Journal: bioRxiv

Article Title: Cis-Regulatory Evolution of CCNB1IP1 Driving Gradual Increase of Cortical Size and Folding in primates

doi: 10.1101/2024.12.08.627376

Figure Lengend Snippet: ( A ) Schematic map showing the evolution of cortical folding and size in primates. Brain images were downloaded from http://brainmuseum.org/ . ( B ) (Top) Pearson’s correlation analysis between the gyrification index (GI) and evolutionary divergence (millions of years ago [MYA]). (Bottom) Pearson’s correlation analysis between log transformed brain weight and evolutionary divergence. ( C ) Nissl staining of E28, E35, and E42 Chinese tree shrew cortices and quantification of the OSVZ thickness during tree shrew brain development. * Each point represents a sample. ( D ) PCA map of the frontal cortex layers of Chinese tree shrews at E35 based on the expression levels of all genes. Bottom panel: cell marker analysis with Sox2 , Pax6 , and Nes for NPCs and Neurond6, Tbr1, and Fezf2 for neurons. ( E ) Immunofluorescence staining of the cortices for the upper layer neuron, deeper layer neuron, and AP markers SATB2 , TBR1 , and SOX2 , respectively, at E35 in Chinese tree shrews. ( F ) Computational pipeline used to identify highly expressed human CCNB1IP1 .

Article Snippet: Primary antibodies: Rabbit monoclonal anti-Ki67 (1:500, Cell Signaling Technology, 9129S); Phospho-Histone H3 (Ser10) Antibody (1:500, Cell Signaling Technology, 9701S); Chicken Polyclonal anti-GFP (1:500, Invitrogen, A10262); Rabbit Polyclonal anti-Pax6 (1:500, Invitrogen, 42-6600); Rabbit Polyclonal anti-TBR2 / Eomes (1:500, Abcam, ab23345); Rat Monoclonal anti-SOX2(1:500, Invitrogen, 53-9811-82); Rabbit Polyclonal anti-SATB2 (1:300, Affinity Biosciences, DF2962); Rabbit Polyclonal anti-TBR1 (1:300, Affinity Biosciences, DF2396); Chicken Polyclonal anti-NeuN (1:500, Millipore ABN91); Mouse anti-nestin (1:500, BD bioscience, Cat#611658); Guinea pig-anti-Ctip2(1:500, Oasis Biofarm, Cat#OB-PGP012) ; Secondary antibodies: Alexa Fluor 594 AffiniPure Donkey Anti-Rabbit (1:800, Jackson ImmunoResearch Labs, 711-585-152); Alexa Fluor 594 AffiniPure Donkey Anti-Rat (1:800, Jackson ImmunoResearch Labs, 712-585-153); Alexa Fluor 488 AffiniPure Donkey Anti-Chicken (1:800, Jackson ImmunoResearch Labs, 703-545-155); Alexa Fluor 488 AffiniPure Donkey Anti-Rabbit (1:800, Jackson ImmunoResearch Labs, 711-545-152); Alexa Fluor 488 AffiniPure Donkey Anti-Rat (1:800, Jackson ImmunoResearch Labs, 712-545-150).

Techniques: Transformation Assay, Staining, Expressing, Marker, Immunofluorescence

Journal: bioRxiv

Article Title: Cis-Regulatory Evolution of CCNB1IP1 Driving Gradual Increase of Cortical Size and Folding in primates

doi: 10.1101/2024.12.08.627376

Figure Lengend Snippet: ( A ) Uniform manifold approximation and projection (UMAP) plot showing cell types identified in the human fetal prefrontal cortex (PFC). ( B ) Expression intensity and percentage of CCNB1IP1 in the identified cell types. ( C ) A t-distributed stochastic neighbour embedding (t-SNE) plot showing cell types identified from the dissected germinal zones of the human fetal cortex. ( D ) Expression intensity and percentage of CCNB1IP1 in the identified cell types. ( E ) (left panel) Expression intensity and percentage of marker genes in identified cell types; (right panel) a t-SNE plot showing that NPCs cluster3 belongs to APs mixed with EOMES expression and NPCs cluster1 belongs to BPs at human 16-18wpc. ( F ) Violin plots showing the expression of pan-RGCs markers PAX6 , SOX2 , IPC marker EOMES and apical RGCs marker PROM1 . ( G ) Violin plots showing the basal RGCs markers MOXD1 , HOPX , and LGALS3BP expression pattern. ( H ) Feature and violin plots demonstrating that CCNB1IP1 expression is higher in BPs than APs at the peak stage of human neurogenesis.

