milk tbs t  (Proteintech)

Journal: International Journal of Molecular Sciences

Article Title: Microcephaly Gene Mcph1 Deficiency Induces p19ARF-Dependent Cell Cycle Arrest and Senescence

doi: 10.3390/ijms25094597

Figure Lengend Snippet: Mcph1-KO embryos exhibit delayed growth during an early stage. ( A ) Images of the whole body of control and Mcph1-KO mice at embryonic period 14.5 (E14.5). Scale bar, 5 mm. Mcph1-Ctr is the control group, and Mcph1-KO is the Mcph1 knockout group. ( B ) Images of the whole body of control and Mcph1-KO mice at embryonic period 11.5 (E11.5). Scale bar, 1 mm.

Article Snippet: The proteins on the gel were then transferred to the PVDF membrane (1620177, Bio-Rad, Hercules, CA, USA), which was blocked with 5% milk (FD6006, FUDE, Hangzhou, China) for 1 h. Primary antibody MCPH1 (1:2000, 4120, Cell Signaling, Danvers, MA, USA), p19ARF (ab80, Abcam, Cambridge, UK), and β-ACTIN (1:5000, A5441, Sigma, St. Louis, MO, USA) were incubated overnight at 4 °C.

Techniques: Control, Knock-Out

Journal: International Journal of Molecular Sciences

Article Title: Microcephaly Gene Mcph1 Deficiency Induces p19ARF-Dependent Cell Cycle Arrest and Senescence

doi: 10.3390/ijms25094597

Figure Lengend Snippet: The metabolism and development processes are mainly affected by Mcph1 disruption. ( A ) Gene mapping analysis of mice in the control group and Mcph1-KO group. The arrow refers to the location of exons 4 and 5. ( B ) The content of Mcph1 in Mcph1-Ctr and Mcph1-KO mice was detected by RT-qPCR. The horizontal coordinate represented the group, the vertical coordinate represented the relative expression of Mcph1 mRNA, and Gapdh was used as a reference. **, p < 0.01. ( C ) The volcano plot of the Mcph1-KO group against the control group. DEGs with a fold change greater than 1.2 and adjusted p value of less than 0.05. Red represents upregulated DEGs, and blue represents downregulated DEGs. Gray indicates genes that are not statistically significant. ( D ) The number and proportion of genes in DEGs. Red shows the proportion of upregulated DEGs, and blue shows downregulated DEGs. ( E ) Classification of biological processes of all DEGs. The metabolic process accounted for 69.8%, and the developmental process accounted for 25.0%. ( F ) Classification of biological processes of upregulated DEGs. The metabolic process accounted for 81%, and the developmental process accounted for 16%. ( G ) Enriched KEGG pathways of all DEGs. KEGG analysis shows the enriched items in all DEGs. ( H ) Enriched KEGG pathways of upregulated DEGs. KEGG analysis shows the enriched items in the upregulated DEGs.

Article Snippet: The proteins on the gel were then transferred to the PVDF membrane (1620177, Bio-Rad, Hercules, CA, USA), which was blocked with 5% milk (FD6006, FUDE, Hangzhou, China) for 1 h. Primary antibody MCPH1 (1:2000, 4120, Cell Signaling, Danvers, MA, USA), p19ARF (ab80, Abcam, Cambridge, UK), and β-ACTIN (1:5000, A5441, Sigma, St. Louis, MO, USA) were incubated overnight at 4 °C.

Techniques: Disruption, Control, Quantitative RT-PCR, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Microcephaly Gene Mcph1 Deficiency Induces p19ARF-Dependent Cell Cycle Arrest and Senescence

