Journal: Biology

Article Title: Beclin 1-Mediated Autophagy Is Potentiated by an Interaction with the Neuronal Adaptor FE65

doi: 10.3390/biology14010097

Figure Lengend Snippet: FE65 enhances autophagic activity. ( A ) The overexpression of FE65 in stable cell lines increased the level of free GFP from the cleavage of GFP-LC3, with or without CQ treatment. ( B ) The knockout of FE65 in cells attenuated the release of free GFP from GFP-LC3, again with or without CQ treatment. GFP-LC3 and GFP were detected in Western blots using an anti-GFP antibody, while the expression of FE65 was confirmed with an anti-FE65 antibody (E20). ( A , B ) The bar charts show the levels of free GFP; error bars represent standard deviation (SD), *** p < 0.001. ( C ) FE65 overexpressing stable cells exhibited increased lipidation of LC3 when treated with or without CQ. Endogenous LC3 was detected by immunoblotting using an anti-LC3 (Proteintech) antibody. ( D ) WT HEK293 and FE65 KO cells were treated with or without CQ. The knockout of FE65 decreased the level of LC3-II. ( C , D ) The bar charts represent the levels of LC3-II; error bars are shown as SD, *** p < 0.001. ( E ) Stable cells overexpressing FE65 showed a decrease in the p62/Beclin 1 ratio, regardless of CQ treatment. Endogenous p62 was detected by immunoblotting using an anti-p62 (Thermofisher) antibody. ( F ) WT HEK293 and FE65 KO cells were treated with or without CQ. The knockout of FE65 resulted in increased p62 accumulation. ( E , F ) The bar charts illustrate the ratio of p62 to Beclin 1, with error bars representing SD, *** p < 0.001, ** p < 0.01. ( G ) GFP-LC3 stably expressing cells were transfected with control and FE65 siRNA. GFP-LC3 puncta were counted after starvation treatment with serum-free medium for 3 h. The bar chart shows the number of puncta per cell. Data were obtained from at least 70 cells per transfection, and the experiment was repeated at least three times. Error bars are SEM, *** p < 0.001. Scale bar, 10 μm. Western blotting also confirmed the knockdown of FE65 in the stable cell line.

Article Snippet: The levels of LC3, p62, FE65, Beclin 1, and tubulin were detected by anti-LC3 antibody (ProteinTech, San Diego, CA, USA), anti-p62 antibody (ProteinTech), anti-FE65 antibody (E20, Santa Cruz, Dallas, TX, USA), anti-Beclin 1 antibody (rat), and anti-tubulin (DM1A, Santa Cruz).

Techniques: Activity Assay, Over Expression, Stable Transfection, Knock-Out, Western Blot, Expressing, Standard Deviation, Transfection, Control, Knockdown

Journal: Biology

Article Title: Beclin 1-Mediated Autophagy Is Potentiated by an Interaction with the Neuronal Adaptor FE65

doi: 10.3390/biology14010097

Figure Lengend Snippet: FE65-Beclin 1 interaction is essential to facilitate Beclin 1-mediated autophagy. ( A ) A GFP-LC3 cleavage assay was performed in WT HEK293 and FE65 KO cells, with and without CQ treatment. The knockout of FE65 decreased the level of free GFP from GFP-LC3 cleavage, and the overexpression of Beclin 1 failed to potentiate GFP-LC3 cleavage in FE65 KO cells. The bar chart shows the level of GFP; error bars represent standard deviation (SD), *** p < 0.001. ( B ) FE65, but not FE65 ΔCt , increased endogenous LC3 lipidation in stably expressing cells, with and without Baf A1 treatment. The bar chart represents the level of LC3-II; error bars are shown as SD, *** p < 0.001, ** p < 0.01. ( C ) FE65-stably transfected cells showed a decrease in the p62/Beclin 1 ratio with and without Baf A1 treatment, whereas FE65 ΔCt did not exhibit this decrease. The bar chart represents the ratio of p62/Beclin 1, with error bars indicating SD. *** p < 0.001, ** p < 0.01, * p < 0.05. ( D , E ) FE65 and FE65 ΔCt were transfected in FE65 KO cells to assess autophagic activity. ( D ) Only FE65 was able to rescue the lipidation of LC3, regardless of CQ treatment. The bar chart illustrates the level of LC3-II; error bars are shown as SD, ** p < 0.01, * p < 0.05. ( E ) FE65 transfection in FE65 KO cells led to a significant decrease in the p62/Beclin 1 ratio, both with and without CQ treatment, while transfection with FE65 ΔCt attenuated this effect. The bar chart represents the ratio of p62/Beclin 1, with error bars indicating SD. ** p < 0.01, * p < 0.05. ( F ) FE65 and FE65 ΔCt were transfected into GFP-LC3 stably expressing cells, with and without Baf A1 treatment. GFP-LC3 puncta were counted, and overexpression of FE65 increased the number of puncta per cell, whereas overexpression of FE65 ΔCt failed to increase GFP-LC3 puncta. The bar chart shows the average number of puncta in cells; data were obtained from at least 40 cells, and the experiments were repeated at least 3 times. Error bars are SEM, *** p < 0.001, ** p < 0.01. Scale bar, 10 μm.