Article Snippet: Primary antibodies: Rabbit monoclonal anti-Ki67 (1:500, Cell Signaling Technology, 9129S); Phospho-Histone H3 (Ser10) Antibody (1:500, Cell Signaling Technology, 9701S); Chicken Polyclonal anti-GFP (1:500, Invitrogen, A10262); Rabbit Polyclonal anti-Pax6 (1:500, Invitrogen, 42-6600); Rabbit Polyclonal anti-TBR2 / Eomes (1:500, Abcam, ab23345); Rat Monoclonal anti-SOX2(1:500, Invitrogen, 53-9811-82); Rabbit Polyclonal anti-SATB2 (1:300, Affinity Biosciences, DF2962); Rabbit Polyclonal anti-TBR1 (1:300, Affinity Biosciences, DF2396); Chicken Polyclonal anti-NeuN (1:500, Millipore ABN91); Mouse anti-nestin (1:500, BD bioscience, Cat#611658); Guinea pig-anti-Ctip2(1:500, Oasis Biofarm, Cat#OB-PGP012) ; Secondary antibodies: Alexa Fluor 594 AffiniPure Donkey Anti-Rabbit (1:800, Jackson ImmunoResearch Labs, 711-585-152); Alexa Fluor 594 AffiniPure Donkey Anti-Rat (1:800, Jackson ImmunoResearch Labs, 712-585-153); Alexa Fluor 488 AffiniPure Donkey Anti-Chicken (1:800, Jackson ImmunoResearch Labs, 703-545-155); Alexa Fluor 488 AffiniPure Donkey Anti-Rabbit (1:800, Jackson ImmunoResearch Labs, 711-545-152); Alexa Fluor 488 AffiniPure Donkey Anti-Rat (1:800, Jackson ImmunoResearch Labs, 712-545-150).

Techniques: Expressing, Marker

Journal: bioRxiv

Article Title: Cis-Regulatory Evolution of CCNB1IP1 Driving Gradual Increase of Cortical Size and Folding in primates

doi: 10.1101/2024.12.08.627376

Figure Lengend Snippet: ( A ) (Top panel) Representative images of cells transfected with CCNB1IP1-GFP and GFP with EdU pulses for 4 h. (Bottom panel) Violin plots of the percentages of GFP + EdU + in GFP + cells. (n=16 vision fields for totally 4 replicates, data represented as mean±SD, * p < 0.05, unpaired Student’s t -test). ( B ) A schematic diagram of the IUE (E13.5-E15.5) strategy. ( C ) (Top panel) Immunofluorescence images of the APs marker PAX6 at E15.5 in the mouse embryonic cortex. *Arrows indicate PAX6 + cells. (Bottom panel) Quantification of PAX6 + cells in electroporated and non-electroporated regions, as shown in (a) (n=3-5 embryos from two IUE experiments, data represented as mean, * p < 0.05, unpaired Student’s t -test). ( D ) (Top panel) Immunofluorescence images of the IPC marker EOMES at E15.5 in the mouse embryonic cortex. (Bottom panel) Quantification of EOMES + cells in electroporated and non-electroporated regions, as indicated in (A), (n=3-5 embryos from two IUE experiments, data represented as mean± SD, * p < 0.05, ** p < 0.001, unpaired Student’s t -test). ( E ) (Top panel) Immunofluorescence of cell cycle marker Ki67 after electroporation at E13.5. (Bottom panel) quantification of Ki67 + cells at electroporated and non-electroporated regions (n=4-5 embryos from two IUE experiments, data represented as mean±SD, ** p < 0.001, *** p < 0.001, unpaired Student’s t -test).