doi: 10.3390/ijms25094597

Figure Lengend Snippet: Analysis of the intersection of Mcph1-KO DEGs and E2F1 target genes. ( A ) The Venn diagram represents the intersecting genes between E2F1 target genes and Mcph1-KO DEGs. There were 12,834 E2F1 target genes, 117 Mcph1-KO DEGs, and 51 overlapping genes. p > 0.05, no statistical significance. Statistical analysis was performed using Fisher’s exact test. ( B ) Correlated intersecting genes heat map. The abundance of 51 genes shared by all Mcph1-KO DEGs and E2F1 target genes was shown. ( C ) Biological processes associated with intersecting genes. GO analysis was performed on intersecting genes, and the top 10 biological processes are shown on the left. ( D ) Expression verification of intersecting genes related to neurodevelopment, such as Satb2 (encoded for special AT-rich sequence-binding protein 2) and Cdkn1c (encoded for p57KIP2). The expression of intersecting genes in control and Mcph1-KO groups was detected by RT-qPCR. *, p < 0.05. ( E ) Heat map of RT-qPCR test results for neurodevelopment-associated intersecting genes. ( F ) The effect of MCPH1 overexpression on Cdkn1c promoter activity was detected by double luciferase assay. *, p < 0.05. P-C-1000 and P-C-2000 were different lengths of Cdkn1c promoter regions.

Article Snippet: The proteins on the gel were then transferred to the PVDF membrane (1620177, Bio-Rad, Hercules, CA, USA), which was blocked with 5% milk (FD6006, FUDE, Hangzhou, China) for 1 h. Primary antibody MCPH1 (1:2000, 4120, Cell Signaling, Danvers, MA, USA), p19ARF (ab80, Abcam, Cambridge, UK), and β-ACTIN (1:5000, A5441, Sigma, St. Louis, MO, USA) were incubated overnight at 4 °C.

Techniques: Expressing, Sequencing, Binding Assay, Control, Quantitative RT-PCR, Over Expression, Activity Assay, Luciferase

Journal: International Journal of Molecular Sciences

Article Title: Microcephaly Gene Mcph1 Deficiency Induces p19ARF-Dependent Cell Cycle Arrest and Senescence

doi: 10.3390/ijms25094597

Figure Lengend Snippet: Growth inhibition and senescence in the primary MEFs without Mcph1 . ( A ) Proliferation analysis of the primary MEFs. The proliferation rate was measured by seeding the same amount (3 × 10 5 ) of primary MEFs of indicated genotypes. The cells were passaged every two days, and the cell numbers were determined before passing. The experiment was repeated three times. *, p < 0.05. ( B ) The proliferation rate was measured by the number of cells in different passages of indicated genotypes. Bars represent the SEM. Statistical analysis was performed using Student’s t -test. *, p < 0.05; **, p < 0.01. ( C ) Flow cytometry was performed with double Annexin V-FITC/PI staining for Mcph1-Ctr and Mcph1-KO MEFs. The percentage of apoptosis (% apoptosis) was measured. Bars represent the SEM. A statistical analysis was performed using Student’s t -test. ( D ) BrdU labeling of P2 primary MEFs in vitro and incubation for 1 h at 37 °C. The cells were stained with anti-BrdU-antibody (red) to determine the proliferation rate of primary MEFs at P2. Scale bars, 50 µm. ( E ) Quantification of BrdU-positive cells percentage of primary MEFs in Mcph1-Ctr and Mcph1-KO group. A statistical analysis was performed using Student’s t -test. ( F ) Knockout of Mcph1 disturbed the cell cycle in primary MEFs. The distribution of the cell cycle was detected by flow cytometry with PI staining. The percentages of the G1, S, and G2/M phases were calculated. The representative charts and quantified results of three independent experiments were shown. Statistical analysis was performed using Student’s t -test. ( G ) SA-β-gal-positive cells could be observed in the primary MEFs. Cells were subcultured and maintained in a growth medium before assay for β-gal activity staining at early passage. Scale, 100 µm. On the right are the staining levels of SA-β-gal-positive cells, which were analyzed digitally by Image J v1.53 k. The percentage of senescence (% β-Galactosidase cells) was measured. Bars represent the SEM. Statistical analysis was performed using Student’s t -test. ( H ) The morphology of control cells and Mcph1-KO cells were examined by phase-contrast microscopy. The P4 primary MEFs were trypsinized by trypsin. Scale bar, 50 µm. The figure on the right shows the quantification of the relative area of cells after trypsinization in control and Mcph1-KO . Bars represent the SEM.

Article Snippet: The proteins on the gel were then transferred to the PVDF membrane (1620177, Bio-Rad, Hercules, CA, USA), which was blocked with 5% milk (FD6006, FUDE, Hangzhou, China) for 1 h. Primary antibody MCPH1 (1:2000, 4120, Cell Signaling, Danvers, MA, USA), p19ARF (ab80, Abcam, Cambridge, UK), and β-ACTIN (1:5000, A5441, Sigma, St. Louis, MO, USA) were incubated overnight at 4 °C.

Techniques: Inhibition, Flow Cytometry, Staining, Labeling, In Vitro, Incubation, Knock-Out, Activity Assay, Control, Microscopy

Journal: International Journal of Molecular Sciences

Article Title: Microcephaly Gene Mcph1 Deficiency Induces p19ARF-Dependent Cell Cycle Arrest and Senescence

doi: 10.3390/ijms25094597

Figure Lengend Snippet: Knockdown of p19Arf can rescue Mcph1-KO cell growth inhibition. ( A ) Protein lysates were extracted from Mcph1-Ctr and Mcph1-KO P2 primary MEFs. The p19ARF was examined by Western blotting using an anti-p19ARF antibody. β-ACTIN was used as a loading control. ( B ) Protein lysates were extracted from Mcph1-Ctr and Mcph1-KO immortalized cells. The MCPH1 protein and p19ARF were examined by Western blotting using an anti-MCPH1 antibody and an anti-p19ARF antibody, respectively. β-ACTIN was used as a loading control. ( C ) The proliferation rate of immortalized cells was measured by the number of cells in different passages of indicated genotypes. shP19 indicates that p19Arf was knocked down in immortalized cells. ( D ) Immortalized cells harvested were fixed and stained with propidium iodide, and their DNA contents were analyzed by flow cytometry. Mcph1-KO arrested the cells in the G1/G0 phase, and depletion of p19Arf rescued them. Each phase was calculated using the cell ModFit LT v3.2 The percentages of cells in G1, S, and G2/M were also shown as indicated.

Article Snippet: The proteins on the gel were then transferred to the PVDF membrane (1620177, Bio-Rad, Hercules, CA, USA), which was blocked with 5% milk (FD6006, FUDE, Hangzhou, China) for 1 h. Primary antibody MCPH1 (1:2000, 4120, Cell Signaling, Danvers, MA, USA), p19ARF (ab80, Abcam, Cambridge, UK), and β-ACTIN (1:5000, A5441, Sigma, St. Louis, MO, USA) were incubated overnight at 4 °C.

Techniques: Knockdown, Inhibition, Western Blot, Control, Staining, Flow Cytometry

Journal: Cells

Article Title: The Central Domain of MCPH1 Controls Development of the Cerebral Cortex and Gonads in Mice.

doi: 10.3390/cells11172715

Figure Lengend Snippet: Figure 1. Generation of the MCPH1 central domain knockout mouse (Mcph1-∆e8). (A) Gene targeting strategy to generate MCPH1 central domain knockout mice. Exon 8 of Mcph1 that encodes the MCPH1 central domain was floxed by two LoxP sites. The expected alleles before and after gene targeting are shown. The expected sizes of wild-type allele (WT), targeted allele (Tg), floxed allele (Flox) and exon 8 deleted allele, after respective enzyme digestion in Southern blotting, are shown. The locations of the Southern blotting probes (P1 for the 5′ homology arm; P2 for the 3′ homology arm) are marked under their respective alleles. Lower panel: Southern blotting to identify the correct homologous recombination events in ES cell clones after digestion with the indicated restriction enzymes and hybridization with the indicated probes. (B) RT-PCR analysis to validate exon 8 deletion in the Mcph1

Article Snippet: Western blotting using an MCPH1 antibody (D38G5, #4120, Cell Signaling Technology) that recognizes the MCPH1’s central domain confirmed that Mcph1-∆e8 indeed was missing this middle domain (Figure 1D).

Techniques: Knock-Out, Southern Blot, Homologous Recombination, Clone Assay, Hybridization, Reverse Transcription Polymerase Chain Reaction

Journal: Cells

Article Title: The Central Domain of MCPH1 Controls Development of the Cerebral Cortex and Gonads in Mice.

doi: 10.3390/cells11172715

Figure Lengend Snippet: Figure 2. Mcph1-∆e8 mice develop primary microcephaly. (A) Macroscopic dorsal view of the mouse

Article Snippet: Western blotting using an MCPH1 antibody (D38G5, #4120, Cell Signaling Technology) that recognizes the MCPH1’s central domain confirmed that Mcph1-∆e8 indeed was missing this middle domain (Figure 1D).

