J , K Treg cells were co-cultured with CD8 + T lymphocytes at a 0.2:1 ratio in indicated groups, and the release of CD8 + T lymphocytes for granzyme B and perforin were detected by ELISA assay; quantitation of granzyme B and perforin were shown ( n = 3). L , M Treg cells were co-cultured with CD8 + T lymphocytes at a 0.2:1 ratio in indicated groups, and Teff-mediated cytoxicity was determined by LDH release assay using a cytotoxicity detection kit ( n = 5). Data represented means ± SD. Statistical difference was evaluated by one-way ANOVA test ( B , C , E , F , H , I , J , K , L , M ). Source data are provided as a Source Data file. (* P < 0.05; ** P < 0.01; *** P < 0.001).

Article Snippet: For human samples, single-cell suspensions from human peripheral blood were gathered and extracted with CD4 + CD25 + CD45RA + Regulatory T Cell Isolation Kit (#130-093-631, Miltenyi Biotec) or CD8 + CD45RA + Effector T Cell Isolation Kit (#130-094-485, Miltenyi Biotec).

Techniques: Cell Culture, Labeling, Flow Cytometry, Quantitation Assay, Enzyme-linked Immunospot, Enzyme-linked Immunosorbent Assay, Lactate Dehydrogenase Assay

Journal: Nature Communications

Article Title: Increased SOAT2 expression in aged regulatory T cells is associated with altered cholesterol metabolism and reduced anti-tumor immunity

doi: 10.1038/s41467-025-56002-w

Figure Lengend Snippet: A – C Representative images of Teffs stained for β-galactosidase (arrows) after co-cultured with Treg cells ( n = 3) ( A ), scale bars: 50 μm; quantitation of β-galactosidase-positive Teffs were shown ( B , C ). D – F The protein expression of P16, P21, P53 in Teff cells in the young and old individuals were monitored by western blotting analysis in indicated groups. The experiment was repeated 3 times independently with similar results. G – J , Treg cells were co-cultured with CD8 + T lymphocytes at a 0.2:1 ratio in indicated groups, and the expression of CD27 or CD28 in human Teffs ( G , H ) and murine Teffs ( I , J ) were detected by flow cytometric analysis ( n = 3). K Schematic representation of Treg cells exerts immunosuppressive path on Teffs (Created in BioRender. Major, Z. (2024) https://BioRender.com/s68z662 ). L , M The content of cholesterol components were measured by the Cholesterol Assay Kit in indicated groups ( n = 5). N – P Representative images of Teffs stained for β-galactosidase (arrows) after co-cultured with Treg cells (N), scale bars: 50 μm; quantitation of β-galactosidase-positive Teffs were shown ( n = 3) ( O , P ). Data represented means ± SD. Statistical difference was evaluated by one-way ANOVA test ( B , C , L , M , O , P ). Source data are provided as a Source Data file. (* P < 0.05; ** P < 0.01; *** P < 0.001).

Article Snippet: For human samples, single-cell suspensions from human peripheral blood were gathered and extracted with CD4 + CD25 + CD45RA + Regulatory T Cell Isolation Kit (#130-093-631, Miltenyi Biotec) or CD8 + CD45RA + Effector T Cell Isolation Kit (#130-094-485, Miltenyi Biotec).

Techniques: Staining, Cell Culture, Quantitation Assay, Expressing, Western Blot, Cholesterol Assay

Journal: Frontiers in Pharmacology

Article Title: Hu-lu-su-pian ameliorates hepatic steatosis by regulating CIDEA expression in AKT-driven MASLD mice

doi: 10.3389/fphar.2024.1503247

Figure Lengend Snippet: HLSP inhibits the CIDEA/FASN/ACC pathway in vitro . (A) HepG2 and Huh-7 cells were transfected with the CIDEA plasmid to overexpress CIDEA and treated with HLSP for 24 h, and then the expression of CIDEA and lipid droplets were detected. (B) Representative oil red O-staining images of treated HepG2 and Huh-7 cells as shown in (A) . (C) Relative mRNA expression levels of lipid metabolism genes (CIDEA, FASN, and ACC) in HepG2 and Huh-7 cells treated as indicated in (A) . (D, E) Protein expression levels of lipid metabolism genes (CIDEA, FASN, and ACC) in HepG2 and Huh-7 cells treated as indicated in (A) . The results are shown in the following table. Mean ± SD; ## P < 0.01 and ### P < 0.001 versus the WT group and * P < 0.05, ** P < 0.01, and *** P < 0.001 versus the AKT group.

