Journal: Applied and Environmental Microbiology

Article Title: Contrasting Microbiome Dynamics of Putative Denitrifying Bacteria in Two Octocoral Species Exposed to Dissolved Organic Carbon (DOC) and Warming

doi: 10.1128/AEM.01886-21

Figure Lengend Snippet: Community compositions and relative abundances of putative denitrifiers in corals X. umbellata and P. flava over the course of the experiment. (a) Relative proportions of denitrifier genera of corals X. umbellata and P. flava inferred by nirS in-silico PCR in relation to the total bacterial community from 16S rRNA gene sequencing. (b) Relative fold changes in copy numbers of nirS gene referenced to 16S rRNA gene and in relation to the day 0 control samples ( n = 3) of corals X. umbellata and P. flava . Values are means ± SD, and the asterisk indicates statistically significant differences (* P < 0.05).

Article Snippet: The 16S rRNA gene amplicon sequencing was conducted at LGC genomics (Berlin, Germany).

Techniques: In Silico, Sequencing

Journal: Nature Communications

Article Title: LSD1 co-repressor Rcor2 orchestrates neurogenesis in the developing mouse brain

doi: 10.1038/ncomms10481

Figure Lengend Snippet: ( a ) X-gal staining to detect Rcor2 expression patterns. Whole-embryo staining at E11.5 stage shows Rcor2 is mainly expressed in the CNS. Scale bar, 1 mm. ( b ) Western blot analysis of Rcor2 expression levels during brain development. The decreased expression of Rcor2 with embryonic development is noteworthy. β-Actin is used as an endogenous control. ( c ) In situ hybridization to detect endogenous Rcor2 mRNA expression patterns in cortical development at E11.5, E13.5, E15.5 and E17.5. Insets show high-magnification image of Rcor2 expression in the neocortex at E13.5. VZ, ventricular zone; SVZ, subventricular zone; CP, cortical plate. Scale bar, 100 μm. ( d ) Confocal images of immunofluorescence to detect cellular localization of Rcor2 in the neocortex at E13.5. Rcor2 localized mainly in the nucleus at interphase and metaphase, and localized between separated chromosomes in anaphase of dividing cells in VZ. Dotted lines circle the shape of nuclei. Scale bar, 5 μm. ( e ) Western blot analysis of Rcor2 expression level in Rcor2 fl/fl and Rcor2 cko brains at E13.5 and E15.5, respectively. Rcor2 expression was depleted in Rcor2 cko brains. β-Actin is used as an endogenous control. ( f ) Representative images of Rcor2 fl/fl and Rcor2 cko brain size at different stages of development. Rcor2 cko mice show severe brain growth retardation at E13.5 and E15.5. Scale bar, 1 mm. ( g ) Representative images of Rcor2 fl/fl and Rcor2 cko cortex at E15.5 by Nissl staining. Structural abnormalities of lamination with reduced cortical thickness are observed in Rcor2 cko cortex. Scale bar, 200 μm. ( h ) RT–qPCR analysis of knockdown efficiencies of the two shRNAs targeting Rcor2. Transcripts were normalized to the control group. Data are shown as mean±s.e.m., t -test, **** P <0.0001, n =3. ( i ) Confocal images of E16.5 cortical sections electroporated with shControl (red), shRcor2-a (red) and shRcor2-b (red) plasmids at E13.5. Knockdown of Rcor2 results in impaired cortical development. IZ, intermediate zone. Scale bar, 20 μm. ( j ) Quantification of the percentage of RFP + cells in different regions of the developing neocortex after electroporation as shown in i . proportion of RFP + cells in different zones ( y axis). Data are shown as mean±s.e.m., t -test, * P <0.05, ** P <0.01, *** P <0.001 and **** P <0.0001, n =3 individual experiments.

Article Snippet: Primary antibodies used were as follows: Rcor2 (1:50, catalogue number NBP1-74099, Novus Biological); Nes (1:300, catalogue number Rat-401, DSHB); Pax6 (1:100, DSHB); Sox2 (1:500, catalogue number sc-17319, Santa Cruz); BrdU (1:500, catalogue number ab6326, Abcam); Ki67 (1:300, catalogue number ab9260, Millipore); Satb2 (1:500, catalogue number ab34735, Abcam); Olig2 (1:200, catalogue number ab109186, Abcam); Shh (1:100, catalogue number sc-33943, Santa Cruz); Ptch1 (1:100, catalogue number sc-6149, Santa Cruz); Dlx2 (1:500, catalogue number ab117546, Abcam); Nkx2.1 (1:300, catalogue number ab76013, Abcam); Tbr1 (1:500, catalogue number ab31940, Abcam); Dcx (1:500, catalogue number ab18723, Abcam); Tbr2 (1:500, catalogue number ab23345, Abcam); Flag M2 (1:1,000, catalogue number F1804, Sigma); Map2 (1:500, catalogue number ab32454, Abcam); and Tuj1 (1:1,000, catalogue number mms-435p, Convance).