Article Snippet: Primary antibodies: Rabbit monoclonal anti-Ki67 (1:500, Cell Signaling Technology, 9129S); Phospho-Histone H3 (Ser10) Antibody (1:500, Cell Signaling Technology, 9701S); Chicken Polyclonal anti-GFP (1:500, Invitrogen, A10262); Rabbit Polyclonal anti-Pax6 (1:500, Invitrogen, 42-6600); Rabbit Polyclonal anti-TBR2 / Eomes (1:500, Abcam, ab23345); Rat Monoclonal anti-SOX2(1:500, Invitrogen, 53-9811-82); Rabbit Polyclonal anti-SATB2 (1:300, Affinity Biosciences, DF2962); Rabbit Polyclonal anti-TBR1 (1:300, Affinity Biosciences, DF2396); Chicken Polyclonal anti-NeuN (1:500, Millipore ABN91); Mouse anti-nestin (1:500, BD bioscience, Cat#611658); Guinea pig-anti-Ctip2(1:500, Oasis Biofarm, Cat#OB-PGP012) ; Secondary antibodies: Alexa Fluor 594 AffiniPure Donkey Anti-Rabbit (1:800, Jackson ImmunoResearch Labs, 711-585-152); Alexa Fluor 594 AffiniPure Donkey Anti-Rat (1:800, Jackson ImmunoResearch Labs, 712-585-153); Alexa Fluor 488 AffiniPure Donkey Anti-Chicken (1:800, Jackson ImmunoResearch Labs, 703-545-155); Alexa Fluor 488 AffiniPure Donkey Anti-Rabbit (1:800, Jackson ImmunoResearch Labs, 711-545-152); Alexa Fluor 488 AffiniPure Donkey Anti-Rat (1:800, Jackson ImmunoResearch Labs, 712-545-150).

Techniques: Transfection, Immunofluorescence, Marker, Electroporation

Journal: bioRxiv

Article Title: Cis-Regulatory Evolution of CCNB1IP1 Driving Gradual Increase of Cortical Size and Folding in primates

doi: 10.1101/2024.12.08.627376

Figure Lengend Snippet: ( A ) Cell cycle distribution analysis after 48 h transfection of GFP and CCNB1IP1-GFP in neuro-2a cells. (n=5-6 replicates, data represented as mean±SD, * p < 0.05, unpaired Student’s t -test). ( B ) Left panel: Representative images showing separate transfection of GFP and CCNB1IP1 (green) with G1-Fucci (red) constructs at 24, 36, 38 h. Right panel: Quantification of GFP + and CCNB1IP1-GFP + co-localization with G1-Fucci + . (n=6-7 replicates, data represented as mean± SD, ** p < 0.01, unpaired Student’s t -test). ( C ) Top panel: Electroporation of CAG-GFP and CAG-CCNB1IP1-GFP constructs at E13.5, followed by EdU at 24 h and analysis at E15.5. *White arrowheads indicate GFP + Edu + double-positive cells. ( D ) Quantification of GFP + and CCNB1IP1-GFP + colocalization with EdU + in the VZ, SVZ, or VZ + SVZ. (n=5-6 embryos from two IUE experiments, data represented as mean±SD, ** p < 0.01, unpaired Student’s t -test). ( E ) Representative images of the mitosis marker Ki67 and quantification of Ki67 + in GFP + cells, (n=4 embryos from one IUE experiment, data represented as mean± SD, * p < 0.05, unpaired Student’s t -test). ( F ) Representative images of the NPCs marker PAX6 immunofluorescence after electroporation with GFP or CCNB1IP1 at E14.5. Quantification of PAX6 + in GFP + cells in the VZ and SVZ (n=3 embryos from one IUE experiments, data represented as mean± SD, * p < 0.05, unpaired Student’s t -test). ( G )(Top) Representative images of the upper layer neuron marker BRN2 at E18.5, following electroporation of GFP or CCNB1IP1 at E14.5. (Bottom) Quantification showing the effects of CCNB1IP1 expression on the number of GFP + and BRN2 + cells. Arrows indicate GFP + BRN2 + cells (n=9 embryos from three IUE experiments, data represented as mean± SD, * p < 0.05, unpaired Student’s t -test).