Article Snippet: The CIDEA plasmid was produced by OriGene (Rockville, MD, USA) and its sequence can be accessed at https://www.origene.com.cn/drawmapbysku?SKU=RC205790 .

Techniques: In Vitro, Transfection, Plasmid Preparation, Expressing, Staining

Journal: Glia

Article Title: CDC42 ‐Effector Proteins Regulate Higher Order Structure of Septins Required for CNS Myelin Integrity

doi: 10.1002/glia.70134

Figure Lengend Snippet: Septins, CDC42, and CDC42‐effector proteins CDC42EP1/CDC42EP2 in oligodendrocytes and myelin. (A, A′) Representative electron micrographs of optic nerves cross‐sectioned at P75 show that mice lacking Septin8 (A′) but not control (A) mice develop pathological myelin outfoldings in the CNS, in agreement with prior quantitative assessment (Patzig et al. ). Myelin outfolding highlighted in red in (A′); asterisk indicates an axon associated with a myelin outfolding. TEM, transmission electron microscopy. (B) Violin plot of the abundance of the indicated transcripts in mature oligodendrocytes (MOL). Re‐analysis of previously published scRNA‐seq data ( GSE75330 ; Marques et al. ). Each datapoint represents one out of 998 cells designated as mature oligodendrocytes (MOL; clusters MOL1‐MOL6 in Marques et al. ) in C57Bl/6 mice. Note that Cdc42 , Cdc42ep1 , and Cdc42ep2 mRNAs are abundant in MOL when compared to Cdc42ep3 , Cdc42ep4 , and Cdc42ep5 mRNAs. For bulk RNA‐seq data of oligodendrocytes immunopanned from mouse cortices (Zhang et al. ), see Figure . (C) Protocol of myelin purification via sucrose density gradient centrifugation and osmotic shocks. For details see (Erwig et al. ). (D) Immunoblots detecting CDC42, CDC42EP1, CDC42EP2, and all myelin septin filament subunits (SEPTIN2, SEPTIN4, SEPTIN7, SEPTIN8) in myelin purified from brains of C57B/6 N‐mice at postnatal day 20 (P20), P45, and P76. Fast Green serves as loading control. n = 2 mice per age. (E) Immunoblotting reveals reduced abundance of CDC42EP1 and CDC42EP2 and validates diminishment of myelin septins in myelin purified from brains of Septin8 −/− mice at P75 compared to controls (Ctrl). Fast Green as loading control. n = 3 mice per genotype.

Article Snippet: Primary antibodies were specific for CDC42 (Santa Cruz, 1:500, #sc‐8401), CDC42EP1 (custom‐made rabbit polyclonal antibody against mouse CDC42EP1 epitope VEKHSNRDRDRDPDH, Pineda, 1:1000), CDC42EP2 (Proteintech Group, 1:300, #11824‐1‐AP), SEPTIN2 (Proteintech Group, 1:500, #11397‐1‐AP), SEPTIN4 (IBL, 1:200, #JP18987), SEPTIN7 (IBL, 1:5000, #18991), SEPTIN8 (Proteintech Group, 1:500, #11769‐1‐AP), or ATP1a3 (Abcam, 1:1000, #ab182571).

Techniques: Control, Transmission Assay, Electron Microscopy, RNA Sequencing, Purification, Gradient Centrifugation, Western Blot

Journal: Glia

Article Title: CDC42 ‐Effector Proteins Regulate Higher Order Structure of Septins Required for CNS Myelin Integrity