Techniques: Staining, Expressing, Western Blot, Control, In Situ Hybridization, Immunofluorescence, Quantitative RT-PCR, Knockdown, Electroporation

Journal: Nature Communications

Article Title: LSD1 co-repressor Rcor2 orchestrates neurogenesis in the developing mouse brain

doi: 10.1038/ncomms10481

Figure Lengend Snippet: ( a ) Immunostaining images of Sox2 at E13.5. Sox2 is dramatically reduced on Rcor2 depletion. VZ, ventricular zone; SVZ, subventricular zone. Scale bar, 20 μm. ( b ) Quantification of Sox2 + cell ratios in VZ/SVZ regions shown in a . Data are shown as mean±s.e.m., t -test, ** P <0.01, n =3 individual experiments. ( c ) Confocal images of Tbr2 expression at E13.5. Tbr2 is dramatically reduced on Rcor2 depletion. Scale bar, 20 μm. ( d ) Quantification of Tbr2 + cell ratios in VZ/SVZ regions shown in c , respectively. Data are shown as mean±s.e.m., t -test, * P <0.05, n =3 individual experiments. ( e ) Immunostaining images of Nestin, Sox2 and Tbr2 in cultured Rcor2 fl/fl and Rcor2 cko NPCs, all of which exhibit significantly reduced expression in the Rcor2 cko NPCs. Scale bar, 20 μm. ( f ) Confocal images of immunofluorescence for Ki67 and PHH3 in Rcor2 fl/fl and Rcor2 cko cortex at E13.5 and E15.5. Ki67 signals (red), but not PHH3 signals (green), are dramatically reduced in Rcor2 cko developing brains. Scale bar, 20 μm. ( g ) Quantification of Ki67 + cells in the VZ/SVZ regions of the developing neocortex as shown in f . Data are shown as mean±s.e.m., t -test, ** P <0.01, n =3 separate stainings from three independent brains. ( h ) Confocal images of BrdU (green) and Ki67 (red) staining in Rcor2 fl/fl and Rcor2 cko cortex 24 and 48 h after BrdU incorporation. Scale bar, 100 μm. ( i ) Quantification of the cell cycle exit by percentage of BrdU + and Ki67 + NPCs divided by BrdU + cells shown in h . Data are shown as mean±s.e.m., t -test, *** P <0.001 and **** P <0.0001, n =3 individual experiments. ( j , k ) Representative images ( j ) and quantification of ( k ) of Rcor2 fl/fl and Rcor2 cko neurosphere sizes. The neurospheres'radius of Rcor2 cko are much smaller than those of Rcor2 fl/fl , t -test, **** P <0.0001, n =12. Scale bar, 50 μm. ( l ) Representative time-lapse imaging of the RGC dividing process in the sections of the cerebral cortex electroporated with RFP-shControl (upper panels) and RFP-shRcor2 (lower panels). The radial glial dividing pattern is abnormal on Rcor2 knockdown, resulting in cell death. Arrows, mother RGCs. Arrowheads, two daughter cells. Scale bar, 50 μm. ( m ) Representative time-lapse images of Rcor2 fl/fl cortex sections electroporated with EGFP-Control (upper panels) and EGFP-Cre (lower panels). Loss of cells is observed with Rcor2 knockout by Cre recombinase electroporation. Scale bar, 50 μm.

Article Snippet: Primary antibodies used were as follows: Rcor2 (1:50, catalogue number NBP1-74099, Novus Biological); Nes (1:300, catalogue number Rat-401, DSHB); Pax6 (1:100, DSHB); Sox2 (1:500, catalogue number sc-17319, Santa Cruz); BrdU (1:500, catalogue number ab6326, Abcam); Ki67 (1:300, catalogue number ab9260, Millipore); Satb2 (1:500, catalogue number ab34735, Abcam); Olig2 (1:200, catalogue number ab109186, Abcam); Shh (1:100, catalogue number sc-33943, Santa Cruz); Ptch1 (1:100, catalogue number sc-6149, Santa Cruz); Dlx2 (1:500, catalogue number ab117546, Abcam); Nkx2.1 (1:300, catalogue number ab76013, Abcam); Tbr1 (1:500, catalogue number ab31940, Abcam); Dcx (1:500, catalogue number ab18723, Abcam); Tbr2 (1:500, catalogue number ab23345, Abcam); Flag M2 (1:1,000, catalogue number F1804, Sigma); Map2 (1:500, catalogue number ab32454, Abcam); and Tuj1 (1:1,000, catalogue number mms-435p, Convance).

Techniques: Immunostaining, Expressing, Cell Culture, Immunofluorescence, Staining, BrdU Incorporation Assay, Imaging, Knockdown, Control, Knock-Out, Electroporation

Journal: Nature Communications

Article Title: LSD1 co-repressor Rcor2 orchestrates neurogenesis in the developing mouse brain

doi: 10.1038/ncomms10481

Figure Lengend Snippet: ( a ) Confocal images of Satb2 and Tbr1 expressions in Rcor2 fl/fl and Rcor2 cko cortex at E15.5, which exhibit significant reduction on Rcor2 knockout. Scale bar, 50 μm. ( b ) Quantification of Satb2 + and Tbr1 + cells in Rcor2 fl/fl and Rcor2 cko cortex at E15.5 in a indicates Satb2 and Tbr1 expressions are decreased on Rcor2 depletion during development. Data are shown as mean±s.e.m., t -test, ** P <0.01, n =3 individual experiments. ( c ) Western blot to analyse Dcx, Satb2 and Tbr1 expressions in Rcor2 fl/fl and Rcor2 cko cortex at E15.5. β-Actin is used as an endogenous control. ( d ) Representative images of Map2 and Tuj1 immunostaining in cultured neurons directly dissociated from Rcor2 fl/fl and Rcor2 cko brain cortex at E15.5. Decreased expression of both markers and reduced neurofilaments can be observed in Rcor2 cko cultured neurons. Scale bar, 20 μm. ( e ) Confocal images of in-vitro cultured Rcor2 fl/fl and Rcor2 cko NPCs 5 days post spontaneous differentiation using neuronal marker Map2 and Tuj1 antibodies, both of which are significantly reduced in the differentiated Rcor2 cko NPCs. Scale bar, 20 μm. ( f , g ) RT–qPCR analysis of neuronal markers expression in both Rcor2 fl/fl and Rcor2 cko neocortex at E15.5 stage ( f ) and in-vitro -cultured Rcor2 fl/fl and Rcor2 cko NPCs 5 days post spontaneous differentiation ( g ). Transcripts were normalized to Rcor2 fl/fl group. Data are shown as mean±s.d., t -test, * P <0.05, ** P <0.01 and *** P <0.001, n =3.

Article Snippet: Primary antibodies used were as follows: Rcor2 (1:50, catalogue number NBP1-74099, Novus Biological); Nes (1:300, catalogue number Rat-401, DSHB); Pax6 (1:100, DSHB); Sox2 (1:500, catalogue number sc-17319, Santa Cruz); BrdU (1:500, catalogue number ab6326, Abcam); Ki67 (1:300, catalogue number ab9260, Millipore); Satb2 (1:500, catalogue number ab34735, Abcam); Olig2 (1:200, catalogue number ab109186, Abcam); Shh (1:100, catalogue number sc-33943, Santa Cruz); Ptch1 (1:100, catalogue number sc-6149, Santa Cruz); Dlx2 (1:500, catalogue number ab117546, Abcam); Nkx2.1 (1:300, catalogue number ab76013, Abcam); Tbr1 (1:500, catalogue number ab31940, Abcam); Dcx (1:500, catalogue number ab18723, Abcam); Tbr2 (1:500, catalogue number ab23345, Abcam); Flag M2 (1:1,000, catalogue number F1804, Sigma); Map2 (1:500, catalogue number ab32454, Abcam); and Tuj1 (1:1,000, catalogue number mms-435p, Convance).

Techniques: Knock-Out, Western Blot, Control, Immunostaining, Cell Culture, Expressing, In Vitro, Marker, Quantitative RT-PCR

Journal: Nature Communications

Article Title: LSD1 co-repressor Rcor2 orchestrates neurogenesis in the developing mouse brain

doi: 10.1038/ncomms10481

Figure Lengend Snippet: ( a ) Schematic overview of strategy to generate an Rcor2 Flag knock-in allele by CRISPR/Cas9. The sgRNA sequence site is shown as a green arrowhead. The start codon of Rcor2 is indicated and capitalized. The oligo donor contained 50 bp homologies on both sides flanking the DSB, in which 3 × Flag sequences are labelled as a red box. ( b ) Western blot analysis to validate FLAG, RCOR2 and LSD1 expressions in Rcor2 Flag knock-in neocortex using Flag-M2 antibody. β-Actin was used as an endogenous control. ( c ) Pie chart depicts distribution of Rcor2 occupancies in genome-wide scale in FLAG ChIP-seq results using Rcor2 Flag knock-in neocortex at E13.5. ( d ) WebLogos of consensus binding motifs of Rcor2 generated by Multiple EM for Motif Elicitation (MEME) motif analysis tool. ( e ) GO analysis for Rcor2-binding regions in genome-wide scale revealed by Flag ChIP-seq results using Rcor2 Flag brain. ( f ) GO analysis for LSD1 occupancy in genome-wide scale revealed by LSD1 ChIP-seq results using Rcor2 Flag brain. ( g ) Density plots analysis of H3K4me1 signal change in promoter regions (−2- to ∼0.5 kb from TSS) on Rcor2 depletion. Compared with all genes, the change of H3K4me1 signal is significantly ( P <0.0005, Kolmogorov–Smirnov test) enhanced in the promoter regions of Shh pathway-related genes on Rcor2 depletion. H3K4me1 signal change on Rcor2 depletion ( x axis); H3K4me1 signal density ( y axis). ( h ) Gene tracks of Rcor2, LSD1 and H3K4me1 enrichments by ChIP-seq analysis at core promoter regions of Dlx2 and upstream regulatory regions of Shh, which are closely related to Shh signalling. ( i ) ChIP–qPCR analysis of Rcor2 Flag and Rcor2 cko cortex at E13.5 using specific FLAG-M2 antibody. Significant enrichments of the Rcor2 at the regulatory regions of Dlx2 and Shh gene locus detected in g in the Rcor2 flag samples are worth noting. Fold enrichments of Rcor2 occupancy compared with input ( y axis). Data are shown as mean±s.d., t -test, *** P <0.001, n =3.