Article Snippet: Primary antibodies: Rabbit monoclonal anti-Ki67 (1:500, Cell Signaling Technology, 9129S); Phospho-Histone H3 (Ser10) Antibody (1:500, Cell Signaling Technology, 9701S); Chicken Polyclonal anti-GFP (1:500, Invitrogen, A10262); Rabbit Polyclonal anti-Pax6 (1:500, Invitrogen, 42-6600); Rabbit Polyclonal anti-TBR2 / Eomes (1:500, Abcam, ab23345); Rat Monoclonal anti-SOX2(1:500, Invitrogen, 53-9811-82); Rabbit Polyclonal anti-SATB2 (1:300, Affinity Biosciences, DF2962); Rabbit Polyclonal anti-TBR1 (1:300, Affinity Biosciences, DF2396); Chicken Polyclonal anti-NeuN (1:500, Millipore ABN91); Mouse anti-nestin (1:500, BD bioscience, Cat#611658); Guinea pig-anti-Ctip2(1:500, Oasis Biofarm, Cat#OB-PGP012) ; Secondary antibodies: Alexa Fluor 594 AffiniPure Donkey Anti-Rabbit (1:800, Jackson ImmunoResearch Labs, 711-585-152); Alexa Fluor 594 AffiniPure Donkey Anti-Rat (1:800, Jackson ImmunoResearch Labs, 712-585-153); Alexa Fluor 488 AffiniPure Donkey Anti-Chicken (1:800, Jackson ImmunoResearch Labs, 703-545-155); Alexa Fluor 488 AffiniPure Donkey Anti-Rabbit (1:800, Jackson ImmunoResearch Labs, 711-545-152); Alexa Fluor 488 AffiniPure Donkey Anti-Rat (1:800, Jackson ImmunoResearch Labs, 712-545-150).

Techniques: Transfection, Construct, Electroporation, Marker, Immunofluorescence, Expressing

Journal: bioRxiv

Article Title: Cis-Regulatory Evolution of CCNB1IP1 Driving Gradual Increase of Cortical Size and Folding in primates

doi: 10.1101/2024.12.08.627376

Figure Lengend Snippet: ( A ) Schematic representation of the generation of human CCNB1IP1 conditional knock-in mice at the Rosa26 locus using CRISPR-Cas9 technology. ( B ) A successful knock-in CCNB1IP1 allele was verified through Southern blotting using 5′ and 3′ probes surrounding the targeting cassette. ( C ) Western blotting analysis of Emx1-Cre;CCNB1IP1 KI/+ cKI mouse cortex, showing high CCNB1IP1 expression compared to controls. ( D ) (Top panel) Representative images of cortical folding (arrows) in cKI Emx1-Cre mice at P1; (Bottom panel) Schematic representation and quantification of the degree of cortical folding in cKI Emx1-Cre mice. Each data point represents a sample, 5 controls and 7 cKI from two nests. ( E ) Immunostaining and quantification of the upper layer marker Brn2 (green) and deeper layer marker Tbr1 (red) in cKI Emx1-Cre mice, indicating significantly increased upper layer neurons. Each data point represents a sample. 5 controls and 7 cKI from two nests. ( F ) A dorsal view of the control and cKI Nestin-cre embryonic brains at E15.5 (left panel) and quantification results showing significant increase of the cortical area in cKI Nestin- cre mice (right panel). Each data point represents a sample, 4 controls and 4 cKI from one nest. ( G-I ) Representative images and quantification of PAX6 + , EOMES + , and Ki67 + cells in cKI Nestin-cre mice. Each data point represents a sample. All statistical data are presented as the mean or mean, * p < 0.05, ** p < 0.001, *** p < 0.001, n.s , not significant, as determined using the unpaired Student’s t -test.

Article Snippet: Primary antibodies: Rabbit monoclonal anti-Ki67 (1:500, Cell Signaling Technology, 9129S); Phospho-Histone H3 (Ser10) Antibody (1:500, Cell Signaling Technology, 9701S); Chicken Polyclonal anti-GFP (1:500, Invitrogen, A10262); Rabbit Polyclonal anti-Pax6 (1:500, Invitrogen, 42-6600); Rabbit Polyclonal anti-TBR2 / Eomes (1:500, Abcam, ab23345); Rat Monoclonal anti-SOX2(1:500, Invitrogen, 53-9811-82); Rabbit Polyclonal anti-SATB2 (1:300, Affinity Biosciences, DF2962); Rabbit Polyclonal anti-TBR1 (1:300, Affinity Biosciences, DF2396); Chicken Polyclonal anti-NeuN (1:500, Millipore ABN91); Mouse anti-nestin (1:500, BD bioscience, Cat#611658); Guinea pig-anti-Ctip2(1:500, Oasis Biofarm, Cat#OB-PGP012) ; Secondary antibodies: Alexa Fluor 594 AffiniPure Donkey Anti-Rabbit (1:800, Jackson ImmunoResearch Labs, 711-585-152); Alexa Fluor 594 AffiniPure Donkey Anti-Rat (1:800, Jackson ImmunoResearch Labs, 712-585-153); Alexa Fluor 488 AffiniPure Donkey Anti-Chicken (1:800, Jackson ImmunoResearch Labs, 703-545-155); Alexa Fluor 488 AffiniPure Donkey Anti-Rabbit (1:800, Jackson ImmunoResearch Labs, 711-545-152); Alexa Fluor 488 AffiniPure Donkey Anti-Rat (1:800, Jackson ImmunoResearch Labs, 712-545-150).

Techniques: Knock-In, CRISPR, Southern Blot, Western Blot, Expressing, Immunostaining, Marker, Control

Journal: International Journal of Molecular Sciences

Article Title: The Generation of Genetically Engineered Human Induced Pluripotent Stem Cells Overexpressing IFN-β for Future Experimental and Clinically Oriented Studies

doi: 10.3390/ijms252212456

Figure Lengend Snippet: A disrupted expression of ectoderm-associated genes in embryonic bodies spontaneously differentiated from IFNB-iPSCs. IFNB-iPSC lines LA8 and LC8 and parental K7-iPSCs were cultured in low-adhesive conditions to stimulate the formation of embryoid bodies (EBs). On day 20, RNA was isolated and gene expressions were analyzed using RT-PCR. Whole-cell lysates of EBs were also analyzed by a Western blot. ( a – c ) Relative expressions of endoderm- ( a ), mesoderm- ( b ), and ectoderm- ( c ) associated markers in iPSCs and 20-day EBs. Data are shown as boxes and whiskers with minimal and maximal values (summarized results of at least 2 independent experiments). The significance of the differences was determined using the two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli. Figures indicate the FDRs for the main comparisons, irrespectively of their significance (i.e., inter-line comparisons on differentiation day 20 and intra-line comparisons between iPSCs and 20-day EBs). ( d ) The Western blot analysis of PAX6 protein in IFNB-EBs and K7-EBs (one experiment). The graph shows the relative densitometric values of PAX6 normalized to HSP90.

Article Snippet: The membrane was blocked with a 5% non-fat dry milk (Cell Signaling Technology, Danvers, MA, USA) in a TNT buffer (10 mM of Tris-HCl, pH 7.5, 150 mM of NaCl, 0.1% Tween-20) and incubated with the following primary antibodies (4 °C, overnight, 1% milk): rabbit anti-human-IFN-β polyclonal antibodies (1:500, FNab10475, FineTest Biotech, Wuhan, Hubei, China), rabbit anti-human-PAX6 polyclonal antibodies (1:500, PAH446Ra01, Cloud-Clone Corp.), mouse anti-human-OCT-4 monoclonal antibody (1:1000, ab184665, Abcam, Cambridge, UK), mouse anti-human-actin-β antibodies (1:10,000, Abcam); and rabbit anti-human-HSP90 polyclonal antibodies (1:10,000, Sigma-Aldrich).

Techniques: Expressing, Cell Culture, Adhesive, Isolation, Reverse Transcription Polymerase Chain Reaction, Western Blot