doi: 10.1002/glia.70134

Figure Lengend Snippet: Cdc42 ‐deletion in oligodendrocytes of adult mice impairs myelin structure and alters protein composition. (A–F) Representative electron micrographs (A, A′) and genotype‐dependent quantification (B–F) of cross‐sectioned optic nerves showing myelin pathology in Cdc42 flox/flox ; Plp CreERT2 (icKO) compared to control (Ctrl) mice 4 months post tamoxifen injection (mo PTI). (A, A′) Myelin pathology highlighted in red; asterisks indicate associated axons. (B) Quantitative analysis of electron micrographs of optic nerves 4, 8, and 10 months PTI reveals normal axon density in Cdc42 ‐icKO mice. Mean ± SEM; datapoints represent individual mice; n = 3–5 mice; multiple unpaired t‐test with Holm‐Šídák correction (4 months PTI p = 0.441, 8 months PTI p = 0.282, 10 months PTI p = 0.875). C Quantitative analysis shows moderately but significantly reduced percentage of myelinated axons in Cdc42 ‐icKO mice 8 months PTI but not 4 or 10 months PTI. Mean ± SEM; datapoints represent individual mice; n = 3–5 mice; multiple unpaired t‐test with Holm‐Šídák correction (4 months PTI p = 0.400, 8 months PTI p = 0.005, 10 months PTI p = 0.537). D Quantitative analysis identifies increased percentage of axon/myelin‐units with myelin outfoldings in Cdc42 ‐icKO mice. Mean ± SEM; datapoints represent individual mice; n = 3–5 mice; multiple unpaired t‐test with Holm‐Šídák correction (4 months PTI p = 0.0135, 8 months PTI p = 0.0163, 10 months PTI p = 0.0005). (E) Quantitative analysis reveals increased percentage of axon/myelin‐units with myelin whorls in Cdc42 ‐icKO mice. Mean ± SEM; datapoints represent individual mice; n = 3–5 mice; multiple unpaired t test with Holm‐Šídák correction (4 months PTI p = 0.003, 8 months PTI p = 0.015, 10 months PTI p = 0.002). F Quantitative analysis shows increased percentage of axon/myelin‐units that display other pathology 8 and 10 months PTI. Mean ± SEM; datapoints represent individual mice; n = 3–5 mice; multiple unpaired t test with Holm‐Šídák correction (4 months PTI p = 0.263, 8 months PTI p = 0.017, 10 months PTI p = 0.0004). (G, H) Differential proteome analysis comparing the relative abundance of proteins in myelin purified from brains of Cdc42 ‐icKO and control (Crtrl) mice 10 months PTI. (G) Heatmap shows mass spectrometric quantification of known myelin constituents in three biological replicates (M1, M2, and M3) as the average of two technical replicates each, compared to the mean of Ctrl. Note that CDC42, myelin septins (SEPTIN2, SEPTIN4, SEPTIN7, SEPTIN8), and the septin‐associated adaptor protein anillin (ANLN) are diminished in myelin when oligodendrocytes lack Cdc42 . (H, H′) Volcano plots summarizing genotype‐dependent quantitative myelin proteome analysis. Data points represent relative abundance of proteins quantified in myelin of Cdc42 ‐icKO compared to Ctrl mice 10 months PTI. Data points are plotted as log2‐transformed fold‐change on the x‐axis against the −log10‐transformed q value on the y‐axis according to two different data acquisition modes (see Methods section for details) that is, MS E (H; 391 proteins) and UDMS E (B′; 535 proteins). The vertical stippled lines mark a log 2 ‐fold change of 0.5 or −0.5 threshold of changed protein abundance in myelin, and the horizontal stippled line indicates a −log 10 ‐transformed q‐value of 1.3 as significance threshold. Data points representing myelin septin subunits (SEPT2, SEPT4, SEPT7, and SEPT8) and CDC42 are highlighted in red with protein names given; their abundance is strongly reduced in Cdc42 ‐icKO compared to Ctrl myelin. Note that CDC42EP1 and CDC42EP2 are not identified by mass spectrometry in the present dataset. For dataset and exact q values see data Table . (I) Immunoblotting validates reduced abundance of CDC42 and myelin septins and reveals diminishment of CDC42EP1 and CDC42EP2 in myelin purified from Cdc42 ‐icKO mice 10months PTI. Fast Green as loading control. Blots show n = 3 mice per genotype.

Article Snippet: Primary antibodies were specific for CDC42 (Santa Cruz, 1:500, #sc‐8401), CDC42EP1 (custom‐made rabbit polyclonal antibody against mouse CDC42EP1 epitope VEKHSNRDRDRDPDH, Pineda, 1:1000), CDC42EP2 (Proteintech Group, 1:300, #11824‐1‐AP), SEPTIN2 (Proteintech Group, 1:500, #11397‐1‐AP), SEPTIN4 (IBL, 1:200, #JP18987), SEPTIN7 (IBL, 1:5000, #18991), SEPTIN8 (Proteintech Group, 1:500, #11769‐1‐AP), or ATP1a3 (Abcam, 1:1000, #ab182571).

