Journal: bioRxiv

Article Title: Cardiovascular Disease-Associated Non-Coding Variants Disrupt GATA4-DNA Binding and Regulatory Functions

doi: 10.1101/2024.09.19.613959

Figure Lengend Snippet: Expression and purification of GATA4 ZF. A) SDS-PAGE (left) and western blot (right) of GATA4 ZF (18.8 kDa). Abbreviations: wash fractions (W), eluted fractions (E), concentrated fractions (C), un-induced protein sample (U), IPTG-induced protein sample (I), supernatant fraction (S), and pellet fraction (P). B) Western Blot of full-length GATA4 expressed in HeLA cells (48.6 kDa).

Article Snippet: HeLa Cervical Adenocarcinoma Human (HeLa) cells (ATCC–CCL-2) were grown in Eagle’s Minimum Essential Medium (EMEM) (ATCC - 30-2003) with 10% Fetal Bovine Serum (FBS) at 37 °C and 5% CO 2 .

Techniques: Expressing, Purification, SDS Page, Western Blot

Journal: bioRxiv

Article Title: Cardiovascular Disease-Associated Non-Coding Variants Disrupt GATA4-DNA Binding and Regulatory Functions

doi: 10.1101/2024.09.19.613959

Figure Lengend Snippet: CVD-associated SNPs alter gene expression and are in eQTL in cardiac tissue. A) Relative luciferase activity in HeLa cells transfected with reporter plasmids containing reference (blue with black circles) and alternate (orange with black squares) of variants rs1506537, rs56992000, rs2941506, and rs2301249. B) Cardiac tissue eQTL analysis of MRPL33 , TOP2B , PGAP3 , and CSK expressed in heart atrial appendage or left ventricle when rs1506537, rs56992000, rs2941506, and rs2301249 occur, respectively. C) University of California Santa Cruz (UCSC) Genome Browser tracks of variants rs2941506 and rs56992000 near TAD boundaries and regulatory elements.

Article Snippet: HeLa Cervical Adenocarcinoma Human (HeLa) cells (ATCC–CCL-2) were grown in Eagle’s Minimum Essential Medium (EMEM) (ATCC - 30-2003) with 10% Fetal Bovine Serum (FBS) at 37 °C and 5% CO 2 .

Techniques: Gene Expression, Luciferase, Activity Assay, Transfection

Journal: bioRxiv

Article Title: The histone demethylase KDM5 is essential for larval growth in Drosophila

doi: 10.1101/297804

Figure Lengend Snippet: (A) Lethality of kdm5 K06801 , kdm5 10424 and kdm5 140 homozygous mutant animals generated from a cross between five female and five male heterozygous parents balanced using CyO-GFP. The column labeled total flies indicates the number of progeny (adult) flies scored from at least three independent crosses. Expected number of progeny is based on Mendelian frequencies and taking into account the lethality of CyO homozygotes, i.e 33% of total adult flies. * p <0.01 (chi-squared test). (B) Position of the NP4707 , 10424 and K06801 P element insertions and molecular mapping of the kdm5 140 deletion. Ab indicates the region used to generated the rabbit polyclonal anti-KDM5 antibody ( S ecombe et al . 2007 ). (C) RT-PCR using primers to the 5’ end of the gene using RNA from whole 3 rd instar larvae. Animals homozygous for kdm5 K06801 or kdm5 10424 show low levels of transcript while kdm5 140 shows none. kdm5 mRNA normalized to wildtype ( w 1118 ) using rp49 . **** p <0.0001. (D) RT-PCR using primers to the 3’ end of the gene using RNA from whole 3 rd instar larvae. kdm5 140 has wildtype levels of the 3’ end of the transcript. **** p <0.0001. ns = not significant. (E) Western from wildtype ( w 1118 ) and kdm5 140 homozygous mutant wing imaginal discs showing KDM5 and alpha tubulin. kdm5 140 animals have no detectable full length or truncated KDM5. *ns indicates non-specific band. (F) Schematic of strain genotype for rescue of kdm5 140 with a genomic rescue transgene. Flies are homozygous for the kdm5 140 mutation on the 2 nd chromosome and homozygous for an 11kb genomic rescue transgene on the 3 rd chromosome. (G) Western blot showing KDM5 protein levels from 3 rd instar larval wing imaginal discs from wildtype ( w 1118 ) and kdm5 140 homozygotes that also have two copies of the kdm5:HA genomic rescue transgene. Anti-KDM5 (top), anti-HA (middle) and anti-histone H3 loading control (bottom). (H) kdm5 140 lethality is rescued by a transgene encoding the kdm5 locus. These data were generated by crossing female and male flies heterozygous for kdm5 140 and homozygous the wildtype genomic rescue transgene (intercross of kdm5 140 /CyO-GFP; kdm5:HA / kdm5:HA males and females).