Article Snippet: Primary antibodies used were as follows: Rcor2 (1:50, catalogue number NBP1-74099, Novus Biological); Nes (1:300, catalogue number Rat-401, DSHB); Pax6 (1:100, DSHB); Sox2 (1:500, catalogue number sc-17319, Santa Cruz); BrdU (1:500, catalogue number ab6326, Abcam); Ki67 (1:300, catalogue number ab9260, Millipore); Satb2 (1:500, catalogue number ab34735, Abcam); Olig2 (1:200, catalogue number ab109186, Abcam); Shh (1:100, catalogue number sc-33943, Santa Cruz); Ptch1 (1:100, catalogue number sc-6149, Santa Cruz); Dlx2 (1:500, catalogue number ab117546, Abcam); Nkx2.1 (1:300, catalogue number ab76013, Abcam); Tbr1 (1:500, catalogue number ab31940, Abcam); Dcx (1:500, catalogue number ab18723, Abcam); Tbr2 (1:500, catalogue number ab23345, Abcam); Flag M2 (1:1,000, catalogue number F1804, Sigma); Map2 (1:500, catalogue number ab32454, Abcam); and Tuj1 (1:1,000, catalogue number mms-435p, Convance).

Techniques: Knock-In, CRISPR, Sequencing, Western Blot, Control, Genome Wide, ChIP-sequencing, Binding Assay, Generated, ChIP-qPCR

Journal: Nature Communications

Article Title: LSD1 co-repressor Rcor2 orchestrates neurogenesis in the developing mouse brain

doi: 10.1038/ncomms10481

Figure Lengend Snippet: ( a , b ) Scatter plot analysis of genome-wide expression profiles of Rcor2 cko versus Rcor2 fl/fl samples at E13.5 ( a ) and E15.5 ( b ). Dots above or below the dash line indicate upregulated or downregulated genes on Rcor2 depletion, respectively. Red dots or green dots highlight the significantly differentially expressed genes on Rcor2 depletion. Raw counts ( x axis); gene expression fold changes on Rcor2 depletion ( y axis). ( c ) Venn diagrams of upregulated genes (left) and downregulated genes (right) in Rcor2 cko samples compared with Rcor2 fl/fl samples. ( d ) The profiles of Rcor2 and H3k4me1 enrichments analysed in ChIP-seq results shown in in regulatory regions of genome-wide scale (red) and of the upregulated genes (purple) according to RNA-seq results. ( e ) Correlation network of overlapped upregulated genes in both E13.5 and E15.5 samples. Lines indicate the correlations between two connected genes with R >0.55. Genes were analysed by GO analysis and divided into different categories.

Article Snippet: Primary antibodies used were as follows: Rcor2 (1:50, catalogue number NBP1-74099, Novus Biological); Nes (1:300, catalogue number Rat-401, DSHB); Pax6 (1:100, DSHB); Sox2 (1:500, catalogue number sc-17319, Santa Cruz); BrdU (1:500, catalogue number ab6326, Abcam); Ki67 (1:300, catalogue number ab9260, Millipore); Satb2 (1:500, catalogue number ab34735, Abcam); Olig2 (1:200, catalogue number ab109186, Abcam); Shh (1:100, catalogue number sc-33943, Santa Cruz); Ptch1 (1:100, catalogue number sc-6149, Santa Cruz); Dlx2 (1:500, catalogue number ab117546, Abcam); Nkx2.1 (1:300, catalogue number ab76013, Abcam); Tbr1 (1:500, catalogue number ab31940, Abcam); Dcx (1:500, catalogue number ab18723, Abcam); Tbr2 (1:500, catalogue number ab23345, Abcam); Flag M2 (1:1,000, catalogue number F1804, Sigma); Map2 (1:500, catalogue number ab32454, Abcam); and Tuj1 (1:1,000, catalogue number mms-435p, Convance).

Techniques: Genome Wide, Expressing, Gene Expression, ChIP-sequencing, RNA Sequencing

Journal: Nature Communications

Article Title: LSD1 co-repressor Rcor2 orchestrates neurogenesis in the developing mouse brain

doi: 10.1038/ncomms10481

Figure Lengend Snippet: ( a ) qPCR analysis of the expression of genes related to the Shh signalling pathway in the cortex of Rcor2 fl/fl and Rcor2 cko brains during development. Significant upregulation of these genes on Rcor2 depletion is noteworthy. Transcripts were normalized to Rcor2 fl/fl group. Data are shown as mean±s.d., t -test, * P <0.05, ** P <0.01 and *** P <0.001, n =3. ( b ) Confocal images of Shh and Ptch1 expressions in Rcor2 fl/fl and Rcor2 cko cortex. Enhanced Shh and Ptch1 signals are observed in Rcor2 cko compared with Rcor2 fl/fl neocortex at E13.5 and E15.5. Insets show high-magnification images of the outlined regions. Scale bars, 50 μm. ( c ) Dlx2 expression in Rcor2 fl/fl and Rcor2 cko cortex detected by immunofluorescence analysis at E15.5. Dlx2 + cells were observed in the neocortex on Rcor2 depletion. Scale bars, 50 μm. ( d ) Confocal images of Shh, Ptch1 and Dlx2 expressions in in-vitro -cultured Rcor2 cko NPCs. Scale bar, 20 μm. ( e ) Western blot analysis of expression levels of Dlx2, Shh and Ptch1 in Rcor2 fl/fl and Rcor2 cko cortex at E15.5. β-Actin is used as an endogenous control.

Article Snippet: Primary antibodies used were as follows: Rcor2 (1:50, catalogue number NBP1-74099, Novus Biological); Nes (1:300, catalogue number Rat-401, DSHB); Pax6 (1:100, DSHB); Sox2 (1:500, catalogue number sc-17319, Santa Cruz); BrdU (1:500, catalogue number ab6326, Abcam); Ki67 (1:300, catalogue number ab9260, Millipore); Satb2 (1:500, catalogue number ab34735, Abcam); Olig2 (1:200, catalogue number ab109186, Abcam); Shh (1:100, catalogue number sc-33943, Santa Cruz); Ptch1 (1:100, catalogue number sc-6149, Santa Cruz); Dlx2 (1:500, catalogue number ab117546, Abcam); Nkx2.1 (1:300, catalogue number ab76013, Abcam); Tbr1 (1:500, catalogue number ab31940, Abcam); Dcx (1:500, catalogue number ab18723, Abcam); Tbr2 (1:500, catalogue number ab23345, Abcam); Flag M2 (1:1,000, catalogue number F1804, Sigma); Map2 (1:500, catalogue number ab32454, Abcam); and Tuj1 (1:1,000, catalogue number mms-435p, Convance).

Techniques: Expressing, Immunofluorescence, In Vitro, Cell Culture, Western Blot, Control

Journal: Nature Communications

Article Title: LSD1 co-repressor Rcor2 orchestrates neurogenesis in the developing mouse brain

doi: 10.1038/ncomms10481

Figure Lengend Snippet: ( a ) Knockdown of Rcor2 impairs cortical neurogenesis, which can be partially rescued by knockdown of Shh during cortical development. In-utero electroporation with RFP-shControl (red)/GFP-shControl (green), RFP-shRcor2 (red)/GFP-shControl (green), RFP-shControl (red)/GFP-shShh (green) or RFP-shRcor2 (red)/GFP-shShh (green) plasmids was performed at E13.5. Cerebral sections were fixed and imaged at E16.5. VZ, ventricular zone; SVZ, subventricular zone; CP, cortical plate. Scale bar, 50 μm. ( b ) Quantification of the percentage of RFP + /GFP + cells in different regions of the developing cortex after electroporation shown in a . Data are shown as mean±s.e.m., t -test, ** P <0.01 and *** P <0.001, n =3 individual experiments. ( c ) Inhibition of Shh by Cyclopamine can partially rescue neurogenesis defects caused by Rcor2 downregulation during cortical development. Rcor2 was knocked down at the lateral ventricle in the brain by in-utero electroporation with RFP-shRcor2 plasmids at E13.5. Cerebral sections were collected at E14.5 and then treated with cyclopamine to inhibit Shh activity for 48 h. Scale bar, 50 μm. ( d ) Quantification of the percentage of RFP + cells in different regions of the developing neocortex after knockdown of Rcor2 or inhibition of Shh shown in c . Data are shown as mean±s.e.m., t -test, * P <0.05, n =3 individual experiments. ( e ) Representative images depicting neurosphere size is partially rescued in the in-vitro -cultured Rcor2 cko NPCs after treatment with Cyclopamine. Scale bar, 50 μm. ( f ) Histogram depicting cell numbers of in-vitro -cultured Rcor2 fl/fl and Rcor2 cko NPCs with or without Cyclopamine treatment for 48 h. Cells (5 × 10 5 ) are seeded initially. Data are shown as mean±s.e.m., t -test, ** P <0.01, n =3. ( g ) Confocal images of Tuj1 expression in the differentiated cells from in-vitro -cultured Rcor2 fl/fl and Rcor2 cko NPCs with or without Cyclopamine treatment. Tuj1 expressions are partially restored in Cyclopamine-treated Rcor2 cko cells. Scale bar, 20 μm. ( h ) Model of Rcor2 function in neurogenesis in the developing neocortex. Rcor2 safeguards cortical neurogenesis by recruiting LSD1 complex to the regulatory regions of Dlx2 and Shh genes, to inhibit the Shh pathway activation during development. The absence of Rcor2 leads to inhibition release of these genes and thus ectopic activation of Shh signalling in the developing neocortex, resulting in cortical neurogenesis defects.

Article Snippet: Primary antibodies used were as follows: Rcor2 (1:50, catalogue number NBP1-74099, Novus Biological); Nes (1:300, catalogue number Rat-401, DSHB); Pax6 (1:100, DSHB); Sox2 (1:500, catalogue number sc-17319, Santa Cruz); BrdU (1:500, catalogue number ab6326, Abcam); Ki67 (1:300, catalogue number ab9260, Millipore); Satb2 (1:500, catalogue number ab34735, Abcam); Olig2 (1:200, catalogue number ab109186, Abcam); Shh (1:100, catalogue number sc-33943, Santa Cruz); Ptch1 (1:100, catalogue number sc-6149, Santa Cruz); Dlx2 (1:500, catalogue number ab117546, Abcam); Nkx2.1 (1:300, catalogue number ab76013, Abcam); Tbr1 (1:500, catalogue number ab31940, Abcam); Dcx (1:500, catalogue number ab18723, Abcam); Tbr2 (1:500, catalogue number ab23345, Abcam); Flag M2 (1:1,000, catalogue number F1804, Sigma); Map2 (1:500, catalogue number ab32454, Abcam); and Tuj1 (1:1,000, catalogue number mms-435p, Convance).

Techniques: Knockdown, In Utero, Electroporation, Inhibition, Activity Assay, In Vitro, Cell Culture, Expressing, Activation Assay

Journal: Gastric Cancer

Article Title: Insulin and the insulin receptor collaborate to promote human gastric cancer

doi: 10.1007/s10120-021-01236-y

Figure Lengend Snippet: Expression of genes that encode, and inhibition of, the insulin and type I IGF receptors in gastric adenocarcinoma. a The affinities of insulin, IGF-1 and IGF-2 for the insulin and type I IGF receptors are shown (adapted from ). Original references are included in the electronic supplementary material (Online Resource 1). Copy number and relative high expression of > 2 SD from the mean for diploid tumours of the genes that encode the insulin receptor ( INSR ) and type I IGF receptor ( IGF1R ) were analysed for 478 gastric adenocarcinomas [ , ]. b Log 2 transformed mRNA relative abundance deduced from absolute transcript abundance by RNA-Seq expectation maximization (RSEM) is shown against the putative copy-number alterations deduced from genomic sequencing data analysed with GISTIC 2.0. Horizontal bars represent the median values, boxes the range of the second and third quartiles of the data and whiskers the range of all data. Expression of INSR and IGF1R , is associated with CNV (Kruskal Wallis; p < 0.0001); statistically significant differences between groups are indicated (Dunn-Boniferroni post hoc; * p < 0.05 ( INSR ); * p < 0.005 ( IGF1R ); ** p < 0.0001 (either)). c Kaplan and Meier curves illustrate correlations analysed by the log rank test between INSR and IGF1R expression, and overall survival for 876 gastric cancer patients . d Kaplan and Meier curves illustrate correlation analysed by the log rank test between INSR expression, and overall survival for 392 gastric cancer patients . e INSR and IGF1R expression calculated as transcripts per million (tpm) in normal ( n = 34) and primary gastric tumour (1° tum.; n = 415) tissue (Student’s t -test; p < 0.0001 and p = 0.06, respectively) and methylation status upstream of INSR and IGF1R transcription start sites (Student’s t -test; p < 0.0002 and p = 0.195, respectively). f MCF-7 breast cancer cells (Br. Ca.), NCI-N87, KATO III, SNU-16, SNU-5, SNU-1, MKN74, NUGC3 and AGS gastric adenocarcinoma cells (Ga. Ad.) and metastatic cells isolated from gastric adenocarcinoma patients, GC1, HC1, NC1 and JW1, were lysed and insulin receptor, type I IGF receptor, β-tubulin or GAPDH were analysed by western transfer. ERBB2 , FGFR2 and MET are amplified and overexpressed in NCI-N87, KATO III and SNU-16, and SNU-5, respectively. SNU-1, MKN74, NUGC3 and AGS and the metastatic patient cells are triple-negative for amplification or overexpression of these oncogenes . g NCI-N87, SNU-16, SNU-5, SNU-1, MKN-74, NUGC3 and AGS were incubated in full, untreated FCS-containing medium without and with 0.5 µM BMS-754807 for three days, lysed and their DNA content measured. Asterisks indicate if there was significantly less DNA in the presence of BMS-754807 than in its absence (Student’s t -test; p < 0.01). h SNU-1, MKN-74, NUGC3 and AGS were incubated in untreated FCS-containing medium and different concentrations of BMS-754807 for three days or as indicated and their DNA content measured. The relative IC values are indicated by blue lines. Asterisks indicate times at which there was significantly less DNA in the presence of BMS-754807 than in its absence (one-way ANOVA; p < 0.0001)

Article Snippet: Cells were lysed in radioimmunoprecipitate (RIPA) buffer and analysed by western transfer as described [ ] with antibodies against: insulin receptor (#3025), type I IGF receptor (#3027), β-tubulin (#5346), phosphorylated Tyr 1150/1151 insulin and Tyr 1135/1136 type I IGF receptors (#3024), Akt (#9272), phosphorylated Ser 473 Akt (#4060), ERK1 and ERK2 (#9102), phosphorylated Thr 202/204 ERK1 and Thr 185/187 ERK2 (#4370), cleaved Asp 214 poly-(ADP-ribose) polymerase-1 (PARP-1) (#9541) (Cell Signaling Technologies), or GAPDH-HRP (sc-25778) (Santa Cruz Biotechnology).

Techniques: Expressing, Inhibition, Transformation Assay, RNA Sequencing, Genomic Sequencing, Methylation, Isolation, Western Blot, Amplification, Over Expression, Incubation

Journal: Gastric Cancer

Article Title: Insulin and the insulin receptor collaborate to promote human gastric cancer

doi: 10.1007/s10120-021-01236-y

Figure Lengend Snippet: Pharmacological inhibition of the insulin and type I receptors induces cell death a SNU-1 were cultured in suspension, whereas MKN74 and NUGC3 were plated onto coverslips, in untreated FCS-containing medium and the indicated concentration of BMS-754807 for three days. Cells were assayed for histone H3 Ser 10 phosphorylation (khaki arrows) by immunofluorescence and counterstained with DAPI. Asterisks indicate that the proportion of cells in the mitotic-phase of the cell cycle is significantly lower after incubation with BMS-754807 (one-way ANOVA; SNU-1, p = 0.0009; MKN74, p = 0.0046; NUGC3, p = 0.0023). NS indicates data that are not significantly different. Pale pink arrows indicate apoptotic cells. b SNU-1 were incubated in untreated FCS-containing with BMS-754807 (BMS) for three or six days. Cleaved caspase-3 or cleaved PARP were analysed by immunofluorescence (pink arrows). Asterisks indicate that the proportion of cells with detectable cleaved PARP is statistically significantly different after incubation with BMS-754807 (one-way ANOVA; p < 0.01). c Metastatic adenocarcinoma cells were isolated from patients and incubated in 20% untreated FCS-containing medium and different concentrations of BMS-754807 for three days after which cleaved PARP and GAPDH were analysed by western transfer. The protein bands were quantified by densitometry and corrected for GAPDH. Asterisks indicate concentrations of BMS-754807 at which there was statistically significantly more cleaved PARP in its presence than in its absence (one-way ANOVA; p < 0.001). d Cells were incubated in full, untreated FCS-containing medium with BMS-754807 for three days and phosphorylated (Phospho.) insulin Tyr 1150/1151 and type I IGF Tyr 1135/1136 receptors (IGF receptors), Akt Ser 473 and cleaved PARP analysed as above, quantified and corrected for corresponding total protein or GAPDH, respectively. Representative images are shown as inserts. Asterisks indicate concentrations of BMS-754807 at which there was statistically significantly less phosphorylated protein, or more cleaved PARP than in its absence (one-way ANOVA; p < 0.001). e SNU-1 and NUGC3 were incubated in serum-free medium for two hours and then in serum-free medium and the indicated concentrations of BMS-754807 in the absence or presence of ligand (lig.) for 15 min. Phosphorylated (Phospho.) insulin Tyr 1150/1151 and type I IGF Tyr 1135/1136 receptors (IGF receptors), Akt Ser 473 or GAPDH were measured. Total corresponding protein is shown underneath the phosphorylated protein images

Article Snippet: Cells were lysed in radioimmunoprecipitate (RIPA) buffer and analysed by western transfer as described [ ] with antibodies against: insulin receptor (#3025), type I IGF receptor (#3027), β-tubulin (#5346), phosphorylated Tyr 1150/1151 insulin and Tyr 1135/1136 type I IGF receptors (#3024), Akt (#9272), phosphorylated Ser 473 Akt (#4060), ERK1 and ERK2 (#9102), phosphorylated Thr 202/204 ERK1 and Thr 185/187 ERK2 (#4370), cleaved Asp 214 poly-(ADP-ribose) polymerase-1 (PARP-1) (#9541) (Cell Signaling Technologies), or GAPDH-HRP (sc-25778) (Santa Cruz Biotechnology).

Techniques: Inhibition, Cell Culture, Suspension, Concentration Assay, Phospho-proteomics, Immunofluorescence, Incubation, Isolation, Western Blot

Journal: Gastric Cancer

Article Title: Insulin and the insulin receptor collaborate to promote human gastric cancer

