Journal: FEBS letters

Article Title: Potential role of LMP2 as an anti-oncogenic factor in human uterine leiomyosarcoma: morphological significance of calponin h1.

doi: 10.1016/j.febslet.2012.05.029

Figure Lengend Snippet: Fig. 1. Biological activity of hLMP2 in uterine leiomyosarcoma (LMS). (A) Phase-contrast micrographs of the parental transformed SKN-CEM9#2 (T type) clone and flat revertants of the SKN-LMP2#122 (F type) clone (magnification 100). Changes in human uterine LMS cell line, SKN-transfectants, SKN-CEM9 (T type) clone, and SKN-LMP2wt (F type) clone xenograft volumes in mice (n = 8). Representative photographs of xenografts in mice (Left). Tumor growth of SKN-LMP2 was markedly reduced in comparison with that of the control transfectant SKN-CEM9 (T type) clone. Tumor growth kinetics after subcutaneous injection of the SKN-CEM9 (T type) clone and SKN-LMP2 (F type) clone (Right). (B) RT-PCR experiments revealed hLMP2, hLMP7, Calponin h1, SRF, cyclin B and b-actin mRNA expression in tumors. Precursor LMP2 or LMP7 (pre-LMP2, pre- LMP7) and mature LMP2 or LMP7 (LMP2, LMP7) are shown. (C) Western blotting revealed LMP2, LMP7, calponin h1, SRF, cyclin B, and b-actin in SKN-transfectant clones. (D) The luciferase reporter vectors containing the hCalponin h1 promoter with wild type SRF binding sites (Calponin-wt-Luc.), mutant SRF binding sites (Calponin-mut-Luc.), or empty luciferase reporter vector (Basic-Luc.) [23] were transiently co-transfected with pSV-b-galactosidase in SKN-transfectants, SKN-CEM9#2, SKN-LMP2#121, or SKN- LMP2#122 clones for the final 48 h, and then luciferase activities were measured. Values were normalized to those obtained with the co-transfected pSV-b-galactosidase expression vector. Each assay was performed at least three times and in triplicate. Luciferase reporter assays showed that LMP2 expression markedly induced calponin h1 promoter activation. Data are presented as the mean from three independent experiments (⁄S.D.). The experiments were performed four times with similar results. SKN transformantsa, CEM9 SKN-CEM9#2; LMP2, SKN-LMP2wt#121, SKN-LMP2wt#122. Detail is shown in SFig. 2, SFig. 3 and STable 3. RT-PCRb, total RNA samples were isolated from the individual xenografted-tumors, which were removed at 5 weeks after xenografting. W.B.c, W.B. are performed with the total cell lysates from SKN transformants.

Article Snippet: LMP2 expression vector was co-transfected into SKN cells with shRNA vector. c shRNA, Calponin h1 shRNA or Scramble shRNA were co-transfected into SKN cells with LMP2wt expression vector using manufacturer’s recomendations (Santa Cruz Biotechnology, Inc., CA, USA). d Morphology.

Techniques: Activity Assay, Transformation Assay, Comparison, Control, Transfection, Injection, Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Clone Assay, Luciferase, Binding Assay, Mutagenesis, Plasmid Preparation, Activation Assay, Isolation

Journal: FEBS letters

Article Title: Potential role of LMP2 as an anti-oncogenic factor in human uterine leiomyosarcoma: morphological significance of calponin h1.

doi: 10.1016/j.febslet.2012.05.029

Figure Lengend Snippet: Fig. 2. Biological activity of calponin h1 in uterine leiomyosarcoma (LMS). (A) Phase-contrast micrographs of the parental transformed SKN-CEM9#1Scr.shRNA (T type) clone, SKN-CEM9#2 calponin h1shRNA (T type) clone, SKN-CEM9#2 (T type) clone, SKN-LMP2#1Scr.shRNA (F type) clone, and SKN-LMP2#2Calponin h1shRNA (T type) clone of the SKN-LMP2 (F type) clone (magnification 60). The growth rates of the SKN-transfectant clones were measured as population doubling time (PDT). (B) Western blotting and RT-PCR experiments revealed calponin h1, precursor LMP2 (pre-LMP2), mature LMP2 (LMP2), and b-actin in SKN-transfectant clones. SKN transformantsa, CEM9#3 Scr.shRNA, CEM9#4 Calponin h1shRNA, LMP2#1 Scr.shRNA, LMP2#2 Calponin h1shRNA, Detail is shown in Table 1 and SFig. 5 and STable 3. (C) Changes in the human uterine LMS cell line, SKN-transfectant, SKN-CEM9#2 (T type) clone, SKN-LMP2wt#2/Calponin h1shRNA (T type) clone, and SKN-LMP2wt#1/ Scr.shRNA (F type) clone xenograft volumes in mice (n = 3). Representative photographs of xenografts in mice (Left). Tumor growth of the SKN-LMP2wt#2/Calponin h1shRNA (T type) clone is mildly increased in comparison with that of the SKN-LMP2wt#1/Scr.shRNA (F type) clone. Tumor growth kinetics after subcutaneous injection of the SKN-transfectant clones (Right). RT-PCR experiments revealed hCalponin h1, hLMP2 and b-actin mRNA expression in tumors (Bottom). Experiments were performed three times with similar results. SKN-CEM9c, SKN- CEM9#2; LMP2wt+Calponin h1shRNAd, SKN-LMP2wt#2/ CalponinshRNA; LMP2wt/Scr.shRNAe, SKN-LMP2wt#1/Scr.shRNA. Details of SKN transfectants are shown in Table 1, SFig. 5 and STable 3. RT-PCRf, total RNA samples were isolated from the individual xenografted-tumors, which were removed from BALB/c nu/numice at 5 weeks after xenografting. Xenograftsg, BALB/c nu/nu mice were inoculated with SKN-CEM9#2, SKN-LMP2wt#2/CalponinshRNA or SKN-LMP2wt#1/Scr.shRNA.

Article Snippet: LMP2 expression vector was co-transfected into SKN cells with shRNA vector. c shRNA, Calponin h1 shRNA or Scramble shRNA were co-transfected into SKN cells with LMP2wt expression vector using manufacturer’s recomendations (Santa Cruz Biotechnology, Inc., CA, USA). d Morphology.

Techniques: Activity Assay, Transformation Assay, shRNA, Transfection, Clone Assay, Western Blot, Reverse Transcription Polymerase Chain Reaction, Comparison, Injection, Expressing, Isolation

Journal: PLoS Genetics

Article Title: The NADPH Metabolic Network Regulates Human αB-crystallin Cardiomyopathy and Reductive Stress in Drosophila melanogaster

doi: 10.1371/journal.pgen.1003544

Figure Lengend Snippet: (A) The abdominally located Drosophila heart (Hrt) is a simple linear tube that is divided into an anterior, conical chamber (CC) and three posterior compartments. A1 = abdominal segment 1; A6–A7 = abdominal segments 6 and 7; Os = ostia inflow tracts (modified from ). (B–F) Cardiac-restricted expression of wild type human CryAB (HG4>wtCryAB) had no significant effect on any measured parameter of cardiac performance in Drosophila . Several indices of cardiac function were influenced by the expression of mutant CryAB R120G (HG4>R120GCryAB). The diameter across the heart tubes during (D) diastole and (E) systole were significantly greater, and (F) percent fractional shortening significantly lower relative to HG4>wtCryAB flies. Knockdown of G6PD in CryAB R120G mutant hearts (HG4>R120GCryAB+G6PDRNAi) substantially improved cardiac performance of the mutant hearts as evidenced by (B) significant decreases in heart period, (C) arrhythmic beating patterns, (E) systolic dimensions and (F) significantly increased fractional shortening relative to HG4>R120GCryAB hearts. * P≤0.05, ** P≤0.01, *** P≤0.001, **** P≤0.0001.

Article Snippet: Despite a lack of reduction in CryAB R120G diastolic diameters in response to Zw (G6PD) RNAi co-expression, systolic diameters in the double mutants were rescued and did not significantly differ from those found in fly hearts expressing wildtype human CryAB .

Techniques: Modification, Expressing, Mutagenesis, Knockdown

Journal: PLoS Genetics

Article Title: The NADPH Metabolic Network Regulates Human αB-crystallin Cardiomyopathy and Reductive Stress in Drosophila melanogaster

doi: 10.1371/journal.pgen.1003544

Figure Lengend Snippet: M-mode traces reveal the positions of the Drosophila heart wall edges (Y direction) over time (X direction). M-modes from hearts expressing wild type CryAB exhibit fairly regular contraction and relaxation cycles. However, movement traces of cardiac tubes expressing mutant CryAB R120G reveal hearts with dilated dimensions, a lower extent of shortening and arrhythmic beating patterns relative to controls. M-modes from hearts expressing mutant CryAB R120G and Zw (G6PD) RNAi suggest that knockdown of G6PD increases heart rate, promotes rhythmic beating patterns and rescues the cardiac tube dimensions of the mutant hearts.

Article Snippet: Despite a lack of reduction in CryAB R120G diastolic diameters in response to Zw (G6PD) RNAi co-expression, systolic diameters in the double mutants were rescued and did not significantly differ from those found in fly hearts expressing wildtype human CryAB .

Techniques: Expressing, Mutagenesis, Knockdown

Journal: PLoS Genetics

Article Title: The NADPH Metabolic Network Regulates Human αB-crystallin Cardiomyopathy and Reductive Stress in Drosophila melanogaster

doi: 10.1371/journal.pgen.1003544

Figure Lengend Snippet: A–C: The phenotypic distribution produced by GMR>CryAB R120G in a w 1118 control strain background (A), and the altered distributions produced by RNAi-mediated knockdown of Zw (B, C). D-H: The CryAB R120G phenotypic distribution in a y w 2 control background (D) compared to phenotypes when Zw is overexpressed (E–H). The number of eyes scored is given as n; P is given for comparison with the appropriate control (A for B, C; D for E–H). Controls were chosen to most closely match the genetic background of the test strains. The CryAB R120G line 16A was used for these tests.

Article Snippet: Despite a lack of reduction in CryAB R120G diastolic diameters in response to Zw (G6PD) RNAi co-expression, systolic diameters in the double mutants were rescued and did not significantly differ from those found in fly hearts expressing wildtype human CryAB .

Techniques: Produced, Control, Knockdown, Comparison

Journal: PLoS Genetics

Article Title: The NADPH Metabolic Network Regulates Human αB-crystallin Cardiomyopathy and Reductive Stress in Drosophila melanogaster

doi: 10.1371/journal.pgen.1003544

Figure Lengend Snippet: The phenotypic distribution in the Canton S control (A) compared to that of deletions that remove Pgd (B, C), a Zw Pgd double mutant (D), and RNAi-mediated knockdown of Pgd (E). The CryAB R120G line 16A was used for these tests. In B–D, heterozygous +/− females were assayed.

Article Snippet: Despite a lack of reduction in CryAB R120G diastolic diameters in response to Zw (G6PD) RNAi co-expression, systolic diameters in the double mutants were rescued and did not significantly differ from those found in fly hearts expressing wildtype human CryAB .

Techniques: Control, Mutagenesis, Knockdown

Journal: PLoS Genetics

Article Title: The NADPH Metabolic Network Regulates Human αB-crystallin Cardiomyopathy and Reductive Stress in Drosophila melanogaster

doi: 10.1371/journal.pgen.1003544

Figure Lengend Snippet: The phenotypic distribution in the w 1118 control (A) compared to RNAi-mediated knockdown of the putative mitochondrial IDH (B, C) or cytoplasmic IDH (D–F), or MEN (G). The CryAB R120G line 16A was used for these tests.

Article Snippet: Despite a lack of reduction in CryAB R120G diastolic diameters in response to Zw (G6PD) RNAi co-expression, systolic diameters in the double mutants were rescued and did not significantly differ from those found in fly hearts expressing wildtype human CryAB .

Techniques: Control, Knockdown

Journal: PLoS Genetics

Article Title: The NADPH Metabolic Network Regulates Human αB-crystallin Cardiomyopathy and Reductive Stress in Drosophila melanogaster

doi: 10.1371/journal.pgen.1003544

Figure Lengend Snippet: (A) The GSH∶GSSG ratio was measured in the heads of female flies with RNAi-mediated knockdown of PGD, G6PD, MEN, or IDH, or overexpression of G6PD (G6PD TG). (B) GSH and GSSG were also measured in whole larvae expressing RNAi or extra transgenic copies under the control of tubulin-Gal4. All data points represent a set of at least 3 independent replicas, and are reported here as the mean ratio of nmol GSH to nmol GSSG (calculated as GSH equivalents) ± SE. Statistical results are for comparison with w 1118 for knockdowns in A; y w 2 for G6PD overexpression in A; and y w 2 for all comparisons in B. (** P<0.05, * P<0.1).

Article Snippet: Despite a lack of reduction in CryAB R120G diastolic diameters in response to Zw (G6PD) RNAi co-expression, systolic diameters in the double mutants were rescued and did not significantly differ from those found in fly hearts expressing wildtype human CryAB .

Techniques: Knockdown, Over Expression, Expressing, Transgenic Assay, Control, Comparison

Journal: Neuron

Article Title: Semaphorin 6D tunes amygdalar circuits for emotional, metabolic, and inflammatory outputs.

doi: 10.1016/j.neuron.2024.06.017

Figure Lengend Snippet: Figure 1. Loss of Sema6d increases anxiety (A) Phenome-wide association of SEMA6D polymorphisms across 4,756 GWASs from the GWAS Atlas. (B) SEMA6D mRNA expression in the amygdala from patients with schizophrenia compared with controls in the PRJNA379666 dataset. TPM, transcripts per kilobase million. Boxes denote the interquartile range (IQR). The median is shown as horizontal bars. Whiskers extend to 1.5 times the IQR. Outliers are shown as individual points. Healthy controls, n = 24; patients with schizophrenia, n = 22.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Blocker Casein in PBS ThermoFisher Scientific Cat# 37582 Cholera Toxin Subunit B (Recombinant), Alexa Fluor 594 Conjugate ThermoFisher Scientific Cat# C34777 Corn oil Sigma-Aldrich Cat# C8267 DAPI Solution Thermo fisher Cat# 62248 FD Rapid GolgiStain Kit FD NeuroTechnologies Cat# PK401 Fluoromount Diagnostic BioSystems Cat# K024 Olive oil Wako Cat# 150-00276 OPAL 520 REAGENT PACK Akoya Biosciences Cat# FP1487001KT OPAL 570 REAGENT PACK Akoya Biosciences Cat# FP1488001KT OPAL 690 REAGENT PACK Akoya Biosciences Cat# FP1497001KT RNAscope(R) Multiplex Fluorescent Reagent Kit v2 Advanced Cell Diagnostics, Inc. Cat# 323100 RNAscope(R) Target ProbeMm-Gad2-C3, Mouse Advanced Cell Diagnostics, Inc. Cat# 439371-C3 RNAscope(R) Target ProbeMm-Plxna4-C2, Mouse Advanced Cell Diagnostics, Inc. Cat# 515491-C2 RNAscope(R) Target ProbeMm-Rbfox3-C3, Mouse Advanced Cell Diagnostics, Inc. Cat# 313311-C3 RNAscope(R) Target ProbeMm-Sema6d-C1, Mouse Advanced Cell Diagnostics, Inc. Cat# 565871 RNAscope(R) Target ProbeMm-Sema6d-O1-C1, Mouse Advanced Cell Diagnostics, Inc. Cat# 1255981-C1 Sodium azide Wako Cat# 195-11092 Tamoxifen Sigma-Aldrich Cat# T5648 Triton X-100 Nacalai Tesque Cat# 35501-02 Type I collagenase Gibco Cat# 17100017 RIPA Buffer Nacalai Tesque Cat# 08714-04 4%-Paraformaldehyde Phosphate Buffer Solution Nacalai Tesque Cat# 09154-85 Critical commercial assays Chromium Next GEM Single Cell 3ʹ GEM, Library & Gel Bead Kit v3.1 10x Genomics Cat# 1000128 Chromium Nuclei Isolation Kit with RNase Inhibitor 10x Genomics Cat# 1000494 LIVE/DEAD Fixable Aqua Dead Cell Stain Kit, for 405 nm excitation Invitrogen Cat# L34957 LIVE/DEAD Fixable Violet Dead Cell Stain Kit, for 405 nm excitation Invitrogen Cat# L34955 Myelin removal beads Miltenyi Biotec Cat# 130-096-733 Neural Tissue Dissociation Kit (P) Miltenyi Biotec Cat# 130-092-628 PowerUp SYBR Green Master Mix ThermoFisher Scientific Cat# A25741 QuantiFast Multiplex PCR Kit Qiagen Cat# 204757 RNeasy Plus Mini Kit Qiagen Cat# 74136 SuperScript IV Reverse Transcriptase ThermoFisher Scientific Cat# 18090010 TruSeq Stranded mRNA Library Prep Illumina Cat# 20020594 Visium Spatial for FFPE Gene Expression Kit, Mouse Transcriptome 10x Genomics Cat# 1000339 Deposited data RNA sequencing data This paper GEO: GSE201216 RNA sequencing data This paper GEO: GSE195825 (Continued on next page) e2 Neuron 112, 1–18.e1–e9, September 4, 2024

Techniques: Expressing