Article Snippet: Antibodies used were anti-pH3 (Cell signaling #9701, 1/1000), anti-histone H3 (Active Motif #39763 or #39163, 1/5000), anti-alpha Tubulin (Developmental Studies Hybridoma Bank, University of Iowa; 1:5000).

Techniques: Mutagenesis, Generated, Labeling, Reverse Transcription Polymerase Chain Reaction, Western Blot, Control

Journal: PLoS ONE

Article Title: Singlet Oxygen Induced Products of Linoleates, 10- and 12-(Z,E)-Hydroxyoctadecadienoic Acids (HODE), Can Be Potential Biomarkers for Early Detection of Type 2 Diabetes

doi: 10.1371/journal.pone.0063542

Figure Lengend Snippet: Abbreviations: a, Blood collection; b, Glycometabolism Markers (glucose, insulin, leptin, adiponectin, IL-6, TNF-α); c, Oxidative stress markers (9-(Z,E)-HODE, 9-(E,E)-HODE, 12-(Z,E)-HODE, 10-(Z,E)-HODE, 13-(Z,E)-HODE, 13-(E,E)-HODE, 5HETE, 12HETE, 15HETE, isoPs, linoleic acid, total cholesterol, 7β-hydroxycholesterol).

Article Snippet: 9-(E,E)-HODE, 13-(E,E)-HODE, 10-(Z,E)-HODE, and 12-(Z,E)-HODE were obtained from Larodan Fine Chemicals AB (Malmo, Sweden).

Techniques:

Journal: PLoS ONE

Article Title: Singlet Oxygen Induced Products of Linoleates, 10- and 12-(Z,E)-Hydroxyoctadecadienoic Acids (HODE), Can Be Potential Biomarkers for Early Detection of Type 2 Diabetes

doi: 10.1371/journal.pone.0063542

Figure Lengend Snippet: (a) Total ion chromatogram, (b) 9-(Z,E)- and (E,E)-HODE, (c) 10- and 12-(Z,E)-HODE, (d) 13-(Z,E)- and (E,E)-HODE, (e) 13-(Z,E)-HODE-d 4 , (f) 5-HETE, (g) 12-HETE, (h) 15-HETE, (i) 8–iso-PGF 2α , and (j) 8-iso-PGF 2α -d 4.

Article Snippet: 9-(E,E)-HODE, 13-(E,E)-HODE, 10-(Z,E)-HODE, and 12-(Z,E)-HODE were obtained from Larodan Fine Chemicals AB (Malmo, Sweden).

Techniques:

Journal: PLoS ONE

Article Title: Singlet Oxygen Induced Products of Linoleates, 10- and 12-(Z,E)-Hydroxyoctadecadienoic Acids (HODE), Can Be Potential Biomarkers for Early Detection of Type 2 Diabetes

doi: 10.1371/journal.pone.0063542

Figure Lengend Snippet: Correlation between fasting plasma levels of (10- and 12-(Z,E)-HODE)/LA in subjects without diabetic type and the insulinogenic index (A) or Matsuda Index 3 (B) during OGTT.

Article Snippet: 9-(E,E)-HODE, 13-(E,E)-HODE, 10-(Z,E)-HODE, and 12-(Z,E)-HODE were obtained from Larodan Fine Chemicals AB (Malmo, Sweden).

Techniques:

Journal: International Journal of Nanomedicine

Article Title: One-step detection of circulating tumor cells in ovarian cancer using enhanced fluorescent silica nanoparticles

doi: 10.2147/IJN.S45059

Figure Lengend Snippet: Measurement of the fluorescent intensity of MNP-SiO 2 (RITC). Notes: Measurement of the fluorescent intensity of MNP-SiO 2 (RITC) by GEN-2 Fluorescence Lifetime Spectrometer-Steady State Quanta Master (Photon Technology International, Birmingham, NJ, USA). We prepared nanoparticles with various amounts of organic dye (2.08 mg, 4.17 mg, 8.35 mg, and 12.52 mg) to increase the fluorescent intensity and optimize the synthesis. Abbreviations: MNP-SiO 2 (RITC), magnetic nanoparticle-SiO 2 (rhodamine B isothiocyanate [RITC]); AU, absorbance units.