Article Snippet: The levels of LC3, p62, FE65, Beclin 1, and tubulin were detected by anti-LC3 antibody (ProteinTech, San Diego, CA, USA), anti-p62 antibody (ProteinTech), anti-FE65 antibody (E20, Santa Cruz, Dallas, TX, USA), anti-Beclin 1 antibody (rat), and anti-tubulin (DM1A, Santa Cruz).

Techniques: Cleavage Assay, Knock-Out, Over Expression, Standard Deviation, Stable Transfection, Expressing, Transfection, Activity Assay

Journal: Hippocampus

Article Title: CA1 pyramidal neuron gene expression mosaics in the Ts65Dn murine model of Down syndrome and Alzheimer’s disease following maternal choline supplementation (MCS)

doi: 10.1002/hipo.22832

Figure Lengend Snippet: qPCR TaqMan gene probes utilized for CA1 region gene expression profiling

Article Snippet: Significance was judged at the level α=0.05, two-sided. table ft1 table-wrap mode="anchored" t5 caption a7 Gene name ID App Mm01344172_m1 Apbb1 Mm01274044_g1 Bace1 Mm00478671_m1 Camk2a Mm01258148_m1 Casp8 Mm00802247_m1 Dyrk1a Mm00432934_m1 Gapdh Mm99999915_g1 Hsf1 Mm01201402_m1 Hprt1 Mm01318747_g1 Mapk13 Mm00442493_m1 p75NTR Mm01309638_m1 Prkaa2 Mm01264790_m1 Sdha Mm01352360_m1 Sesn2 Mm004060686_m1 Synj1 Mm01215509_m1 Open in a separate window qPCR TaqMan gene probes utilized for CA1 region gene expression profiling

Techniques: Expressing

Journal: Hippocampus

Article Title: CA1 pyramidal neuron gene expression mosaics in the Ts65Dn murine model of Down syndrome and Alzheimer’s disease following maternal choline supplementation (MCS)

doi: 10.1002/hipo.22832

Figure Lengend Snippet: Expression levels impacted by perinatal MCS in ~11 MO MCS-treated offspring included select AD and DS response genes. A. Adam10 is upregulated 1.52 fold (2N versus Ts) in Ts65Dn mice and downregulated in Ts+ mice (Ts+ versus Ts) to 2N levels by MCS. B. Dyrk1a is upregulated 1.30 fold (2N versus Ts) in Ts65Dn mice and downregulated in Ts+ mice relative to 2N and Ts levels by MCS treatment, while Dyrk1a levels in Ts+ are not significantly different from 2N+ levels. C. Eif2ak2 is upregulated 1.35 fold (2N versus Ts) in Ts65Dn mice and downregulated in Ts+ mice (Ts+ versus Ts) to 2N levels by MCS. D. Mapk13 is upregulated 1.22 fold (2N versus Ts) in Ts65Dn mice and downregulated in Ts+ mice relative to 2N and Ts levels by MCS treatment, while Mapk13 levels in Ts+ are not significantly different from 2N+ levels. E. Apbb1 is downregulated 1.64 fold (2N versus Ts) in Ts65Dn mice and upregulated in Ts+ mice (Ts+ versus Ts) to 2N levels by MCS. F. Bace1 is downregulated 1.92 fold (2N versus Ts) in Ts65Dn mice and upregulated in Ts+ mice (Ts+ versus Ts) to 2N levels by MCS. G. Casp8 is downregulated 1.58 fold (2N versus Ts) in Ts65Dn mice and upregulated in Ts+ mice (Ts+ versus Ts) to 2N levels by MCS. H. Mapt2N6D is downregulated 1.87 fold (2N versus Ts) in Ts65Dn mice and upregulated in Ts+ mice (Ts+ versus Ts) to 2N levels by MCS. I. Mapt2N6P is downregulated 2.12 fold (2N versus Ts) in Ts65Dn mice and upregulated in Ts+ mice (Ts+ versus Ts) to significantly higher levels than Mapt2N6P expression in 2N and 2N+ mice. Key: *, p<0.01; **, p<0.005; ***, p<0.001. Upward arrowhead, significant upregulation; downward arrowhead, significant downregulation.

Article Snippet: Significance was judged at the level α=0.05, two-sided. table ft1 table-wrap mode="anchored" t5 caption a7 Gene name ID App Mm01344172_m1 Apbb1 Mm01274044_g1 Bace1 Mm00478671_m1 Camk2a Mm01258148_m1 Casp8 Mm00802247_m1 Dyrk1a Mm00432934_m1 Gapdh Mm99999915_g1 Hsf1 Mm01201402_m1 Hprt1 Mm01318747_g1 Mapk13 Mm00442493_m1 p75NTR Mm01309638_m1 Prkaa2 Mm01264790_m1 Sdha Mm01352360_m1 Sesn2 Mm004060686_m1 Synj1 Mm01215509_m1 Open in a separate window qPCR TaqMan gene probes utilized for CA1 region gene expression profiling

Techniques: Expressing

Journal: Hippocampus

Article Title: CA1 pyramidal neuron gene expression mosaics in the Ts65Dn murine model of Down syndrome and Alzheimer’s disease following maternal choline supplementation (MCS)

doi: 10.1002/hipo.22832

Figure Lengend Snippet: qPCR for select genes using subregional CA1 dissections of admixed cells in ~11 MO mice. A. Apbb1 and Casp8 were upregulated in Ts65Dn mice and displayed significant reduction in MCS treated trisomic offspring. App is upregulated in Ts65Dn mice, and partially recovers to 2N levels by MCS treatment. Bace1 showed no significant differences based on diet or genotype. B. Dyrk1a and Prkaa2 were upregulated in trisomic mice, and displayed normalization (Dyrk1a trend-level p<0.067) with MCS treatment in Ts65Dn offspring. Hsf1 and Mapk13 were downregulated due to MCS treatment in trisomic mice (Mapk13 trend-level p<0.11). Sesn2 showed no significant differences based on diet or genotype. Key: *, p<0.05; **, p<0.01; ***, p<0.001. Upward arrowhead, significant upregulation; downward arrowhead, significant downregulation. Diamond, qPCR results validate microarray findings.

Article Snippet: Significance was judged at the level α=0.05, two-sided. table ft1 table-wrap mode="anchored" t5 caption a7 Gene name ID App Mm01344172_m1 Apbb1 Mm01274044_g1 Bace1 Mm00478671_m1 Camk2a Mm01258148_m1 Casp8 Mm00802247_m1 Dyrk1a Mm00432934_m1 Gapdh Mm99999915_g1 Hsf1 Mm01201402_m1 Hprt1 Mm01318747_g1 Mapk13 Mm00442493_m1 p75NTR Mm01309638_m1 Prkaa2 Mm01264790_m1 Sdha Mm01352360_m1 Sesn2 Mm004060686_m1 Synj1 Mm01215509_m1 Open in a separate window qPCR TaqMan gene probes utilized for CA1 region gene expression profiling

Techniques: Microarray

Journal: Journal of Alzheimer's Disease Reports

Article Title: Identifying Alzheimer's disease genes in apolipoprotein E −/− mice brains with confirmed Porphyromonas gingivalis entry

doi: 10.1177/25424823251332874

Figure Lengend Snippet: Differential expression of genes induced by P. gingivalis FDC 381 (Pg) entry in the ApoE−/− mice versus the sham control groups in the cerebral region of the brain.

Article Snippet: amyloid beta precursor protein binding family B member 1 , Apbb1-Mm00679084_m1 , Other , No change.

Techniques: Quantitative Proteomics, Control, Binding Assay, Membrane, Activity Assay, Glycoproteomics, Protein Binding, Ubiquitin Proteomics

Journal: Research

Article Title: Essential Regulation of Spermatogonial Stem Cell Fate Decisions and Male Fertility by APBB1 via Interaction with KAT5 and GDF15 in Humans and Mice

doi: 10.34133/research.0647

Figure Lengend Snippet: Single-cell transcriptome profiles of testicular cells from normal adult men. (A) UMAP plot showed the annotated testicular cell types. Each dot represented a single testicular cell, and it was colored based on the cell types. (B) Expression of the selected markers identifying major testicular cell types on the UMAP plot. Blue or gray represented a higher or lower expression level as shown on the color key at the right bottom. (C) UMAP plot indicated the annotated different types of spermatogonia. Each dot represented a cell with colors according to cell types. The black solid line showed the SSC differentiation trajectory simulated by Monocle 3. Arrow indicated the direction of differentiation. (D) A violin plot illustrated the expression of selected genes used to identify spermatogonial types. (E) Left: Heat map showed the differentially expressed genes (DEGs) for each cluster in (C). The scaled gene expression levels were colored according to Z score at upper right. Right: Corresponding top 5 GO (biological process) terms enriched in the DEGs of each spermatogonial cluster. (F) Expression of SSC top 10 DEGs projects to spermatogonial developmental trajectory. APBB1 was found to be enriched in SSCs, and its level was progressively decreased with developmental trajectory. The solid black line was the average expression level of each gene along the developmental trajectory. Each dot represented a cell, and its Y value denoted the expression level with colors pursuant to the cell types.

Article Snippet: The Stra8 -GFP-Cre and Apbb1 flox/flox mice were obtained from Cyagen (Suzhou, China).

Techniques: Expressing, Gene Expression

Journal: Research

Article Title: Essential Regulation of Spermatogonial Stem Cell Fate Decisions and Male Fertility by APBB1 via Interaction with KAT5 and GDF15 in Humans and Mice

doi: 10.34133/research.0647

Figure Lengend Snippet: Expression patterns of APBB1 in adult and fetal human testes. (A) H&E staining and APBB1 immunohistochemistry of human testes with normal spermatogenesis. APBB1 was found to be expressed in spermatogonia along the basement membrane of the seminiferous tubules. Scale bars, 50 μm. (B) Western blot detection of APBB1 expression in 3 testes with normal spermatogenesis. (C) Immunofluorescence images of APBB1 with germ cell marker (DDX4), SSC marker (GFRA1), differentiating spermatogonial marker (KIT), and proliferating cell marker (PCNA) in normal human testis. The green signal was APBB1, and the red signals represented different marker molecules, respectively. Cell nuclei were counterstained with DAPI. The bar graphs on the right showed the proportion of APBB1 coexpressed with the marker molecules, respectively. APBB1 was predominantly expressed in quiescent SSCs. White arrows indicated double-positive cells. Scale bars, 50 μm. (D) Left: H&E staining of fetal human testes. Scale bars, 50 μm. Middle: Immunofluorescence images of APBB1 with germ cell marker (DDX4), gonocyte marker (KIT), and proliferating cell marker (PCNA) in fetal testes. The green signal was APBB1, while the red signals represented different marker molecules, respectively. Cellular nuclei were counterstained with DAPI. White arrows denoted double-positive cells. Scale bars, 20 μm. Right: Bar graph demonstrated the coexpression proportion of APBB1 with different markers.

Article Snippet: The Stra8 -GFP-Cre and Apbb1 flox/flox mice were obtained from Cyagen (Suzhou, China).

Techniques: Expressing, Staining, Immunohistochemistry, Membrane, Western Blot, Immunofluorescence, Marker

Journal: Research

Article Title: Essential Regulation of Spermatogonial Stem Cell Fate Decisions and Male Fertility by APBB1 via Interaction with KAT5 and GDF15 in Humans and Mice

doi: 10.34133/research.0647

Figure Lengend Snippet: Effect of APBB1 knockdown on proliferation and apoptosis of human SSCs. (A) qPCR detection of APBB1 mRNA levels in human SSCs transfected with APBB1 shRNAs. Three shRNAs were engineered and interfered with APBB1 expression, and APBB1-KD1 had the best inhibitory effect. (B and C) Western blot assay of APBB1 protein levels in human SSCs transfected with APBB1 shRNAs. The protein levels were lowest in human SSCs by APBB1-KD1 transfection. (D) CCK8 assays were performed to analyze the proliferation ability of human SSCs transfected with APBB1 shRNAs from 1 to 5 d. Cell proliferation was significantly enhanced at days 3 to 5 after APBB1 inhibition. Each small circle represented the data of one detection. (E) EdU incorporation assay was conducted to detect DNA synthesis in human SSCs at 48 h after APBB1 knockdown. Cell nuclei were counterstained with DAPI. (F) Rain cloud plots showed the percentages of EdU-positive cells in (E). Cellular DNA synthesis was significantly increased upon APBB1 knockdown. Each dot represented the result of one picture analysis, and 50 replicates were counted per group. (G) Western blot analysis revealed the decreased expression levels of proteins, including PLZF, CCNE1, CCND1, PCNA, and THY1, in regulating SSC proliferation of human SSCs after APBB1 knockdown. (H) Bar graph indicated the relative levels of each protein in (G) compared to the NC group. (I) Flow cytometry detection of apoptotic cells in human SSCs after APBB1 knockdown. (J) Bar graph displayed the percentages of apoptotic cells in human SSCs after APBB1 knockdown. * P < 0.05; ** P < 0.01.

Article Snippet: The Stra8 -GFP-Cre and Apbb1 flox/flox mice were obtained from Cyagen (Suzhou, China).

Techniques: Knockdown, Transfection, Expressing, Western Blot, Inhibition, DNA Synthesis, Flow Cytometry

Journal: Research

Article Title: Essential Regulation of Spermatogonial Stem Cell Fate Decisions and Male Fertility by APBB1 via Interaction with KAT5 and GDF15 in Humans and Mice

doi: 10.34133/research.0647

Figure Lengend Snippet: RNA sequencing analysis of downstream genes and enrichment pathways of APBB1 in human SSCs. (A) Wayne diagram showed the number of highly expressed genes identified in human SSCs by the APBB-KD1 and NC groups. (B) Bar graph indicated the number of DEGs in human SSCs between the APBB-KD1 and NC groups. Three hundred ninety-three genes were significantly up-regulated and 63 genes were remarkably down-regulated. (C) Volcano plot illustrated the distribution of all identified genes in (A). Top 20 DEGs were labeled. Green, gray, and red represented the down-regulated, not changed, and up-regulated genes, respectively. (D) Left: Heatmap showed the expression and trends of all genes. The scaled gene expression levels were colored according to Z score at upper right. All genes were categorized into 5 clusters, and genes in cluster 2 were significantly down-regulated in human SSCs by APBB1-KD1, where genes in cluster 3 were significantly up-regulated in human SSCs by APBB1-KD1. Top 20 DEGs were labeled. Right: Top 5 GO terms enriched in genes of each cluster. WNT signaling pathway was significantly up-regulated in human SSCs by APBB1 knockdown. (E) All DEGs were analyzed for KEGG enrichment, and top 10 KEGG terms were listed. (F) The qPCR was used to validate the expression of 6 randomly selected DEGs of RNA sequencing. The expression changes of these genes by qPCR were consistent with the RNA sequencing results. (G) Western blot detected the elevated expression of GDF15 protein. * P < 0.05.

Article Snippet: The Stra8 -GFP-Cre and Apbb1 flox/flox mice were obtained from Cyagen (Suzhou, China).

Techniques: RNA Sequencing, Labeling, Expressing, Gene Expression, Knockdown, Western Blot

Journal: Research

Article Title: Essential Regulation of Spermatogonial Stem Cell Fate Decisions and Male Fertility by APBB1 via Interaction with KAT5 and GDF15 in Humans and Mice

doi: 10.34133/research.0647

Figure Lengend Snippet: GDF15 alleviated phenotypic changes caused by APBB1 knockdown in human SSCs. (A) The qPCR assay displayed the GDF15 transcripts in human SSCs by 3 GDF15 shRNAs. The most substantial decrease in GDF15 mRNA levels was observed in human SSCs after GDF15-KD3 transfection. (B and C) Western blot detection of GDF15 protein levels in human SSCs by 3 GDF15 shRNAs. GDF15-KD3 had the best inhibitory effect on protein levels. (D) CCK8 detected cell proliferation in human SSCs treated with NC, APBB1-KD1, and APBB1-KD1 + GDF15-KD3 from 1 to 5 d. GDF15 inhibition antagonized the increase in SSC proliferation induced by APBB1 knockdown at days 3 to 5 post-transfection. (E and F) Western blot detected the expression level of PCNA proteins in human SSCs treated with NC, APBB1-KD1, and APBB1-KD1 + GDF15-KD3 at 48 h. GDF15 knockdown antagonized the up-regulation of PCNA levels caused by APBB1 inhibition. (G and H) EdU incorporation assay evaluated DNA synthesis in human SSCs treated with NC, APBB1-KD1, and APBB1-KD1 + GDF15-KD3 at 48 h. Cell nuclei were counterstained with DAPI. Scale bars, 20 μm. GDF15 knockdown reversed the increase in DNA synthesis induced by APBB1 inhibition. (I and J) Western blot detection of alterations in WNT and MAPK signaling pathways in human SSCs treated with NC, APBB1-KD1, and APBB1-KD1 + GDF15-KD3. APBB1 knockdown up-regulated β-catenin, a key protein of the WNT pathway, and enhanced the phosphorylation of MEK and ERK1/2, important proteins of the MAPK pathway. Simultaneous knockdown of GDF15 and APBB1 antagonized this change. (K and L) TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling) assay detected the apoptosis of human SSCs treated with NC, APBB1-KD1, and APBB1-KD1 + GDF15-KD3. Simultaneous knockdown of GDF15 and APBB1 antagonized this change. Cellular nuclei were counterstained with DAPI. Scale bars, 20 μm. * P < 0.05; ** P < 0.01.

Article Snippet: The Stra8 -GFP-Cre and Apbb1 flox/flox mice were obtained from Cyagen (Suzhou, China).

Techniques: Knockdown, Transfection, Western Blot, Inhibition, Expressing, DNA Synthesis, Protein-Protein interactions, Phospho-proteomics, TUNEL Assay, End Labeling

Journal: Research

Article Title: Essential Regulation of Spermatogonial Stem Cell Fate Decisions and Male Fertility by APBB1 via Interaction with KAT5 and GDF15 in Humans and Mice

doi: 10.34133/research.0647

Figure Lengend Snippet: Prediction and validation of APBB1-interacting proteins in human SSCs. (A) Wayne diagram illustrated the intersection of 3 databases on the prediction of APBB1-interacting proteins. All 3 databases predicted that APBB1 interacted with APP and KAT5. (B) Violin plot demonstrated the distribution of APBB1, APP, and KAT in testicular single-cell atlas shown in (A). (C) Co-immunoprecipitation (Co-IP) assay revealed that APBB1 interacted with KAT5 in human SSCs. (D) Immunohistochemistry of APBB1 (green fluorescence) and KAT5 (red fluorescence) in normal human testes. Cellular nuclei were counterstained with DAPI. White arrows indicated double-positive cells. Scale bars, 20 μm. (E) Bar graph demonstrated the percentages of APBB1 coexpressed with KAT5 in normal human testes in (D). APBB1 and KAT5 were colocalized in most SSCs. (F) The qPCR results showed that the mRNA level of KAT5 was decreased by APBB1 knockdown. (G and H) Western blot revealed KAT5 protein levels in human SSCs after APBB1 knockdown. Bar graphs indicated the relative levels of KAT5 in the APBB1-KD1 human SSCs compared with the NC group. (I and J) Western blot detected the alterations in β-catenin, p-ERK1/2, p-MEK, and GDF15 in human SSCs treated with NC, APBB1-KD1, and APBB1-KD1 + KAT5-OE. APBB1 knockdown increased levels of β-catenin, phosphorylation of MEK and ERK1/2, and GDF15. Simultaneous knockdown of APBB1 and overexpression of KAT5 antagonized this change. (K and L) Western blot implicated protein levels of KAT5 in NC, APBB1-KD1, and APBB1-KD1 + GDF15-KD3 groups. * P < 0.05.

Article Snippet: The Stra8 -GFP-Cre and Apbb1 flox/flox mice were obtained from Cyagen (Suzhou, China).

Techniques: Biomarker Discovery, Co-Immunoprecipitation Assay, Immunohistochemistry, Fluorescence, Knockdown, Western Blot, Phospho-proteomics, Over Expression

Journal: Research

Article Title: Essential Regulation of Spermatogonial Stem Cell Fate Decisions and Male Fertility by APBB1 via Interaction with KAT5 and GDF15 in Humans and Mice

doi: 10.34133/research.0647

Figure Lengend Snippet: Effect of Apbb1 knockout on fertility and spermatogenesis in mice. (A) Bar graph showed the number of pups per litter in Apbb1 −/− and wild-type mice. Apbb1 knockout impaired male fertility in mice. (B) Images of testes from the Apbb1 −/− and wild-type mice. The bar graphs indicated the ratio of testes to body weight for each group of mice. Apbb1 knockout caused significant reduction in testicular volume in mice. (C) H&E staining of testes from wild-type and Apbb1 −/− mice. The pentagram indicated the seminiferous tubules with abnormal spermatogenesis in Apbb1 −/− mice. Scale bars, 50 μm. (D) Dot plot denoted the percentages of different types of abnormal seminiferous tubules. (E) H&E staining of the epididymis of wild-type and Apbb1 −/− mice. Apbb1 −/− mice had the markedly reduced sperm concentration in many tubules in the tail of the epididymis, with some tubules almost absent of sperm. Pentagrams implied epididymal tubules with abnormal sperm concentration. Scale bars, 50 μm. (F) Dot plot showed the percentages of epididymal ducts with reduced spermatozoa in (E). (G) The table displayed the parameters of sperm concentration and motility in wild-type and Apbb1 −/− mice assessed by CASA. Sperm concentration was significantly decreased in Apbb1 −/− mice. (H) Images of sperm Papanicolaou staining for morphological assessment of sperm in wild-type and Apbb1 −/− mice. Scale bars, 20 μm. (I) The table revealed sperm morphology assessment in wild-type and Apbb1 −/− mice in (H). Sperm tail damage was significantly elevated in Apbb1 −/− mice. (J) Transmission electron microscopy images of the principal piece of the sperm tail in wild-type and Apbb1 −/− mice. Scale bars were 500 nm and 100 nm in the left and right images, respectively. (K) Bar graph showed the proportion of principal piece anomalies in wild-type and Apbb1 −/− mice. (L) Immunohistochemistry of Plzf (green) and Kit (red) in wild-type and Apbb1 −/− mice. Cellular nuclei were counterstained with DAPI. Scale bars, 20 μm. (M) Dot plot showed the ratio of Kit + /Plzf + cells in wild-type and Apbb1 −/− mice. This ratio was significantly reduced in Apbb1 knockout mice, reflecting the reduced spermatogonial differentiation. * P < 0.05, *** P < 0.001.

Article Snippet: The Stra8 -GFP-Cre and Apbb1 flox/flox mice were obtained from Cyagen (Suzhou, China).