Techniques: Control, Injection, Purification, Transformation Assay, Quantitative Proteomics, Mass Spectrometry, Western Blot

Journal: Glia

Article Title: CDC42 ‐Effector Proteins Regulate Higher Order Structure of Septins Required for CNS Myelin Integrity

doi: 10.1002/glia.70134

Figure Lengend Snippet: Myelination in mice upon deletion of Cdc42ep1 , Cdc42ep2 , or both genes in oligodendrocytes. (A–C) qRT‐PCR to determine the abundance of Cdc42 , Cdc42ep1 , and Cdc42ep2 mRNAs in the white matter (corpora callosa) of Cdc42ep1 flox/flox ; Cnp Cre/wt (EP1 cKO, A), Cdc42ep2 flox/flox ; Cnp Cre/wt (EP2 cKO, B), Cdc42ep1 flox/flox ; Cdc42ep2 flox/flox ; Cnp Cre/wt (dcKO, C), and respective control (Ctrl) mice at age 20 weeks. Cdc42ep1 ‐mRNA was strongly reduced in EP1‐cKO and dcKO mice, Cdc42ep2 ‐mRNA was strongly reduced in EP2‐cKO and dcKO mice, while the abundance of Cdc42 ‐mRNA was unaltered. Mean ± SEM; data points represent individual mice; n = 4–5 mice. Data passed the Shapiro–Wilk test for normality and multiple unpaired t‐tests with parametric design; no assumption about consistent standard deviation. A Cdc42ep1 p = 0.0008, Cdc42ep2 p = 0.282, Cdc42 p = 0.246. (B) Cdc42ep1 p = 0.310, Cdc42ep2 p = 0.00009, Cdc42 p = 0.626. C Cdc42ep1 p = 0.000001, Cdc42ep2 p = 0.0008, Cdc42 p = 0.284. (D) Immunoblotting shows that CDC42EP1 is virtually undetectable in myelin purified from brains of EP1‐cKO or dcKO mice, and that CDC42EP2 is virtually undetectable in myelin purified from brains of EP2‐cKO or dcKO mice. Na + /K + ‐transporting ATPase subunit‐α3 (ATP1a3) serves as loading control. Age P75; blots show n = 2 mice per genotype. (E–N) Representative electron micrographs (E–H) and genotype‐dependent quantification (I–N) of cross‐sectioned optic nerves reveal myelin outfoldings as the main pathology in Cdc42ep1 flox/flox ; Cdc42ep2 flox/flox ; Cnp Cre/wt (dcKO) mice at age 20 weeks. (E–H) Myelin outfoldings highlighted in red; asterisks indicate associated axons. I Quantification shows unchanged density of axons. Mean ± SEM; datapoints represent individual mice; n = 5–6 mice; one‐way ANOVA (Ctrl vs. EP1 cKO p = 0.994; Ctrl vs. EP2 cKO p = 0.805; Ctrl vs. dcKO p = 0.367). J Quantification reveals unchanged percentage of myelinated axons. Mean ± SEM; datapoints represent individual mice; n = 5–6 mice; one‐way ANOVA (Ctrl vs. EP1 cKO p = 0.486; Ctrl vs. EP2 cKO p = 0.945; Ctrl vs. dcKO p = 0.916). (K–K′) g‐ratio analysis indicates unchanged myelin sheath thickness. Datapoints represent axon/myelin‐units (K) or individual mice (K′); n = 3 mice; mean ± SEM (K′); one‐way ANOVA (Ctrl vs. EP1 cKO p = 0.726; Ctrl versus EP2 cKO p = 0.156; Ctrl vs. dcKO p > 0.999). (L) Quantification reveals increased percentage of axon/myelin‐units with myelin outfoldings in EP1 cKO and dcKO mice. n = 5–6 mice; mean ± SEM; one‐way ANOVA (Ctrl vs. EP1 cKO p = 0.044; Ctrl vs. EP2 cKO p = 0.372; Ctrl versus dcKO p < 0.0001). (M) Quantification shows unchanged percentage of axon/myelin‐units with myelin whorls. Mean +/− SEM; n = 5–6 mice; one‐way ANOVA (Ctrl vs. EP1 cKO p = 0.064; Ctrl vs. EP2 cKO p = 0.174; Ctrl versus dcKO p = 0.155). (N) Quantification reveals increased percentage of axon/myelin‐units displaying other pathology in dcKO mice. Mean ± SEM; datapoints represent individual mice; n = 5–6 mice; one‐way ANOVA (Ctrl vs. EP1 cKO p = 0.557; Ctrl vs. EP2 cKO p = 0.902; Ctrl vs. dcKO p = 0.006).