Article Snippet: To confirm the MUC1 antibody binding on the MNP-SiO 2 (RITC), we used an S-3100 ultraviolet-visible (UV-Vis) spectrophotometer (Scinco Co, Ltd, Seoul, Korea).

Techniques: Fluorescence

Journal: International Journal of Nanomedicine

Article Title: One-step detection of circulating tumor cells in ovarian cancer using enhanced fluorescent silica nanoparticles

doi: 10.2147/IJN.S45059

Figure Lengend Snippet: Scheme of MUC1 antibody conjugation on the surface of MNP-SiO 2 (RITC) nanoparticles. Abbreviations: MUC1, mucin 1 cell surface-associated antibody; MNP-SiO 2 (RITC), magnetic nanoparticle-SiO 2 (rhodamine B isothiocyanate); PEG, polyethylene glycol; APS, (3-aminopropyl)triethoxysilane; EDC, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride; DIEA, N,N-diisopropylethylamine; NMP, N-methyl-2-pyrrolidone.

Article Snippet: To confirm the MUC1 antibody binding on the MNP-SiO 2 (RITC), we used an S-3100 ultraviolet-visible (UV-Vis) spectrophotometer (Scinco Co, Ltd, Seoul, Korea).

Techniques: Conjugation Assay

Journal: International Journal of Nanomedicine

Article Title: One-step detection of circulating tumor cells in ovarian cancer using enhanced fluorescent silica nanoparticles

doi: 10.2147/IJN.S45059

Figure Lengend Snippet: TEM images of MNP-SiO 2 (RITC) and MUC1-MNP-SiO 2 (RITC). Notes: ( A ) MNP-SiO 2 (RITC). ( B ) MUC1-MNP-SiO 2 (RITC). Abbreviations: TEM, transmission electron microscopy; MUC1, mucin 1 cell surface-associated antibody; MNP-SiO 2 (RITC), magnetic nanoparticle-SiO 2 (rhodamine B isothiocyanate).

Article Snippet: To confirm the MUC1 antibody binding on the MNP-SiO 2 (RITC), we used an S-3100 ultraviolet-visible (UV-Vis) spectrophotometer (Scinco Co, Ltd, Seoul, Korea).

Techniques: Transmission Assay, Electron Microscopy

Journal: International Journal of Nanomedicine

Article Title: One-step detection of circulating tumor cells in ovarian cancer using enhanced fluorescent silica nanoparticles

doi: 10.2147/IJN.S45059

Figure Lengend Snippet: UV-Vis spectra of MNP-SiO 2 (RITC) and MUC1-MNP-SiO 2 (RITC). Notes: Both MNP-SiO 2 (RITC) (black line) and MUC1-MNP-SiO 2 (RITC) (red line) show the same fluorescence peak of RITC (580 nm) (black arrow) but only MUC1-MNP-SiO 2 (RITC) shows a protein peak (280 nm) (red arrow). Abbreviations: UV-Vis, ultraviolet-visible spectra; MNP-SiO 2 (RITC), magnetic nanoparticle-SiO 2 (rhodamine B isothiocyanate); MUC1, mucin 1 cell surface-associated antibody.

Article Snippet: To confirm the MUC1 antibody binding on the MNP-SiO 2 (RITC), we used an S-3100 ultraviolet-visible (UV-Vis) spectrophotometer (Scinco Co, Ltd, Seoul, Korea).

Techniques: Fluorescence

Journal: International Journal of Nanomedicine

Article Title: One-step detection of circulating tumor cells in ovarian cancer using enhanced fluorescent silica nanoparticles

doi: 10.2147/IJN.S45059

Figure Lengend Snippet: Fluorescence microscopy image of HEK293T and OVCAR-3 cells incubated with MUC1-MNP-SiO 2 (RITC) nanoprobe. Notes: ( A – D ) HEK293T cells (MUC1 negative cell lines). ( E – H ) OCVCAR-3 cells (MUC1 positive cell lines). The magnification of the upper row of panels is 100×. The magnification of the lower row of panels is 400×. Abbreviations: HEK293T, human embryonic kidney cell line; OVCAR-3, human ovarian cancer cell line; MUC1, mucin 1 cell surface-associated antibody; MNP-SiO 2 (RITC), magnetic nanoparticle-SiO 2 (rhodamine B isothiocyanate).