doi: 10.1007/s10120-021-01236-y

Figure Lengend Snippet: Cell survival effects of insulin, IGF-1 and IGF-2 in gastric adenocarcinoma. a NUGC3 were incubated in the absence or presence of 0.5 µM staurosporine without or with 50 ngml −1 ligand for 4 h, fixed and evidence of cell death analysed by immunofluorescent detection of cleaved Asp 214 PARP (pink arrows). The proportions of cells in which cell death had been induced are shown as means ± SEM (two-way ANOVA; p < 0.001). b Metastatic cells isolated from patients were added to poly-HEMA-coated wells in serum-free medium to prevent attachment and induce anoikis and incubated in serum-free medium in the absence or presence of ligand (lig.) minus or plus BMS-754807 for 24 h after which cleaved PARP, phosphorylated Akt Ser 473, Akt and GAPDH were measured by western transfer. Asterisks indicate if there was statistically significantly less cleaved PARP or more phosphorylated Ser 473 Akt after incubation in the presence of ligand, or more cleaved PARP or less phosphorylated Ser 473 Akt after incubation in the presence of ligand and BMS-754807, than in the presence of ligand alone (two-way ANOVA; p < 0.01). NS indicates data are not significantly different. c SNU-1 and NUGC3 were incubated with staurosporine and different concentrations of ligand prior to analysis of cleaved PARP, phosphorylated Akt Ser 473, Akt and GAPDH by western transfer. d The primary sequences of the insulin receptor in the second fibronectin type III domain (FnIII-2’) that differentiate isoform B from isoform A are shown (adapted from ). Insulin is shown positioned in the binding pocket of isoform B with the amino acid residues in FnIII-2’encoded by exon 11 that are absent in isoform A arrowed (adapted from ). The molecular masses of the IGF ligands and their affinities for insulin receptor isoforms B and A are listed . Please see electronic supplementary material for further details (online resource 1). e RNA extracted from gastric cancer cells and metastatic patient samples incubated in untreated FCS-containing medium was reverse transcribed and cDNA amplified with primers designed to detect insulin receptor isoform B (211 bp), insulin receptor isoform A (207 bp), isoform B and isoform A simultaneously (187 bp and 151 bp, respectively) or 18S rRNA (68 bp)

Article Snippet: Cells were lysed in radioimmunoprecipitate (RIPA) buffer and analysed by western transfer as described [ ] with antibodies against: insulin receptor (#3025), type I IGF receptor (#3027), β-tubulin (#5346), phosphorylated Tyr 1150/1151 insulin and Tyr 1135/1136 type I IGF receptors (#3024), Akt (#9272), phosphorylated Ser 473 Akt (#4060), ERK1 and ERK2 (#9102), phosphorylated Thr 202/204 ERK1 and Thr 185/187 ERK2 (#4370), cleaved Asp 214 poly-(ADP-ribose) polymerase-1 (PARP-1) (#9541) (Cell Signaling Technologies), or GAPDH-HRP (sc-25778) (Santa Cruz Biotechnology).

Techniques: Incubation, Isolation, Western Blot, Binding Assay, Reverse Transcription, Amplification

Journal: Gastric Cancer

Article Title: Insulin and the insulin receptor collaborate to promote human gastric cancer

doi: 10.1007/s10120-021-01236-y

Figure Lengend Snippet: Knockdown of the insulin receptor induces cell death in gastric cancer cells. a NUGC3 were transfected with scrambled oligonucleotide (scr.), siINSR2 or siIGF1R2 and analysed as described in the legend to Fig. . High magnification images facilitate visualisation of apoptotic nuclei (pale pink arrows). Cells were analysed next for the presence of cleaved PARP (pink arrows). Asterisks indicate that the proportion of apoptotic cells, or cells with cleaved PARP, is significantly higher after transfection with siINSR2 than with the scrambled oligonucleotide (**) or siIGF1R2 (*) (one-way ANOVA; p < 0.0001). NS indicates data are not significantly different. b SNU-1, MKN-74 and NUGC3 cells were transfected with scrambled oligonucleotide (scr.) or siINSR2 (siR) and cultured as indicated in untreated-FCS-containing medium prior to analysis of insulin receptor, cleaved PARP or GAPDH by western transfer. c SNU-1 and NUGC3 were transfected with scrambled oligonucleotide, siINSR2 or siINSR3 and cultured for three days in untreated-FCS-containing medium prior to measurement of insulin receptor, cleaved PARP or β-tubulin by western transfer. Asterisks (**) indicate that there was significantly less insulin receptor or more cleaved PARP after transfection with siINSR2 or siINSR3 than with scrambled oligonucleotide (one-way ANOVA; p < 0.001). d SNU-1, MKN-74, NUGC3 and AGS, and patient samples, HC1 and NC1, were transfected with scrambled oligonucleotide (scr.) or siIGF1R2, cultured for three days in untreated-FCS-containing medium and type I IGF receptor, cleaved PARP and GAPDH analysed. e NUGC3 were transfected with scrambled oligonucleotide (scr.), siINSR2, siIGF1R2 or both siINSR2 and siIGF1R2 and cultured in untreated-FCS-containing medium for three days prior to measurement of type I IGF receptor, insulin receptor, cleaved PARP or GAPDH by western transfer. There was significantly more cleaved PARP after transfection with siINSR2, or siINSR2 and siIGF1R2 (one-way ANOVA; p < 0.001)

Article Snippet: Cells were lysed in radioimmunoprecipitate (RIPA) buffer and analysed by western transfer as described [ ] with antibodies against: insulin receptor (#3025), type I IGF receptor (#3027), β-tubulin (#5346), phosphorylated Tyr 1150/1151 insulin and Tyr 1135/1136 type I IGF receptors (#3024), Akt (#9272), phosphorylated Ser 473 Akt (#4060), ERK1 and ERK2 (#9102), phosphorylated Thr 202/204 ERK1 and Thr 185/187 ERK2 (#4370), cleaved Asp 214 poly-(ADP-ribose) polymerase-1 (PARP-1) (#9541) (Cell Signaling Technologies), or GAPDH-HRP (sc-25778) (Santa Cruz Biotechnology).

Techniques: Knockdown, Transfection, Cell Culture, Western Blot