Article Snippet: Primary antibodies were specific for CDC42 (Santa Cruz, 1:500, #sc‐8401), CDC42EP1 (custom‐made rabbit polyclonal antibody against mouse CDC42EP1 epitope VEKHSNRDRDRDPDH, Pineda, 1:1000), CDC42EP2 (Proteintech Group, 1:300, #11824‐1‐AP), SEPTIN2 (Proteintech Group, 1:500, #11397‐1‐AP), SEPTIN4 (IBL, 1:200, #JP18987), SEPTIN7 (IBL, 1:5000, #18991), SEPTIN8 (Proteintech Group, 1:500, #11769‐1‐AP), or ATP1a3 (Abcam, 1:1000, #ab182571).

Techniques: Quantitative RT-PCR, Control, Standard Deviation, Western Blot, Purification

Journal: Glia

Article Title: CDC42 ‐Effector Proteins Regulate Higher Order Structure of Septins Required for CNS Myelin Integrity

doi: 10.1002/glia.70134

Figure Lengend Snippet: Progressive formation of myelin outfoldings when both Cdc42ep1 and Cdc42ep2 are lacking from oligodendrocytes. (A–K) Representative electron micrographs (A–F) and genotype‐dependent quantification (G–K) of cross‐sectioned optic nerves show that myelin outfoldings are the main pathology in Cdc42ep1 flox/flox ; Cdc42ep2 flox/flox ; Cnp Cre/wt (dcKO) mice. (A–F) Mice were analyzed at ages P45 (A, B), 20 weeks (C, D), and 1 year (E, F). Myelin outfoldings highlighted in red; asterisks indicate associated axons. (G) Quantification indicates unchanged density of axons. Mean ± SEM; datapoints represent individual mice; n = 3–5 mice; multiple unpaired t test with Holm‐Šídák correction (P45 p = 0.075, 20 weeks p = 0.142, 1 year p = 0.161). (H) Quantification shows unchanged percentage of myelinated axons. Mean ± SEM; datapoints represent individual mice; n = 3–5 mice; multiple unpaired t test with Holm‐Šídák correction (P45 p = 0.281, 20 weeks p = 0.393, 1 year p = 0.095). (I) Quantification reveals increased frequency of axon/myelin‐units with outfoldings in dcKO mice at ages P45, 20 weeks, and 1 year. Mean ± SEM; datapoints represent individual mice; n = 3–5 mice; multiple unpaired t‐test with Holm‐Šídák correction (P45 p = 0.001, 20 weeks p = 0.009, 1 year p = 0.019). (J) Quantification indicates unchanged percentage of axon/myelin‐units with myelin whorls. Mean ± SEM; datapoints represent individual mice; n = 3–5 mice; multiple unpaired t test with Holm‐Šídák correction (P45 p = 0.051, 20 weeks p = 0.066, 1 year p = 0.160). (K) Quantification reveals increased percentage of axon/myelin‐units with other pathology in dcKO mice at 1 year of age. Mean ± SEM; datapoints represent individual mice; n = 3–5 mice; multiple unpaired t test with Holm‐Šídák correction (P45 p = 0.261, 20 weeks p = 0.070, 1 year p = 0.013). (G‐K) Note that the data for Ctrl and dcKO at age 20 weeks are the same as in Figure and are shown here for comparability.

Article Snippet: Primary antibodies were specific for CDC42 (Santa Cruz, 1:500, #sc‐8401), CDC42EP1 (custom‐made rabbit polyclonal antibody against mouse CDC42EP1 epitope VEKHSNRDRDRDPDH, Pineda, 1:1000), CDC42EP2 (Proteintech Group, 1:300, #11824‐1‐AP), SEPTIN2 (Proteintech Group, 1:500, #11397‐1‐AP), SEPTIN4 (IBL, 1:200, #JP18987), SEPTIN7 (IBL, 1:5000, #18991), SEPTIN8 (Proteintech Group, 1:500, #11769‐1‐AP), or ATP1a3 (Abcam, 1:1000, #ab182571).