Article Snippet: To confirm the MUC1 antibody binding on the MNP-SiO 2 (RITC), we used an S-3100 ultraviolet-visible (UV-Vis) spectrophotometer (Scinco Co, Ltd, Seoul, Korea).

Techniques: Fluorescence, Microscopy, Incubation

Journal: International Journal of Nanomedicine

Article Title: One-step detection of circulating tumor cells in ovarian cancer using enhanced fluorescent silica nanoparticles

doi: 10.2147/IJN.S45059

Figure Lengend Snippet: Fluorescence microscopy image of the indirect blood model including OVCAR-3 cells incubated with MUC1-MNP-SiO 2 (RITC). Notes: ( A – C ) the magnification in these panels is 100× ( D – F ) the magnification in these panels is 400×. The magnification in these panels is 400×. The red signal detected on the surface of OVCAR-3 cells. Abbreviations: OVCAR-3, human ovarian cancer cell line; MUC1, mucin 1 cell surface-associated antibody; MNP-SiO 2 (RITC), magnetic nanoparticle-SiO 2 (rhodamine B isothiocyanate).

Article Snippet: To confirm the MUC1 antibody binding on the MNP-SiO 2 (RITC), we used an S-3100 ultraviolet-visible (UV-Vis) spectrophotometer (Scinco Co, Ltd, Seoul, Korea).

Techniques: Fluorescence, Microscopy, Incubation

Journal: International Journal of Nanomedicine

Article Title: One-step detection of circulating tumor cells in ovarian cancer using enhanced fluorescent silica nanoparticles

doi: 10.2147/IJN.S45059

Figure Lengend Snippet: Flow cytometry analysis of the indirect blood samples including OVCAR-3 cells after incubation with MUC1-MNP-SiO 2 (RITC). Notes: ( A ) Unspiked blood samples. ( B ) Positive control (5000 OVCAR-3 cells). ( C ) Indirect blood samples with 100 OVCAR-3 cells. ( D ) Indirect blood samples with 1000 OVCAR-3 cells. ( E ) Indirect blood samples with 10,000 OVCAR-3 cells. Numbers of positive cells are shown in parentheses and indicated by red dots. Abbreviations: OVCAR-3, human ovarian cancer cell line; MUC1, mucin 1 cell surface-associated antibody; P1, population 1; MNP-SiO 2 (RITC), magnetic nanoparticle-SiO 2 (rhodamine B isothiocyanate); SSC-A, side scatter area; PE-A, phycoerythrin area.

Article Snippet: To confirm the MUC1 antibody binding on the MNP-SiO 2 (RITC), we used an S-3100 ultraviolet-visible (UV-Vis) spectrophotometer (Scinco Co, Ltd, Seoul, Korea).

Techniques: Flow Cytometry, Incubation, Positive Control

Journal: International Journal of Nanomedicine

Article Title: One-step detection of circulating tumor cells in ovarian cancer using enhanced fluorescent silica nanoparticles

doi: 10.2147/IJN.S45059

Figure Lengend Snippet: Flow cytometry analysis of the indirect blood samples including DAPI staining of OVCAR-3 cells after incubation with MUC1-MNP-SiO 2 (RITC). Notes: ( A ) Unspiked blood samples. ( B ) Indirect blood samples with 100 OVCAR-3 cells. ( C ) Indirect blood samples with 1000 OVCAR-3 cells. ( D ) Indirect blood samples with 10,000 OVCAR-3 cells. Numbers of positive cells are shown in parentheses. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole fluorescent stain; OVCAR-3, human ovarian cancer cell line; MUC1, mucin 1 cell surface-associated antibody; MNP-SiO 2 (RITC), magnetic nanoparticle-SiO 2 (rhodamine B isothiocyanate); DAPI-A, 4’,6-diamidino-2-phenylindole area; PE-A, phycoerythrin area; P1, population 1; P2, population 2; P3, population 3.

Article Snippet: To confirm the MUC1 antibody binding on the MNP-SiO 2 (RITC), we used an S-3100 ultraviolet-visible (UV-Vis) spectrophotometer (Scinco Co, Ltd, Seoul, Korea).

