Journal: The EMBO Journal

Article Title: Cyclin-dependent kinase inhibitor p18 regulates lineage transitions of excitatory neurons, astrocytes, and interneurons in the mouse cortex

doi: 10.1038/s44318-024-00325-9

Figure Lengend Snippet: ( A ) Plasmids to induce KD of p18 and p27 mixed with pCAG-H2B-TagRFP were delivered at E15.5 to AldH1l1 -EGFP mice. EdU was administered 2 h before fixation at E16.5. Arrowheads; double-positive cells to EdU and H2B-TagRFP. Bar, 50 µm. ( B ) The expression of AldH1l1 -EGFP prepared in ( A ). Arrowheads; double-positive cells to AldH1l1 -EGFP and H2B-TagRFP. Bar, 50 µm. ( C ) Quantification of the stained images shown in ( A ) ( n = 3 brains each) and ( B ) ( n = 6 sections from 3 brains each). Total counted cells of Control, p18 KD, p27 KD, Double KD; 331, 433, 363, 420 . One-way ANOVA with Tukey’s test; error bars show mean ± SEM. From left to right, * P = 0.02035068, * P = 0.03126960, ** P = 0.00955672 (a), * P = 0.01734001, ** P = 0.00268600, *** P = 0.00000817 (b). ( D ) Plasmids to induce KD of p18 and p27 mixed with pCAG-H2B-TagRFP were delivered at E14.5 and then fixed at E18.5. Sections were stained using TAG-1 antibody to recognize axon fibers as the border between the upper and lower positions. Bar, 50 µm. ( E ) Quantification of the stained images shown in ( D ) ( n = 4 brains each). Total counted cells of Control, p18 KD, p27 KD; 544, 614, 429 . One-way ANOVA with Tukey’s test; error bars show mean ± SEM. From left to right, * P = 0.04727532, * P = 0.01171081. ( F ) Plasmids to induce OE of p18 mixed with pCAG-H2B-TagRFP were delivered at E15.5 to AldH1l1 -EGFP mice and then fixed at E16.5-P0. Arrowheads; double-positive cells to AldH1l1 -EGFP and H2B-TagRFP. Bar, 50 µm. ( G ) Quantification of the stained images shown in ( F ) ( n = 3 brains each). Total counted cells of Control, p18 OE ; 256, 274 (E16.5); 400, 449 (E17.5); 390, 473 (E18.5); 253, 365 (P0). Two-tailed t test; error bars show mean ± SEM. From left to right, ** P = 0.00752188, * P = 0.03272830, ** P = 0.00392106, * P = 0.01368411. ( H ) pCAG-mKO2 or pCAG-p18-P2A-mKO2 were transfected into cultured NS cells prepared from AldH1l1 -EGFP mouse brains, followed by induction to astrocytes using the conditions indicated on the left. Arrowheads; double-positive cells to AldH1l1 -EGFP and H2B-TagRFP. Bar, 20 µm. ( I ) Quantification of the stained images shown in ( H ) ( n = 3, independent culture each). Total counted cells of Control, p18 OE; 220, 296 (FBS + BMP4 + LIF + ); 140, 170 (FBS- BMP4-LIF-); 195, 380 (FBS-BMP4 + LIF + ). Two-tailed t test; error bars show mean ± SEM. From left to right, * P = 0.04486357, * P = 0.02332944, * P = 0.00392106, *** P = 0.00050115. .

Article Snippet: Then, the mKO2 coding sequence amplified from pmKO2-S1 (#AM-V0141M, MBL) using primers containing the P2A sequence (Kim et al, ) was inserted by replacing EGFP using BamH1 and Not1.

Techniques: Expressing, Staining, Control, Two Tailed Test, Transfection, Cell Culture

Journal: The EMBO Journal

Article Title: Cyclin-dependent kinase inhibitor p18 regulates lineage transitions of excitatory neurons, astrocytes, and interneurons in the mouse cortex

doi: 10.1038/s44318-024-00325-9

Figure Lengend Snippet: ( A ) Constructs for Tg mouse generation. Arrows indicate primers for genotyping. ( B ) To label proliferating neural progenitors, EdU was injected into pregnant R26R-p18-P2A-mKO2/Nes-CreER T2 mice 4 h prior to tamoxifen administration at E15.5, followed by fixation at E16.5. Sections were stained using AldH1l1 (a) and Ngn2 (b) antibodies with EdU detection. Arrowheads; double-positive cells to EdU and AldH1l1 (a) or Ngn2 (b). Bar, 100 µm. ( C ) Quantification of the stained images shown in ( B ) ( n = 3 brains each). Total counted cells of mKO2-, mKO2 + ; 571, 523 (AldH1l1); 488, 444 (Ngn2). Two-tailed t test; error bars show mean ± SEM. * P = 0.02391804 (a), * P = 0.01248425 (b). ( D ) Mice were prepared as indicated in ( B ), and then pups were fixed at P10. Sections were stained using Satb2, AldH1l1, and Sox9 antibodies with EdU detection. Arrowheads; double-positive cells to EdU and Satb2 (yellow) or AldH1l1 (light blue). Bar, 100 µm. ( E ) Quantification of images in ( D ). The bars indicate the proportions of Satb2 (a)-, AldH1l1 (b)-, and Sox9 (c)-positive cells among EdU-positive cells in each BIN. t tests were performed between the control and p18 in the same BINs ( n = 3–4 brains each). Total counted cells of mKO2-, mKO2 + ; 385, 486 (Satb2 + /EdU + , AldH1l1 + /EdU + ); 305, 345 (Sox9 + /EdU + ). Two-tailed t test; error bars show mean ± SEM. From top to bottom, * P = 0.04270011, * P = 0.04262900 (a), *** P = 0.00024186 (b), ** P = 0.00392151 (c). .

Article Snippet: Then, the mKO2 coding sequence amplified from pmKO2-S1 (#AM-V0141M, MBL) using primers containing the P2A sequence (Kim et al, ) was inserted by replacing EGFP using BamH1 and Not1.

Techniques: Construct, Injection, Staining, Two Tailed Test, Control

Journal: The EMBO Journal

Article Title: Cyclin-dependent kinase inhibitor p18 regulates lineage transitions of excitatory neurons, astrocytes, and interneurons in the mouse cortex

doi: 10.1038/s44318-024-00325-9

Figure Lengend Snippet: ( A ) Differential expression analysis comparing mKO2- (Control) and mKO2+ (p18 OE) at each stage. At E13.5 and E16.5, 1,211 and 273 DEGs were identified, respectively. There were changes in the expression levels of genes related to “regulation of neural differentiation” at E13.5, whereas no terms related to brain development were found at E16.5. ( B ) The expression levels of neural markers ( Map2 and Nptx1 ) and astrocyte markers ( Aldh1l1 and Gfap ) in mKO2- and mKO2+ mice at E13.5 and E16.5. FDR was calculated by DESeq2. From top to bottom, left to right, *** P = = 1.44e-11, *** P = 7.15e-7 (Map2), *** P = 3.63e-11, *** P = 1.28e-10 (Nptx1), ** P = 0.000234, *** P = 1.05e-9, *** P = 6.14e-7, * P = 0.00120 (AldH1l1), *** P = 1.36e-5, *** P = 1.58e-5, * P = 0.00936 (Gfap). ( C ) The expression levels of p27 ( Cdkn1b ) in mKO2- and mKO2+ mice at E13.5 and E16.5 ( n = 3 for each sample). FDR was calculated by DESeq2; error bars show mean ± SD. No statistically significant differences were detected. ( D ) Schema of the analysis pipeline for the cell type deconvolution. ( E ) UMAPs of scRNA-seq from the developing mouse brain (La Manno et al, ). Subtypes of Neuron ( Mapt + , Ly6h + ), Intermediate progenitor ( Eomes + ), Radial glia ( Nes + , Vim + ) and GABAergic neuron ( Gad1 + , Gad2 + ) were indicated by their marker genes. ( F ) Full deconvolution result using BayesPrism. Each dot represents a replicate of each sample. ( G ) The expression level of the top 50 variable genes found in Fig. at E13.5 and E16.5. Representative genes are indicated by a thick black line.

Article Snippet: Then, the mKO2 coding sequence amplified from pmKO2-S1 (#AM-V0141M, MBL) using primers containing the P2A sequence (Kim et al, ) was inserted by replacing EGFP using BamH1 and Not1.

Techniques: Quantitative Proteomics, Control, Expressing, Marker

Journal: The EMBO Journal

Article Title: Cyclin-dependent kinase inhibitor p18 regulates lineage transitions of excitatory neurons, astrocytes, and interneurons in the mouse cortex

doi: 10.1038/s44318-024-00325-9

Figure Lengend Snippet: ( A ) Positions of CpG (green) and the Stat3 binding site at the promoter region of GFAP (yellow) investigated in a previous study (Takizawa et al, ). ( B ) Representative results showing methylated sites (black) among CpG positions (circle) in the cortical VZ of p18-P2A-mKO2-negative or -positive mice. Tamoxifen was administered to pregnant R26R-p18-P2A-mKO2/Nes-CreER T2 mice at E12.5 and 15.5, and then embryonic brains were dissected at E13.5 and 16.5. After the preparation of fresh brain sections, the genomic DNA from the VZ isolated by LMD was prepared for bisulfite assay. The yellow color indicates the Stat3 binding site. ( C ) Quantification of the methylation status at the Stat3 binding site at E13.5 and E16.5 in mKO2- and mKO2+ mice ( n = 3 for E13.5 and 4 for E16.5 brains). Two-tailed t -test; error bars show mean ± SEM. No statistically significant differences were detected.

Article Snippet: Then, the mKO2 coding sequence amplified from pmKO2-S1 (#AM-V0141M, MBL) using primers containing the P2A sequence (Kim et al, ) was inserted by replacing EGFP using BamH1 and Not1.

Techniques: Binding Assay, Methylation, Isolation, Two Tailed Test

Journal: The EMBO Journal

Article Title: Cyclin-dependent kinase inhibitor p18 regulates lineage transitions of excitatory neurons, astrocytes, and interneurons in the mouse cortex

doi: 10.1038/s44318-024-00325-9

Figure Lengend Snippet: Primers for plasmids generated in this study.

Article Snippet: Then, the mKO2 coding sequence amplified from pmKO2-S1 (#AM-V0141M, MBL) using primers containing the P2A sequence (Kim et al, ) was inserted by replacing EGFP using BamH1 and Not1.

Techniques: Generated, shRNA

Journal: The EMBO Journal

Article Title: Cyclin-dependent kinase inhibitor p18 regulates lineage transitions of excitatory neurons, astrocytes, and interneurons in the mouse cortex

doi: 10.1038/s44318-024-00325-9

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Then, the mKO2 coding sequence amplified from pmKO2-S1 (#AM-V0141M, MBL) using primers containing the P2A sequence (Kim et al, ) was inserted by replacing EGFP using BamH1 and Not1.

Techniques: Mutagenesis, Recombinant, Sequencing, SYBR Green Assay, Transfection, Software, Amplification, Imaging, Plasmid Preparation, Reporter Assay, Modification

Journal: Nature Communications

Article Title: HuR controls glutaminase RNA metabolism

doi: 10.1038/s41467-024-49874-x

Figure Lengend Snippet: MIA PaCa-2 expression levels of ELAVL1 and GLS from RNA-Seq data ( a , mean and S.E.M.) and heatmap ( b ) with bidirectional clustering of singscores from KEGG Metabolic Pathways and scaled ELAVL1 expression. The letter ‘Q’ marks pathways that involve glutamine from (Control n = 4; Knockout n = 3). The dendrograms represent full bidirectional complete clustering based on correlation values. c Chromosome 2 indicating GLS loci and exon structure for KGA and GAC isoforms. Bellow, exon differential expression after ELAVL1 knockdown in MIA PaCa-2 or HeLa cells. Asterisks denote FDR < 0.05. d , above, Western blot of doxycycline-inducible ELAVL1 -silenced breast cancer cell lines (MDA-MB-231 and BT549) using two shRNAs, displaying a marked decrease in KGA protein level, accompanied by an increase in GAC. d , below, Western blot of constitutively silenced ELAVL1 breast cancer cell lines, grouped by telaglenastat resistance, which was defined elsewhere . Arrows indicate KGA and GAC isoforms and HuR-specific bands and western blots repeated at least two times with reproducible results. e Scheme for CRISPR-mediated knock-in of the mKO2-P2A-BleoR cassette into GLS exon 19 to produce the KGA-mKO2 fusion protein. f Increase and decrease in cellular fluorescence after ELAVL1 ectopic expression (bottom) or sh ELAVL1 -mediated transient silencing (top), respectively, in HEK293T knock-in cells, number independent cells evaluated per condition >1433. Representative images on the right. The scale bar is 50 μm. g Cell cycle analysis of BT549 knock-in cells KGA levels (evaluated by mKO2 fluorescence) are enhanced in S-G2/M phases, as expected , . Representative images in bottom part, with HiLo lookup table for DNA staining, one cell with higher DNA content and higher KGA content and the opposite example below, confirming the functionality of the system. Box plots represent the interquartile range; the vertical curve is the kernel density of the distribution, and the dark horizontal line denotes the median. Each dot represents an individual cell. Statistical significance derived from bootstrapped DEXSeq ( c ) or Two-sided Welch’s t -test ( f ). * p < 0.05, ** p < 0.01, *** p < 0.0001.

Article Snippet: Primary antibodies used for immunofluorescence and immunoblotting were Vinculin (Abcam ab18058), Actin (Abcam ab3280), GLS (Abcam ab156876), GAC (RheaBiotech IM-0322), KGA (RheaBiotech IM-0519), HuR (Molecular Probes mp21277 and Cell Signaling 12582), and V5 (Life Technologies 46-1157). pLKO.1 puro was a gift from Bob Weinberg (Addgene plasmid # 8453); pLKO.1-blast, used in GAC silencing experiment was a gift from Keith Mostov (Addgene plasmid # 26655); Tet-pLKO-puro was a gift from Dmitri Wiederschain (Addgene plasmid # 21915); psPAX2 was a gift from Didier Trono (Addgene plasmid # 12260); pMD2.G was a gift from Didier Trono (Addgene plasmid # 12259); pUMVC was a gift from Robert A. Weinberg (Addgene plasmid # 8449); pCMV-VSV-G was a gift from Robert Weinbeg (Addgene plasmid # 8454); pQC mKorange IX was a gift from Connie Cepko (Addgene plasmid # 37344), previously modified by replacing the mKO2 coding sequence with an empty MCS , generating pQC MCS IRES Puro (Addgene # 110343) and pQC V5 MCS IRES Puro (Addgene # 110342).

Techniques: Expressing, RNA Sequencing, Control, Knock-Out, Quantitative Proteomics, Knockdown, Western Blot, CRISPR, Knock-In, Fluorescence, Cell Cycle Assay, Staining, Derivative Assay

Journal: Nature Communications

Article Title: HuR controls glutaminase RNA metabolism

doi: 10.1038/s41467-024-49874-x

Figure Lengend Snippet: a Relative quantification of BT549 mRNA levels following doxycycline-inducible silencing of ELAVL1 , using qPCR for two distinct shRNA sequences. b HuR was immunoprecipitated (western blot above, arrows indicate HuR and IgG heavy chain). The control IgG were used from rabbit, in opposing to the mouse anti-HuR antibody. Quantification of mRNA derived from 3′UTR KGA regions immunoprecipitated bound in HuR from ( c ) PC-3 cells using agarose gel and ( d ) from BT549 using qRT-PCR. Quantification of mRNA derived from 3′UTR GAC regions immunoprecipitated bound in HuR from ( e ) PC-3 cells using agarose gel and ( f ) from BT549 using qRT-PCR. Agarose gel from PC-3 ( g ) and q-RT-PCR from BT549 ( h ) revealed that HuR binds to its mRNA (as already published elsewhere ) in addition to GLS intron 14. i In vitro FRAP analysis of recombinant mKO2-HuR incubated with in vitro transcribed control RNA or intron 14. The bar plot (right) denotes T 1/2 recovery times. Statistical significance derived from Two-sided Welch’s t-test ( a , d , f , h , and i ), error bars are SEM; qRT-PCR assays were evaluated in triplicate; otherwise, each point represents a replicate. * p < 0.05, ** p < 0.01, *** p < 0.0001.

Article Snippet: Primary antibodies used for immunofluorescence and immunoblotting were Vinculin (Abcam ab18058), Actin (Abcam ab3280), GLS (Abcam ab156876), GAC (RheaBiotech IM-0322), KGA (RheaBiotech IM-0519), HuR (Molecular Probes mp21277 and Cell Signaling 12582), and V5 (Life Technologies 46-1157). pLKO.1 puro was a gift from Bob Weinberg (Addgene plasmid # 8453); pLKO.1-blast, used in GAC silencing experiment was a gift from Keith Mostov (Addgene plasmid # 26655); Tet-pLKO-puro was a gift from Dmitri Wiederschain (Addgene plasmid # 21915); psPAX2 was a gift from Didier Trono (Addgene plasmid # 12260); pMD2.G was a gift from Didier Trono (Addgene plasmid # 12259); pUMVC was a gift from Robert A. Weinberg (Addgene plasmid # 8449); pCMV-VSV-G was a gift from Robert Weinbeg (Addgene plasmid # 8454); pQC mKorange IX was a gift from Connie Cepko (Addgene plasmid # 37344), previously modified by replacing the mKO2 coding sequence with an empty MCS , generating pQC MCS IRES Puro (Addgene # 110343) and pQC V5 MCS IRES Puro (Addgene # 110342).

Techniques: Quantitative Proteomics, shRNA, Immunoprecipitation, Western Blot, Control, Derivative Assay, Agarose Gel Electrophoresis, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, In Vitro, Recombinant, Incubation