Techniques:

Journal: Glia

Article Title: CDC42 ‐Effector Proteins Regulate Higher Order Structure of Septins Required for CNS Myelin Integrity

doi: 10.1002/glia.70134

Figure Lengend Snippet: Neuropathological analysis of white matter upon oligodendroglial deletion of Cdc42ep1 , Cdc42ep2 , or both genes. (A–O) Representative light micrographs and genotype‐dependent quantification of secondary neuropathology. Shown are micrographs of a white matter tract (hippocampal fimbria) of Cdc42ep1 flox/flox ; Cdc42ep2 flox/flox ; Cnp Cre/wt (dcKO; B, E, H, K, and N) and control (Ctrl; A, D, G, J, and M) mice at age 1 year immunolabeled for the astrocyte marker GFAP (A, B), the microglia markers IBA1 (D, E) and MAC3/LAMP2 (G, H), the marker amyloid precursor protein (APP) to label axonal swellings (J,K), and the oligodendrocyte cell body marker ASPA (M,N). White arrowheads point at immunopositive cells. Scale bars 50 μm. (C) Quantification shows unchanged GFAP‐immunopositive area. Mean ± SEM; datapoints represent individual mice; n = 4 mice; one‐way ANOVA (Ctrl vs. EP1 cKO p = 0.996; Ctrl vs. EP2 cKO p = 0.820; Ctrl vs. dcKO p = 0.518). F Quantification shows unchanged IBA1‐immunopositive area. Mean +/‐SEM; datapoints represent individual mice; n = 4 mice; one‐way ANOVA (Ctrl vs. EP1 cKO p = 0.703; Ctrl vs. EP2 cKO p = 0.999; Ctrl vs. dcKO p = 0.057). I Quantification shows unchanged MAC3/LAMP2‐immunopositive area. Mean ± SEM; datapoints represent individual mice; n = 4 mice; Kruskal–Wallis test (Ctrl vs. EP1 cKO p = 247; Ctrl vs. EP2 cKO p > 0.999; Ctrl vs. dcKO p > 0.999). (L) Quantification shows unchanged number of APP‐immunopositive axonal swellings. Mean ± SEM; datapoints represent individual mice; n = 4 mice; Kruskal–Wallis test (Ctrl vs. EP1 cKO p > 0.999; Ctrl versus EP2 cKO p = 0.492; Ctrl vs. dcKO p = 0.065). (O) Quantification shows unchanged number of ASPA‐immunopositive oligodendrocytes. Mean ± SEM; datapoints represent individual mice; n = 4 mice; one‐way ANOVA (Ctrl vs. EP1 cKO p = 0.882; Ctrl vs. EP2 cKO p = 0.597; Ctrl vs. dcKO p = 0.823). (P) qRT‐PCR to determine the abundance of neuropathology associated ( Gfap , Iba1 , and Lamp2 ) and oligodendroglial ( Aspa and Mog ) transcripts in the white matter (corpora callosa) of dcKO and Ctrl mice at 20 weeks of age. Mean ± SEM; datapoints represent individual mice; n = 5 mice; data passed the Shapiro–Wilk test for normality; multiple unpaired t‐test with parametric design; no assumption about consistent SDs and p = 0.05 threshold for p value comparisons ( Gfap p = 0.977, Iba1 p = 0.405, Lamp2 p = 0.949, Aspa p = 0.1149, Mog p = 0.145). (Q–R) Representative immunofluorescence labelling of the paranode marker CASPR on longitudinal spinal cord sections of Cdc42ep1 flox/flox ; Cdc42ep2 flox/flox ; Cnp Cre/wt (dcKO) and Ctrl mice (Q) to assess the density of paranodes, and genotype‐dependent quantification (R). R Quantification shows unchanged density of CASPR‐immunopositive paranodes. Mean ± SEM; datapoints represent individual mice; n = 5 mice; unpaired t‐test (Ctrl vs. dcKO p = 0.956).

Article Snippet: Primary antibodies were specific for CDC42 (Santa Cruz, 1:500, #sc‐8401), CDC42EP1 (custom‐made rabbit polyclonal antibody against mouse CDC42EP1 epitope VEKHSNRDRDRDPDH, Pineda, 1:1000), CDC42EP2 (Proteintech Group, 1:300, #11824‐1‐AP), SEPTIN2 (Proteintech Group, 1:500, #11397‐1‐AP), SEPTIN4 (IBL, 1:200, #JP18987), SEPTIN7 (IBL, 1:5000, #18991), SEPTIN8 (Proteintech Group, 1:500, #11769‐1‐AP), or ATP1a3 (Abcam, 1:1000, #ab182571).

Techniques: Control, Immunolabeling, Marker, Quantitative RT-PCR, Immunofluorescence

Journal: Glia

Article Title: CDC42 ‐Effector Proteins Regulate Higher Order Structure of Septins Required for CNS Myelin Integrity

doi: 10.1002/glia.70134

Figure Lengend Snippet: Myelin protein composition is altered when Cdc42ep1 and Cdc42ep2 are lacking from oligodendrocytes. (A, B) Differential proteome analysis comparing the relative abundance of proteins in myelin purified from brains of Cdc42ep1 flox/flox ; Cdc42ep2 flox/flox Cnp Cre/wt (dcKO) and control (Ctrl) mice at 20 weeks of age. A heatmap shows mass spectrometric quantification of known myelin constituents in three biological replicates (M1, M2, and M3) as the average of two technical replicates each, compared to the mean of Ctrl. Note that the abundance of myelin septins (SEPTIN2, SEPTIN4, SEPTIN7, and SEPTIN8), the septin‐associated adaptor protein anillin (ANLN), and CDC42 in myelin is reduced when oligodendrocytes lack Cdc42ep1 and Cdc42ep2 . (B) Volcano plots summarizing genotype‐dependent quantitative myelin proteome analysis. Data points represent relative abundance of proteins quantified in myelin of dcKO compared to Ctrl mice. Data points are plotted as log2‐transformed fold‐change on the x‐axis against the −log10‐transformed q value on the y‐axis according to two different data acquisition modes (see Section for details) that is, MS E (B; 459 proteins) and UDMS E (B′; 728 proteins). The vertical stippled lines mark a log 2 ‐fold change of 0.5 or −0.5 threshold of changed protein abundance in myelin, and the horizontal stippled line indicates a −log 10 ‐transformed q‐value of 1.3 as significance threshold. Data points representing myelin septin subunits (SEPTIN2, SEPTIN4, SEPTIN7, SEPTIN8), ANLN, and CDC42 are highlighted in orange with protein names given; their abundance is strongly reduced in dcKO compared to Ctrl myelin. Note that CDC42EP1 is identified by mass spectrometry in EP2 cKO and Ctrl samples in the present UDMS E data set; CDC42EP2 is not identified. For dataset and exact q‐values see data Table . (C) Immunoblot validates reduced abundance of myelin septins and CDC42 and virtual absence of CDC42EP1 and CDC42EP2 in myelin purified from Cdc42ep1 flox/flox ; Cdc42ep2 flox/flox ; Cnp Cre/wt (dcKO) mice compared to control (Ctrl) mice. Fast Green serves as loading control. (D) qRT‐PCR to determine the abundance of mRNAs encoding myelin septin subunits ( Septin2 , Septin4 , Septin7 , and Septin8 ) and the septin‐associated adaptor anillin ( Anln ) in the white matter (corpora callosa) of dcKO and Ctrl mice at 26 weeks of age. Mean ± SEM; datapoints represent individual mice; n = 5 mice per genotype. Data did not pass Shapiro–Wilk test for normality; unpaired Mann–Whitney test with nonparametric design and 0.05 threshold for p value comparisons ( Septin2 p = 0.547, Septin4 p = 0.690, Septin7 p > 0.999, Septin8 p = 0.134, and Anln p = 0.547).