Techniques: Flow Cytometry, Staining, Incubation

Journal: bioRxiv

Article Title: HDAC Inhibitors recapitulate Human Disease-Associated Microglia Signatures in vitro

doi: 10.1101/2024.10.11.617544

Figure Lengend Snippet: Bulk RNAseq data from the HMC3 DAM model. A. Heatmaps showing the expression of Cluster 11 (left), Microglia 13 (middle) and DAM1/DAM2 (right) marker gene sets in bulk RNAseq data generated 24hrs following exposure to DMSO (control), Entinostat (red; 10µM) or Vorinostat (green; 1µM). Each column represents a single sample, each row a single gene represented in the respective marker set. Pairwise differential testing between DMSO control and each of the treatment conditions (Entinostat, 10µM; Vorinostat, 1µM) was conducted using a Wald test with the Benjamini-Hochberg correction (FDR alpha < 0.05). The legend represents Z scores, with lower scores indicated in red and higher scores indicated in blue. Data represents n=3 independent experiments for each treatment group with each n for all compounds being performed at the same time. B. Volcano Plots depicting the distribution of differentially expressed genes from different signatures (DAM1, DAM2 , Cluster 11 , Microglia 13 ) for each treatment condition (Entinostat or Vorinostat) in comparison to DMSO control. HMC3 microglia were treated for 24hrs with DMSO as control, Entinostat (10µM) or Vorinostat (1µM) followed by bulk RNA-Seq. Volcano plots depict all genes present in each marker set (DAM1: 10 genes; DAM2: 20 genes; Cluster 11: 89 genes, Microglia 13: 127 genes) plotted based on log2FC (fold change expression) and -log10(p value) with the ones significantly upregulated marked in red and labelled with the gene name. Plots are organized from Cluster 11 (top left), to DAM1 (bottom left), to DAM2 (top right) to Microglia 13 (bottom right). C. PCA plot of bulk RNAseq results from HMC3 microglia treated with DMSO, Vorinostat or Entinostat . Principal component analysis (PCA) was calculated on log-normalized bulk RNA-Seq data derived from compound-treated HMC3 microglia following 24hrs of exposure to DMSO (control; blue), Entinostat (10µM; red) or Vorinostat (1µM; green). Data represents n=3 independent experiments for each of the treatment group with each n for all compounds being performed at the same time. D. Pie chart depicting the number of significantly upregulated genes by Entinostat or Vorinostat for each of the queried marker signatures DAM1, DAM2, Cluster 11 , Microglia 13 . The number of significantly upregulated genes across all three replicates for each treatment group (Entinostat or Vorinostat) in comparison to DMSO control was identified and converted to a percentage of marker genes upregulated/ marker set. Data for DAM1 are depicted in purple, for DAM2 depicted in red, for Cluster 11 depicted in violet and for Microglia 13 depicted in teal. E. Signature-specific markers induced by Vorinostat and Entinostat. Markers significantly induced by Vorinostat and Entinostat for each signatured are depicted for DAM1 (purple), DAM2 (red), Cluster 11 (violet), Microglia 13 (teal).

Article Snippet: The next day, microglia were treated with the respective concentrations of Vorinostat (1µM; Ambeed; Cat #: A234507), Cholic acid (10µM; Cayman chemical; Cat #: 20250), Flavokavain B (10µM; ChromaDex; Cat #: ASB-00006058- 005), Wiskostatin (1µM; Cayman chemical; Cat #: 15047), Trimipramine (10µM; Cayman chemical; Cat #: 15921), Naftopidil (10µM; APExBIO; Cat #: 57149-07-2), Ramipril (10µM; APExBIO; Cat #: B2208), Valporic acid (90µg/ml Sigma; Cat #: PHR1061), Geranylgeraniol (10µM; Sigma; Cat #: 24034-73-9), Entinostat (10µM; Ambeed; Cat #: A122285), Amiodarone hydrochloride (10µM; Sigma; Cat #: A8423), Temozolomide (100µM; APExBIO; Cat #: B1399) or DMSO (Sigma-Aldrich, Cat #:472301) as control and incubated for 6hrs and 24hrs before harvest for RNA extraction.