Techniques:

Journal: Cells

Article Title: The Central Domain of MCPH1 Controls Development of the Cerebral Cortex and Gonads in Mice.

doi: 10.3390/cells11172715

Figure Lengend Snippet: Figure 3. Reduction of neuroprogenitors in Mcph1-∆e8 embryonic cortex. (A) Immunofluorescence

Article Snippet: Western blotting using an MCPH1 antibody (D38G5, #4120, Cell Signaling Technology) that recognizes the MCPH1’s central domain confirmed that Mcph1-∆e8 indeed was missing this middle domain (Figure 1D).

Techniques:

Journal: Cells

Article Title: The Central Domain of MCPH1 Controls Development of the Cerebral Cortex and Gonads in Mice.

doi: 10.3390/cells11172715

Figure Lengend Snippet: Figure 4. Proliferation defects of neuroprogenitors in the Mcph1-∆e8 embryonic cortex. (A) Immunostain- ing of the E15.5 embryonic cortex after EdU pulse labeling for 1 hr using antibodies against EdU (green) and pS28-H3 (a mitotic marker, red). The nucleus is counterstained by DAPI (blue). (B) Quantification of the percentages of EdU+ and pS28-H3+ cells among the total DAPI+ cells. (C) Double staining of the E15.5

Article Snippet: Western blotting using an MCPH1 antibody (D38G5, #4120, Cell Signaling Technology) that recognizes the MCPH1’s central domain confirmed that Mcph1-∆e8 indeed was missing this middle domain (Figure 1D).

Techniques: Labeling, Marker, Double Staining

Journal: Cells

Article Title: The Central Domain of MCPH1 Controls Development of the Cerebral Cortex and Gonads in Mice.

doi: 10.3390/cells11172715

Figure Lengend Snippet: Figure 5. Mcph1-∆e8 cells exhibit PCC and defective DDR. (A) Representative images of PCC in Mcph1-∆e8 MEFs. Primary MEF cells were stained with a pS10-H3 antibody (red) and counterstained with DAPI (blue). (B) Quantification of the percentages of PCC cells (prophase cells lacking the pS10-H3 signal) in control and Mcph1-∆e8 primary MEF cells. More than 250 prophase cells were scored in each group of the indicated genotype. (C) Western blot analysis of p-Chk1 and γH2AX in control and Mcph1-∆e8 MEFs with or without HU treatment. β-actin was used as a loading control. (D) The quantification of the indicated protein intensities from panel (C). Unpaired Student’s t-test was used for statistical analysis. ***, p < 0.001.

Article Snippet: Western blotting using an MCPH1 antibody (D38G5, #4120, Cell Signaling Technology) that recognizes the MCPH1’s central domain confirmed that Mcph1-∆e8 indeed was missing this middle domain (Figure 1D).

Techniques: Staining, Control, Western Blot

Journal: Cells

Article Title: The Central Domain of MCPH1 Controls Development of the Cerebral Cortex and Gonads in Mice.

doi: 10.3390/cells11172715

Figure Lengend Snippet: Figure 6. Defective gonad development in Mcph1-∆e8 mice. (A) Macroscopic view of testes from a control and a Mcph1-∆e8 mouse (6 weeks of age). The ratios of the testis weight (TW) to body weight (BW) from 6-week-old control and Mcph1-∆e8 mice are shown in the lower panel. (B) H&E staining of testis sections from 6-week-old control and Mcph1-∆e8 male mice. The epididymis (left panel) and seminiferous tubules (right panel) are shown. Yellow, green, red and blue arrowheads mark spermatogonia, pachytene spermatocytes, round spermatids and elongated spermatids, respectively.

Article Snippet: Western blotting using an MCPH1 antibody (D38G5, #4120, Cell Signaling Technology) that recognizes the MCPH1’s central domain confirmed that Mcph1-∆e8 indeed was missing this middle domain (Figure 1D).

Techniques: Control, Staining