Techniques: Knock-Out, Staining, Concentration Assay, Transmission Assay, Electron Microscopy, Immunohistochemistry

Journal: Research

Article Title: Essential Regulation of Spermatogonial Stem Cell Fate Decisions and Male Fertility by APBB1 via Interaction with KAT5 and GDF15 in Humans and Mice

doi: 10.34133/research.0647

Figure Lengend Snippet: Apbb1 knockdown promoted SSC self-renewal and inhibited differentiation in vivo. (A) Top panel: GFP fluorescence images of testes from recipient mice after transplantation of SSCs with Apbb1 shRNA (Apbb1-KD) and negative control shRNA (NC). Bottom panel: GFP immunofluorescence images of testicular sections from recipient mice after transplantation of SSCs with Apbb1-KD and NC. Scale bars, 100 μm. (B) Bar graph demonstrated the GFP fluorescence intensity of receptor mouse testes calculated by ImageJ in (A). The total testicular GFP fluorescence intensity was higher in the Apbb1-KD group, which implies more SSC colonization. (C) Bar graph illustrated the number of SSC colonies formed after transplantation in (A). (D) H&E staining of testicular sections from recipient mice after transplantation of SSCs with Apbb1-KD and NC. Spermatogenesis reconstruction was inhibited after SSC transplantation in the Apbb1-KD group. (E) Concentration and motility of recipient mouse spermatozoa assessed by CASA after 2 months of SSC transplantation. Sperm concentration was significantly reduced after SSC transplantation in the Apbb1-KD group. (F) Immunofluorescence images of SSC (Plzf + ) and spermatocytes (γH2ax + ). Nuclei were counterstained with DAPI. Scale bars, 100 μm. (G) Dot plot showed the ratio of Plzf + γH2ax + double-positive tubules to Plzf-positive tubules in (F). This ratio was significantly decreased, suggesting that differentiation of spermatogonia was impaired in the Apbb1-KD group. (H) Dot plot illustrated the mean number of SSCs (Plzf + ) in seminiferous tubules with Apbb1-KD and NC. The mean number of SSCs was significantly increased, which implicates that self-renewal of SSCs is elevated after Apbb1 knockdown. (I) Schematic diagram illustrated the influence of Apbb1 knockdown on the self-renewal and differentiation of SSCs in vivo. * P < 0.05, *** P < 0.001.

Article Snippet: The Stra8 -GFP-Cre and Apbb1 flox/flox mice were obtained from Cyagen (Suzhou, China).

Techniques: Knockdown, In Vivo, Fluorescence, Transplantation Assay, shRNA, Negative Control, Immunofluorescence, Staining, Concentration Assay

Journal: Research

Article Title: Essential Regulation of Spermatogonial Stem Cell Fate Decisions and Male Fertility by APBB1 via Interaction with KAT5 and GDF15 in Humans and Mice

doi: 10.34133/research.0647

Figure Lengend Snippet: RNA sequencing analyzed transcriptomic changes in Apbb1 −/− testes. (A) Dot plot showed principal components analysis (PCA) of top 1,000 genes in Apbb1 −/− and control mice. Each point represented a sample. (B) The qPCR verified the expression level of randomly selected DEGs. (C) Volcano plot illustrated distribution of all genes. The genes that were significantly down-regulated were shown in blue, whereas those that were significantly up-regulated were indicated in orange. Top 10 DEGs were labeled. (D) Heatmap displayed the expression levels of top 200 DEGs in wild-type and Apbb1 −/− mice. Top 20 DEGs were labeled. The scaled gene expression levels were colored according to Z score at right. (E) Sankey plot indicated top 5 GO terms enriched in top 50 DEGs. Left: Top 50 DEGs in wild-type and Apbb1 −/− mice. Middle: Top 5 GO terms enriched in these DEGs. Each term was connected to the contained genes through a channel. Right: Each circle represented a GO term, colored by −log 10 ( P value), and sized pursuant to the number of gene counts.

Article Snippet: The Stra8 -GFP-Cre and Apbb1 flox/flox mice were obtained from Cyagen (Suzhou, China).

Techniques: RNA Sequencing, Control, Expressing, Labeling, Gene Expression

Journal: Research

Article Title: Essential Regulation of Spermatogonial Stem Cell Fate Decisions and Male Fertility by APBB1 via Interaction with KAT5 and GDF15 in Humans and Mice