Techniques: Expressing, Marker, Generated, Control, Comparison, RNA Sequencing Assay, Derivative Assay

Journal: bioRxiv

Article Title: HDAC Inhibitors recapitulate Human Disease-Associated Microglia Signatures in vitro

doi: 10.1101/2024.10.11.617544

Figure Lengend Snippet: Bulk RNA-Seq of the human iPSC-derived microglia (iMG) DAM model. A. Volcano Plots depicting the distribution of differentially expressed genes from different signatures (Cluster 11 , Microglia 13 , iMG Cluster 2+8 ) for Vorinostat treatment in comparison to DMSO control. iPSC-derived microglia at Day 28-29 of differentiation were treated for 24hrs with DMSO as control or Vorinostat (0.1µM) followed by bulk RNAseq. Volcano plots depict all genes present in each marker set (Cluster 11: 89 genes, Microglia 13: 127 genes, iMG Cluster 2+8: 134 genes) plotted based on log2FC (fold change expression) and -log10(p value) with the ones significantly upregulated marked in red and of the most significantly changed genes, a selection of nine genes was labeled with the gene name. Plots are organized from Cluster 11 (left), to Microglia 13 (middle), to iMG Cluster 2+8 (right). B. Heatmaps showing the expression of Cluster 11 (left), Microglia 13 (middle) and iMG Cluster 2+8 (right) marker sets in bulk RNAseq data generated 24hrs following compound treatment with DMSO (control) or Vorinostat (green; 0.1µM). Each column represents a single sample, each row a single gene represented in the respective marker set. Pairwise differential testing between DMSO control and each of the treatment conditions (Entinostat, 10µM; Vorinostat, 1µM) was conducted using a Wald test with the Benjamini-Hochberg correction (FDR alpha < 0.05). The legend represents Z scores, with lower scores indicated in red and higher scores indicated in blue. Data represents n=5 independent experiments per treatment group from one batch of iPSC-derived human microglia. For data replication in a second batch see Supple. . C. Venn diagram depicting significantly induced markers across the signatures for Cluster 11 , Microglia 13 and iMG Cluster 2+8 in Vorinostat-treated iMGs. Each circle shows significantly induced markers from each marker set - Cluster 11 (violet), Microglia 13 (green), Dolan et al. (red). Overlays of circles depict induced marker genes shared across different combinations of marker sets. Percentage indicates ratio of each marker set in relation to the total number of significantly induced markers across all three signatures. D. MITF expression in HMC3 and iMG DAM models. Violin plots depict the expression of the transcription factor MITF in transcripts per million (TPM) across treatment conditions in HMC3 microglia (top; DMSO (blue), Vorinostat (1µM; green), Entinostat (10µM; red); n=3/group) and iMG (bottom; DMSO (blue), Vorinostat (0.1µM; green); n=6 per group, one iMG batch; for data replication see Suppl. ). For statistical analysis of HMC3 data, one-ay ANOVA followed by Dunnett’s multiple comparisons test was performed. For iMG data, unpaired t-test was performed. Each dot represents a replicate, central interrupted line represents the median and fine dotted lines represent the interquartile range. *p.adj ≤ 0.05, **p.adj ≤ 0.01, ***p.adj ≤ 0.001 test

Article Snippet: The next day, microglia were treated with the respective concentrations of Vorinostat (1µM; Ambeed; Cat #: A234507), Cholic acid (10µM; Cayman chemical; Cat #: 20250), Flavokavain B (10µM; ChromaDex; Cat #: ASB-00006058- 005), Wiskostatin (1µM; Cayman chemical; Cat #: 15047), Trimipramine (10µM; Cayman chemical; Cat #: 15921), Naftopidil (10µM; APExBIO; Cat #: 57149-07-2), Ramipril (10µM; APExBIO; Cat #: B2208), Valporic acid (90µg/ml Sigma; Cat #: PHR1061), Geranylgeraniol (10µM; Sigma; Cat #: 24034-73-9), Entinostat (10µM; Ambeed; Cat #: A122285), Amiodarone hydrochloride (10µM; Sigma; Cat #: A8423), Temozolomide (100µM; APExBIO; Cat #: B1399) or DMSO (Sigma-Aldrich, Cat #:472301) as control and incubated for 6hrs and 24hrs before harvest for RNA extraction.