Article Snippet: Primary antibodies were specific for CDC42 (Santa Cruz, 1:500, #sc‐8401), CDC42EP1 (custom‐made rabbit polyclonal antibody against mouse CDC42EP1 epitope VEKHSNRDRDRDPDH, Pineda, 1:1000), CDC42EP2 (Proteintech Group, 1:300, #11824‐1‐AP), SEPTIN2 (Proteintech Group, 1:500, #11397‐1‐AP), SEPTIN4 (IBL, 1:200, #JP18987), SEPTIN7 (IBL, 1:5000, #18991), SEPTIN8 (Proteintech Group, 1:500, #11769‐1‐AP), or ATP1a3 (Abcam, 1:1000, #ab182571).

Techniques: Purification, Control, Transformation Assay, Quantitative Proteomics, Mass Spectrometry, Western Blot, Quantitative RT-PCR, MANN-WHITNEY

Journal: Glia

Article Title: CDC42 ‐Effector Proteins Regulate Higher Order Structure of Septins Required for CNS Myelin Integrity

doi: 10.1002/glia.70134

Figure Lengend Snippet: Disorganized higher order structure of myelin septin filaments when oligodendrocytes lack Cdc42ep1 and Cdc42ep2 . (A, B) Representative light micrographs of teased fibers dissected from dorsal spinal cords of Cdc42ep1 flox/flox ; Cdc42ep2 flox/flox (Ctrl, A) and Cdc42ep1 flox/flox ; Cdc42ep2 flox/flox ; Cnp Cre/wt (dcKO, B) mice at 26 weeks of age. All myelin septin subunits (SEPTIN2, SEPTIN4, SEPTIN7, and SEPTIN8) were collectively immunolabeled (shown in red) together with the axonal marker SMI312 (in blue) and imaged using a confocal microscope. Higher order septin structures of up to 12 μm length along the axon are readily detected in Ctrl (arrowheads in A), while septin‐immunopositive structures are more frequent but shorter and fragmented in dcKO mice (arrowheads in B). (C, D) Representative micrographs of selected myelin septins (SEPTIN2, SEPTIN4, and SEPTIN8) collectively immunolabeled on expanded spinal cord, imaged via multiphoton imaging, and 3‐dimensionally reconstructed. Objects are false‐colored in red, green, and blue according to their position along the z‐axis. Objects located in the same z‐plane are assigned the same color. (C, C′) Overview of myelin septin immunolabeling in expanded Ctrl spinal cord (C) and magnified view of the boxed region (C′). (D, D′) Overview of myelin septin immunolabeling in expanded dcKO spinal cord (D) and magnified view of the boxed region (D′). Note that higher order septin structures are readily detected in Ctrl (C′). In dcKO mice, septin‐immunopositive structures are more frequent but shorter and fragmented (D′). (E, G) Representative micrographs and schematics of myelin septins (SEPTIN2, SEPTIN4, SEPTIN8) collectively immunolabeled on cross‐sectioned spinal cord, superresolution imaging at 100× magnification (E), and genotype‐dependent quantification (F, G). Myelin septins are shown in magenta (arrows pointing at puncta in E), the marker for compact myelin PLP is in blue (outlined by stippled lines in E). (F) Genotype‐dependent quantification of the relative frequency of distinct septin puncta per axon/myelin‐profile indicate a shift towards a larger number of myelin septin puncta in control (gray) compared to dcKO (green) spinal cord. Ninety‐two axon/myelin profiles from five control mice and 100 axon/myelin profiles from five dcKO mice; number of myelin septin puncta by Chi‐squared test p = 6.151e−09. (G) Quantification of the mean signal area covered by septin puncta indicates that septin puncta are enlarged in dcKO mice. Mean ± SEM; datapoints represent individual mice; n = 5 mice per genotype; Students t test ( p = 0.0482).

Article Snippet: Primary antibodies were specific for CDC42 (Santa Cruz, 1:500, #sc‐8401), CDC42EP1 (custom‐made rabbit polyclonal antibody against mouse CDC42EP1 epitope VEKHSNRDRDRDPDH, Pineda, 1:1000), CDC42EP2 (Proteintech Group, 1:300, #11824‐1‐AP), SEPTIN2 (Proteintech Group, 1:500, #11397‐1‐AP), SEPTIN4 (IBL, 1:200, #JP18987), SEPTIN7 (IBL, 1:5000, #18991), SEPTIN8 (Proteintech Group, 1:500, #11769‐1‐AP), or ATP1a3 (Abcam, 1:1000, #ab182571).

Techniques: Immunolabeling, Marker, Microscopy, Imaging, Control