doi: 10.34133/research.0647

Figure Lengend Snippet: Expression of APBB1 in patients with OA and NOA. (A) Prediction of pathogenicity for APBB1 mutation sites. Four different softwares were used to evaluate each mutation site and summarized the results in a table. Four potential APBB1 pathogenic mutation sites were detected. (B) H&E staining of testes from OA (normal spermatogenesis) and NOA patients (c.1940C>G), pentagram indicated tubules with male germ cell loss, and triangles denoted tubules with impaired germ cell maturation. Scale bar, 50 μm. (C) Western blot was performed to detect APBB1 expression in testes from OA and NOA patients, including maturation arrest at spermatogonia (Spg MA), maturation arrest at spermatocytes (Spc MA), and hypospermatogenesis (HS). (D) Bar graphs demonstrated the relative levels of APBB1 protein in testes from OA and NOA patients, including Spg MA, Spc MA, and HS. The expression level of APBB1 was significantly down-regulated in Spg MA and Spc MA. (E) Immunofluorescence images of APBB1 and GFRA1 (SSC marker) in seminiferous tubules of testes from OA and NOA patients, including Spg MA, Spc MA, and HS. White arrows indicated double-positive SSCs. Scale bars, 20 μm. (F) Bar plot showed the positive rate of APBB1 in SSCs of seminiferous tubules from OA and NOA patients, including Spg MA, Spc MA, and HS. The percentage of APBB1-positive SSC was significantly reduced in NOA patients with Spg MA or Spc MA. Each dot represented a counting result. * P < 0.05.

Article Snippet: The Stra8 -GFP-Cre and Apbb1 flox/flox mice were obtained from Cyagen (Suzhou, China).

Techniques: Expressing, Mutagenesis, Staining, Western Blot, Immunofluorescence, Marker

Journal: Cell

Article Title: A highly conserved program of neuronal microexons is misregulated in autistic brains

doi: 10.1016/j.cell.2014.11.035

Figure Lengend Snippet: A) Heatmap of PSI changes (ΔPSIs) between time points during differentiation of ESCs to glutamatergic neurons in vitro (Hubbard et al., 2013). Yellow/pink indicate increased/decreased PSI at a given transition (T1 to T5). Unsupervised clustering detects eight clusters of exons based on their dynamic PSI regulation (clusters I-VIII, legend). Right, top: scheme of the neuronal differentiation assay time points of sample collection, and analyzed transitions. Right, bottom: PSIs for each microexons (grey lines) in five selected clusters; red lines show the median for the cluster at each time point. B) Representative RT-PCR assays monitoring AS patterns of microexons during neuronal differentiation in Ap1s2 (9 nt), Mef2d (21 nt), Apbb1 (6 nt), Ap1b1 (21nt), Enah (12 nt) and Shank2 (9 and 21 nt). See also Figure S3.

Article Snippet: LUMIER assay HEK-293T cells were transiently transfected using Polyfect (Qiagen) with Renilla Luciferase (RL)-tagged Apbb1, with or without inclusion of the microexon, or with a version consisting of two alanine substitutions, together with 3Flag-tagged Kat5.

Techniques: In Vitro, Differentiation Assay, Reverse Transcription Polymerase Chain Reaction

Journal: Cell

Article Title: A highly conserved program of neuronal microexons is misregulated in autistic brains

doi: 10.1016/j.cell.2014.11.035

Figure Lengend Snippet: A) Structural alignment of APBB1-PTB1 (pink) and APBB1-PTB2 (cyan) domains. Residues located at the protein-binding interface of APBB1-PTB2 are shown in blue. Inset shows the microexon residues in APBB1-PTB1 (E462-R463). B) Upon superimposition of APBB1-PTB1 (pink) and APBB1-PTB2 (cyan) domains, the microexon (magenta) is located close to the APBB1-PTB2 binding partner (APP protein fragment, blue), suggesting the microexon in PTB1 may affect protein binding. C) Quantification of LUMIER-normalized luciferase intensity ratio (NLIR) values for RL-tagged Apbb1, with or without the microexon, or with a mutated version consisting of two Alanine substitutions (ALA-mic.), co-immunoprecipitated with 3Flag-tagged Kat5. D, E) 293T cells were transfected HA-tagged Apbb1 (D) or AP1S2 (E) constructs, with or without the respective microexon, together with 3Flag-tagged Kat5 (D) or AP1B1 (E), as indicated. Immunoprecipitation was performed with anti-Flag (D) or anti-HA (E) antibody, and the immunoprecipitates were blotted with anti-HA or anti-Flag antibody, as indicated. Results shown in (E) were confirmed in a biological replicate experiment (Figure S6D). p-values in C and D correspond to t-tests for four and three replicates, respectively; error bars indicate standard error. Asterisk in panel E indicates a band corresponding to the light chain of the HA antibody.

Article Snippet: LUMIER assay HEK-293T cells were transiently transfected using Polyfect (Qiagen) with Renilla Luciferase (RL)-tagged Apbb1, with or without inclusion of the microexon, or with a version consisting of two alanine substitutions, together with 3Flag-tagged Kat5.

Techniques: Protein Binding, Binding Assay, Luciferase, Immunoprecipitation, Transfection, Construct