Techniques: RNA Sequencing Assay, Derivative Assay, Comparison, Control, Marker, Expressing, Selection, Labeling, Generated

Journal: bioRxiv

Article Title: Functional Characterization of a Novel Long Non-Coding RNA in Leishmania braziliensis Identified Through Computational Screening for Conserved RNA Structures

doi: 10.1101/2025.01.14.632970

Figure Lengend Snippet: Characterization of LbrM2903_26_lncRNA45 (lncRNA45). (A) IGV representation of genomic regions on chromosome 26 of L. braziliensis MHOMBR75M2903, showing lncRNA44 and lncRNA45 (magenta), two coding sequences (CDSs) - hypothetical protein LBRM2903_260014500.1 and NMD3 family protein LBRM2903_260014600.1 (purple), and the predicted locus 709 (blue). (B) RNA-seq coverage (red stacking bars) of lncRNA45 (magenta) in axenic amastigotes (AXA), metacyclic promastigotes (META), and procyclic promastigotes (PRO). The target locus 709 is indicated in blue. (C) Detection of lncRNA45 as a transcript smaller than 500 bp in total RNA extract from L. braziliensis M2903 procyclic promastigotes, by northern blotting using a radiolabeled specific probe. rRNA served as a loading control. (D) Characterization of lncRNA45 size and processing pathways using RNA circularization. Total RNA from L. braziliensis M2903 promastigotes was extracted and treated with tobacco acidic phosphatase for decapping (TAP+) or left untreated (TAP-), followed by circularization with T4 RNA ligase. Complementary circular DNA (ccDNA) was synthesized by reverse transcription using a primer binding 200 nt from the 5’ end of lncRNA45. PCR amplification with primers lncRNA45_cF1 and lncRNA45_cR1 (shown in gray) was performed using ccDNA as a template, and PCR products were cloned into pGEM-T for sequencing (Hang, Deng et al., 2015). Sequences from three cDNA clones derived from TAP- (dark green) and four from TAP+ (light green) were analyzed. Poly(A) tails (red dots) were detected in 3 out of 7 transcript profiles, all derived from TAP+ templates. The most representative transcript profile among the clones is indicated by a black arrow.

Article Snippet: The reactions were incubated for 2 minutes on ice and after that 30 unities (U) of T4 RNA ligase (NEB) enzyme were added to the system together with 1x T4 Buffer, 1mM ATP and 40U of RNAse inhibitor RNasin® (Promega) in a final volume of 50µL.

Techniques: RNA Sequencing Assay, Northern Blot, Control, Synthesized, Reverse Transcription, Binding Assay, Amplification, Clone Assay, Sequencing, Derivative Assay

Journal: bioRxiv

Article Title: RGG peptide induces the disassembly of disease-relevant FUS and TDP43 condensates

doi: 10.1101/2025.03.19.643735

Figure Lengend Snippet: (A) Graph representing the distribution of FUS-P525L protein in nucleus and cytoplasm. The fluorescent intensities of the nucleus and cytoplasm were calculated from the experiment as performed in , and the ratio is plotted here. A student-paired t-test was used to calculate the significance value (n=6). (B) Incucyte images (real-time cell death analysis) representing the cellular uptake of propidium iodide (PI) in different conditions in HeLa cells. Scale bar=100um. (C) Graph representing the number of propidium iodide (PI) positive cells (dead cells) from the experiment as performed in B. The time on the x-axis reflects the time-point after transfection in hours. The significance was calculated using 2-way ANOVA with multiple comparisons (n=4). Error bars in all graphs represent mean ±SEM, and the same color points in A depict the data from a single experimental set. *, **, ***, and **** denote p-value≤0.05, ≤0.01, ≤0.001, and ≤0.0001, respectively.

Article Snippet: The cell death analysis was performed using the IncuCyte S3 live-cell analysis instrument (Sartorius), and the change in the number of PI-positive cells (dead cells) in different conditions was plotted in the graph.

Techniques: Transfection

Journal: bioRxiv

Article Title: RGG peptide induces the disassembly of disease-relevant FUS and TDP43 condensates

doi: 10.1101/2025.03.19.643735

Figure Lengend Snippet: Incucyte images representing the cellular uptake of propidium iodide (PI) in different conditions in HeLa cells. Scale bar=100um. The images are part of and .

Article Snippet: The cell death analysis was performed using the IncuCyte S3 live-cell analysis instrument (Sartorius), and the change in the number of PI-positive cells (dead cells) in different conditions was plotted in the graph.

Techniques: