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: eLife

Article Title: Regulation of store-operated Ca 2+ entry by IP 3 receptors independent of their ability to release Ca 2+

doi: 10.7554/eLife.80447

Figure Lengend Snippet: ( A ) Confocal images of hNPCs (passage 6) stained for DAPI and neural stem cell proteins: Pax6 and Ki67 (proliferation marker). Scale bars, 50 μm. ( B ) WB for IP 3 R1 of hNPCs expressing non-silencing (NS) or IP 3 R1-shRNA. ( C ) Summary results (mean ±s.d., n=3) show IP 3 R1 expression relative to actin. ** p < 0.01, Student’s t -test with unequal variances. ( D ) Changes in [Ca 2+ ] c evoked by thapsigargin (Tg, 10 µM) in Ca 2+ -free HBSS and then restoration of extracellular Ca 2+ (2 mM) in hNPCs expressing NS or IP 3 R1-shRNA. Mean ± s.e.m. from hree independent experiments, each with four replicates that together included 100–254 cells. Inset shows the target of Tg. ( E–G ) Summary results (individual cells, median (bar), 25th and 75th percentiles (box) and mean (circle)) show Ca 2+ signals evoked by Tg or Ca 2+ restoration ( E ), rate of Ca 2+ entry ( F ) and resting [Ca 2+ ] c ( G ). *** p < 0.001, Mann-Whitney U-test. ( H ) Changes in [Ca 2+ ] c evoked by Tg (10 µM) in Ca 2+ -free HBSS and after restoring extracellular Ca 2+ (2 mM) in neurons (differentiated hNPCs) expressing NS or IP 3 R1-shRNA. Mean ± s.e.m. from three experiments with ~200 cells. ( I,J ) Summary results (presented as in E-G) show Ca 2+ signals evoked by Tg or Ca 2+ restoration ( I ) and rate of Ca 2+ entry ( J ). *** p < 0.001. Mann-Whitney U-test. See also . Source data in . Figure 1—source data 1. Loss of IP 3 R1 attenuates SOCE in human neural stem cells.

Article Snippet: The primary antibodies used were to: IP 3 R1 (1:1000, ThermoFisher, Cat# PA1-901, RRID: AB_2129984 ); β-actin (1:5000, BD Biosciences, Cat# 612656, RRID: AB_2289199 ); STIM1 (1:1000, Cell Signaling Technology, Cat# 5668 S, RRID: AB_10828699 ); Orai1 (1:500, ProSci, Cat# PM-5205, RRID: AB_10941192 ); IP 3 R2 (1:1000, custom made by Pocono Rabbit Farm and Laboratory; ); and IP 3 R3 (1:500, BD Biosciences, Cat# 610313, RRID: AB_397705 ).

Techniques: Staining, Marker, Expressing, shRNA, MANN-WHITNEY

Journal: eLife

Article Title: Regulation of store-operated Ca 2+ entry by IP 3 receptors independent of their ability to release Ca 2+

doi: 10.7554/eLife.80447

Figure Lengend Snippet: ( A ) WB for IP R1-3 of SH-SY5Y cells expressing non-silencing (NS) or IP R1-shRNA. ( B ) Summary results (mean ± s.d., n=4) show IP R expression relative to actin normalized to control NS cells. ** p < 0.01, Student’s t -test with unequal variances. ( C ) Ca 2+ signals evoked by carbachol (CCh, 3 µM) in SH-SY5Y cells expressing NS or IP R1-shRNA. Mean ± s.e.m. from three experiments with 70–90 cells. ( D ) Summary results show peak changes in [Ca 2+ ] c (Δ[Ca 2+ ] c ) evoked by CCh. *** p < 0.001, Mann-Whitney U-test. ( E ) Ca 2+ signals evoked by thapsigargin (Tg, 10 µM) in Ca 2+ -free HBSS and then after restoration of extracellular Ca 2+ (2 mM) in cells expressing NS or IP R1-shRNA. Mean ± s.e.m. from three experiments with ~50 cells. ( F, G ) Summary results (individual cells, mean ± s.e.m., n=3, ~50 cells) show peak changes in [Ca 2+ ] c evoked by Ca 2+ restoration (Δ[Ca 2+ ] c ) ( F ) and rate of Ca 2+ entry ( G ). *** p < 0.001, Mann-Whitney U-test. ( H ) Ca 2+ signals evoked by Tg and then Ca 2+ restoration in cells expressing NS-shRNA, or IP R1-shRNA alone or with IP R1 or IP R3. Traces show mean ± s.e.m. (50–115 cells from three experiments). ( I, J ) Summary results (mean ± s.e.m, 50–115 cells from three experiments) show peak increases in [Ca 2+ ] c (Δ[Ca 2+ ] c ) evoked by Ca 2+ restoration ( I ) and rates of Ca 2+ entry ( J ) evoked by restoring extracellular Ca 2+ . ( K ) Effects of thapsigargin (Tg, 10 µM) in Ca 2+ -free HBSS and then after Ca 2+ restoration (2 mM) in cells expressing IP R1-shRNA alone or with IP R1 or mCh-STIM1. Traces show mean ± s.e.m. (100–150 cells from three experiments). ( L, M ) Summary results (mean ± s.e.m.) show peak increase in [Ca 2+ ] c after Ca 2+ restoration (Δ[Ca 2+ ] c ) ( L ) and rate of Ca 2+ entry ( M ). Different letters indicate significant differences (panels I , J, L, M), p <0.001, one-way ANOVA with pair-wise Tukey’s test. See also – . Source data in . Figure 2—source data 1. Loss of IP 3 R1 attenuates SOCE in SH-SY5Y cells.

Article Snippet: The primary antibodies used were to: IP 3 R1 (1:1000, ThermoFisher, Cat# PA1-901, RRID: AB_2129984 ); β-actin (1:5000, BD Biosciences, Cat# 612656, RRID: AB_2289199 ); STIM1 (1:1000, Cell Signaling Technology, Cat# 5668 S, RRID: AB_10828699 ); Orai1 (1:500, ProSci, Cat# PM-5205, RRID: AB_10941192 ); IP 3 R2 (1:1000, custom made by Pocono Rabbit Farm and Laboratory; ); and IP 3 R3 (1:500, BD Biosciences, Cat# 610313, RRID: AB_397705 ).

Techniques: Expressing, shRNA, MANN-WHITNEY

Journal: eLife

Article Title: Regulation of store-operated Ca 2+ entry by IP 3 receptors independent of their ability to release Ca 2+

doi: 10.7554/eLife.80447

Figure Lengend Snippet: ( A ) SOCE is activated when loss of Ca 2+ from the ER through IP 3 Rs activates STIM1 ( i ). Our results suggest an additional role for IP 3 Rs (ii). ( B ) SH-SY5Y cells expressing IP 3 R1-shRNA alone or with IP 3 R1 or IP 3 R1 DA were stimulated with thapsigargin (Tg, 1 µM) in Ca 2+ -free HBSS before restoring extracellular Ca 2+ (2 mM). Traces show mean ± s.e.m, for 100–150 cells from three experiments. ( C ) Cells expressing IP 3 R1-shRNA and IP 3 R1 DA were treated with NS-siRNA or Orai1-siRNA before measuring Tg-evoked Ca 2+ entry. Traces show mean ± s.e.m. for 85–100 cells from three experiments. ( D ) Summary results (mean ± s.e.m.) show peak increases in [Ca 2+ ] c (Δ[Ca 2+ ] c ) evoked by Ca 2+ restoration. ( E ) Tg-evoked Ca 2+ entry in cells expressing IP 3 R1-shRNA with IP 3 R1, IP 3 R1 RQ or IP 3 R1 RQ/KQ . Traces show mean ± s.e.m, for 90–150 cells from three experiments. ( F ) Summary results (mean ± s.e.m.) show peak increases in [Ca 2+ ] c (Δ[Ca 2+ ] c ) evoked by Ca 2+ restoration. Different letter codes (panels D , F ) indicate significantly different values, p<0.001, for multiple comparison one-way ANOVA and pair-wise Tukey’s test and for two genotype comparison Mann Whitney U-test. See also . Source data in . Figure 3—source data 1. Regulation of SOCE by IP 3 R requires IP 3 binding but not a functional pPore in SH-SY5Y cells.

Article Snippet: The primary antibodies used were to: IP 3 R1 (1:1000, ThermoFisher, Cat# PA1-901, RRID: AB_2129984 ); β-actin (1:5000, BD Biosciences, Cat# 612656, RRID: AB_2289199 ); STIM1 (1:1000, Cell Signaling Technology, Cat# 5668 S, RRID: AB_10828699 ); Orai1 (1:500, ProSci, Cat# PM-5205, RRID: AB_10941192 ); IP 3 R2 (1:1000, custom made by Pocono Rabbit Farm and Laboratory; ); and IP 3 R3 (1:500, BD Biosciences, Cat# 610313, RRID: AB_397705 ).

Techniques: Expressing, shRNA, MANN-WHITNEY, Binding Assay, Functional Assay

Journal: eLife

Article Title: Regulation of store-operated Ca 2+ entry by IP 3 receptors independent of their ability to release Ca 2+

doi: 10.7554/eLife.80447

Figure Lengend Snippet: ( A, B ) SH-SY5Y cells expressing IP 3 R1-shRNA alone ( A ) or with IP 3 R1 DA ( B ) were treated with a low concentration of CPA (2 µM) in Ca 2+ -free HBSS to partially deplete the ER of Ca 2+ and sub-maximally activate SOCE (see ). Carbachol (CCh, 1 µM) was then added to stimulate IP 3 formation through muscarinic receptors, and extracellular Ca 2+ (2 mM) was then restored. Traces (mean ± s.e.m of 68–130 cells from three experiments) show responses with and without the CCh addition. ( C ) Summary results show the peak increases in [Ca 2+ ] c (Δ[Ca 2+ ] c ) after addition of CCh (CCh-induced Ca 2+ release) and then after restoring extracellular Ca 2+ (SOCE). ( D–F ) SH-SY5Y cells wild type (WT) ( D ) and expressing NS-shRNA ( E ) or IP 3 R1-shRNA ( F ) were treated with YM-254890 (YM, 1 µM, 5 min) in Ca 2+ -free HBSS to inhibit Gαq and then with thapsigargin (Tg, 1 µM) before restoring extracellular Ca 2+ (2 mM). Traces show mean ± s.e.m of ~120 cells from three experiments. ( G–I ) Similar analyses of HEK cells. Summary results (mean ± s.e.m, 50–100 cells from three experiments) are shown in ( I ). Different letter codes (panels C and I) indicate significantly different values within the store Ca 2+ release or SOCE groups, p<0.001, one-way ANOVA and pair-wise Tukey’s test. See also . Source data in . Figure 4—source data 1. Receptor-regulated IP 3 production stimulates SOCE in cells with empty Ca 2+ stores and expressing pore-dead IP 3 R.

Article Snippet: The primary antibodies used were to: IP 3 R1 (1:1000, ThermoFisher, Cat# PA1-901, RRID: AB_2129984 ); β-actin (1:5000, BD Biosciences, Cat# 612656, RRID: AB_2289199 ); STIM1 (1:1000, Cell Signaling Technology, Cat# 5668 S, RRID: AB_10828699 ); Orai1 (1:500, ProSci, Cat# PM-5205, RRID: AB_10941192 ); IP 3 R2 (1:1000, custom made by Pocono Rabbit Farm and Laboratory; ); and IP 3 R3 (1:500, BD Biosciences, Cat# 610313, RRID: AB_397705 ).

Techniques: Expressing, shRNA, Concentration Assay

Journal: eLife

Article Title: Regulation of store-operated Ca 2+ entry by IP 3 receptors independent of their ability to release Ca 2+

doi: 10.7554/eLife.80447

Figure Lengend Snippet: ( A–E ) PLA analyses of interactions between STIM1 and Orai1 in SH-SY5Y cells expressing NS-shRNA ( A ) or IP 3 R1-shRNA alone ( B ) or with IP 3 R1 ( C ), IP 3 R1 DA ( D ) or IP 3 R1 RQ/KQ ( E ). Confocal images are shown for control cells or after treatment with thapsigargin (Tg, 1 µM) in Ca 2+ -free HBSS. PLA reaction product is red, and nuclei are stained with DAPI (blue). Scale bars, 5 µm. Summary results show the surface area of the PLA spots for 8–10 cells from two independent analyses. Individual values, median (bar) and 25th and 75th percentiles (box). *** p < 0.001, Student’s t -test with unequal variances. See also . Source data in . Figure 5—source data 1. IP 3 Rs promote interaction of STIM1 with Orai1.

Article Snippet: The primary antibodies used were to: IP 3 R1 (1:1000, ThermoFisher, Cat# PA1-901, RRID: AB_2129984 ); β-actin (1:5000, BD Biosciences, Cat# 612656, RRID: AB_2289199 ); STIM1 (1:1000, Cell Signaling Technology, Cat# 5668 S, RRID: AB_10828699 ); Orai1 (1:500, ProSci, Cat# PM-5205, RRID: AB_10941192 ); IP 3 R2 (1:1000, custom made by Pocono Rabbit Farm and Laboratory; ); and IP 3 R3 (1:500, BD Biosciences, Cat# 610313, RRID: AB_397705 ).

Techniques: Expressing, shRNA, Staining

Journal: eLife

Article Title: Regulation of store-operated Ca 2+ entry by IP 3 receptors independent of their ability to release Ca 2+

doi: 10.7554/eLife.80447

Figure Lengend Snippet: ( A–B ) Representative TIRF images of mVenus STIM1 co-transfected with either wild type mcherry-rat IP 3 R1 ( A ) or IP 3 R1 RQ/KQ (ligand binding mutant), ( B ) in wild type SH-SY5Y cells before (Basal) and after CPA induced store depletion (CPA treated) at 4 min and 7 min. On the right are shown RGB profile plots of STIM1 (green) and IP 3 R1, wild type or mutant (magenta) corresponding to the rectangular selections (Cell 1 and Cell 2). Scale bar is 10 µm.( C–D ) Changes in number of IP 3 R1 ( C ) and STIM1 ( D ) puncta upon CPA-induced store depletion over a period of 10 min in the indicated genotypes. Mean ± s.e.m from seven cells from n=6 independent experiments. ( E ) Summary result (mean ± s.e.m) showing the change in the number of maximum STIM1 puncta formed after CPA-induced store depletion in the indicated genotypes. Mean ± s.e.m. of seven cells from n=6 independent experiments. Different letters indicate significant differences, p<0.05, Mann-Whitney U-test. See also . Source data in . Figure 6—source data 1. Ligand-bound IP 3 R1 supports SOCE-dependent STIM1 movement to ER-PM contact sites.

Article Snippet: The primary antibodies used were to: IP 3 R1 (1:1000, ThermoFisher, Cat# PA1-901, RRID: AB_2129984 ); β-actin (1:5000, BD Biosciences, Cat# 612656, RRID: AB_2289199 ); STIM1 (1:1000, Cell Signaling Technology, Cat# 5668 S, RRID: AB_10828699 ); Orai1 (1:500, ProSci, Cat# PM-5205, RRID: AB_10941192 ); IP 3 R2 (1:1000, custom made by Pocono Rabbit Farm and Laboratory; ); and IP 3 R3 (1:500, BD Biosciences, Cat# 610313, RRID: AB_397705 ).

Techniques: Transfection, Ligand Binding Assay, Mutagenesis, MANN-WHITNEY

Journal: eLife

Article Title: Regulation of store-operated Ca 2+ entry by IP 3 receptors independent of their ability to release Ca 2+

doi: 10.7554/eLife.80447

Figure Lengend Snippet: ( A ) SOCE is activated when loss of Ca 2+ from the ER, usually mediated by opening of IP 3 Rs when they bind IP 3 , causes STIM to unfurl cytosolic domains (2). The exposed cytosolic domains of STIM1 reach across a narrow gap between the ER and PM at a MCS to interact with PIP 2 and Orai1 in the PM. Binding of STIM1 to Orai1 causes pore opening, and SOCE then occurs through the open Orai1 channel. We show that IP 3 Rs when they bind IP 3 also facilitate interactions between Orai1 and STIM, perhaps by stabilizing the MCS (1). Receptors that stimulate IP 3 formation thereby promote both activation of STIM (by emptying Ca 2+ stores) and independently promote interaction of active STIM1 with Orai1. ( B ) Other mechanisms, including ryanodine receptors (RyR), can also release Ca 2+ from the ER. We suggest that convergent regulation of SOCE by IP 3 R with bound IP 3 allows receptors that stimulate IP 3 formation to selectively control SOCE.

Article Snippet: The primary antibodies used were to: IP 3 R1 (1:1000, ThermoFisher, Cat# PA1-901, RRID: AB_2129984 ); β-actin (1:5000, BD Biosciences, Cat# 612656, RRID: AB_2289199 ); STIM1 (1:1000, Cell Signaling Technology, Cat# 5668 S, RRID: AB_10828699 ); Orai1 (1:500, ProSci, Cat# PM-5205, RRID: AB_10941192 ); IP 3 R2 (1:1000, custom made by Pocono Rabbit Farm and Laboratory; ); and IP 3 R3 (1:500, BD Biosciences, Cat# 610313, RRID: AB_397705 ).

Techniques: Binding Assay, Activation Assay

Journal: Emerging Microbes & Infections

Article Title: Multiple introductions of highly pathogenic avian influenza H5N1 viruses into Bangladesh

doi: 10.1038/emi.2014.11

Figure Lengend Snippet: Avian influenza A (H5N1) virus isolates from live bird markets in Bangladesh from December 2010 to March 2013

Article Snippet: Within the HA phylogenetic tree, isolates A/Ck/BD/15079/12 (H5N1) and A/Ck/BD/15083/12 (H5N1), both original samples collected on the same day in January 2012 from the same live bird market in Dhaka city, fell within clade 2.3.2.1, but did not cluster together with the rest of the H5N1 isolates from Bangladesh.

Techniques: Virus, Isolation

Journal: Emerging Microbes & Infections

Article Title: Multiple introductions of highly pathogenic avian influenza H5N1 viruses into Bangladesh

doi: 10.1038/emi.2014.11

Figure Lengend Snippet: Antigenic analysis of H5N1 influenza viruses from Bangladesh in the hemagglutination inhibition assays

Article Snippet: Within the HA phylogenetic tree, isolates A/Ck/BD/15079/12 (H5N1) and A/Ck/BD/15083/12 (H5N1), both original samples collected on the same day in January 2012 from the same live bird market in Dhaka city, fell within clade 2.3.2.1, but did not cluster together with the rest of the H5N1 isolates from Bangladesh.

Techniques: HI Assay

Journal: Emerging Microbes & Infections

Article Title: Multiple introductions of highly pathogenic avian influenza H5N1 viruses into Bangladesh

doi: 10.1038/emi.2014.11

Figure Lengend Snippet: Amino-acid residues characteristic of the H5N1 viruses isolated from Bangladesh

Article Snippet: Within the HA phylogenetic tree, isolates A/Ck/BD/15079/12 (H5N1) and A/Ck/BD/15083/12 (H5N1), both original samples collected on the same day in January 2012 from the same live bird market in Dhaka city, fell within clade 2.3.2.1, but did not cluster together with the rest of the H5N1 isolates from Bangladesh.

Techniques: Isolation, Residue, Virus, Binding Assay, Infection, Marker

Journal: Emerging Microbes & Infections

Article Title: Multiple introductions of highly pathogenic avian influenza H5N1 viruses into Bangladesh

doi: 10.1038/emi.2014.11

Figure Lengend Snippet: Phylogenetic tree of the HA genes of HPAI H5N1 viruses from Bangladesh, generated by neighbor-joining method in MEGA 5. Numbers at the branches indicate bootstrap values; only values >70 are shown. ▴ indicates Bangladesh isolates clade 2.2.2; ▪ indicates Bangladesh isolates from the A/Hubei/1/10-like lineage of clade 2.3.2.1; • indicates Bangladesh isolates from the A/Hong Kong/6841/10-like lineage of clade 2.3.2.1.

Article Snippet: Within the HA phylogenetic tree, isolates A/Ck/BD/15079/12 (H5N1) and A/Ck/BD/15083/12 (H5N1), both original samples collected on the same day in January 2012 from the same live bird market in Dhaka city, fell within clade 2.3.2.1, but did not cluster together with the rest of the H5N1 isolates from Bangladesh.

Techniques: Generated

Journal: Emerging Microbes & Infections

Article Title: Multiple introductions of highly pathogenic avian influenza H5N1 viruses into Bangladesh

doi: 10.1038/emi.2014.11

Figure Lengend Snippet: Phylogenetic tree of the NA gene of H5N1 viruses from Bangladesh, generated by neighbor-joining method in MEGA 5. Numbers at the branches indicate bootstrap values; only values >70 are shown. ▴ indicates Bangladesh isolates clade 2.2.2; ▪ indicates Bangladesh isolates from the A/Hubei/1/10-like lineage of clade 2.3.2.1; • indicates Bangladesh isolates from the A/Hong Kong/6841/10-like lineage of clade 2.3.2.1.

Article Snippet: Within the HA phylogenetic tree, isolates A/Ck/BD/15079/12 (H5N1) and A/Ck/BD/15083/12 (H5N1), both original samples collected on the same day in January 2012 from the same live bird market in Dhaka city, fell within clade 2.3.2.1, but did not cluster together with the rest of the H5N1 isolates from Bangladesh.

Techniques: Generated

Journal: Emerging Microbes & Infections

Article Title: Multiple introductions of highly pathogenic avian influenza H5N1 viruses into Bangladesh

doi: 10.1038/emi.2014.11

Figure Lengend Snippet: Phylogenetic tree of the M gene of H5N1 viruses from Bangladesh, generated by neighbor-joining method in MEGA 5. Numbers at the branches indicate bootstrap values; only values >70 are shown. ▴ indicates Bangladesh isolates clade 2.2.2; ▪ indicates Bangladesh isolates from the A/Hubei/1/10-like lineage of clade 2.3.2.1; • indicates Bangladesh isolates from the A/Hong Kong/6841/10-like lineage of clade 2.3.2.1.

Article Snippet: Within the HA phylogenetic tree, isolates A/Ck/BD/15079/12 (H5N1) and A/Ck/BD/15083/12 (H5N1), both original samples collected on the same day in January 2012 from the same live bird market in Dhaka city, fell within clade 2.3.2.1, but did not cluster together with the rest of the H5N1 isolates from Bangladesh.

Techniques: Generated

Journal: Emerging Microbes & Infections

Article Title: Multiple introductions of highly pathogenic avian influenza H5N1 viruses into Bangladesh

doi: 10.1038/emi.2014.11

Figure Lengend Snippet: Phylogenetic tree of the PB2 gene of H5N1 viruses from Bangladesh, generated by neighbor-joining method in MEGA 5. Numbers at the branches indicate bootstrap values; only values >70 are shown. ▴ indicates Bangladesh isolates clade 2.2.2; ▪ indicates Bangladesh isolates from the A/Hubei/1/10-like lineage of clade 2.3.2.1; • indicates Bangladesh isolates from the A/Hong Kong/6841/10-like lineage of clade 2.3.2.1.

Article Snippet: Within the HA phylogenetic tree, isolates A/Ck/BD/15079/12 (H5N1) and A/Ck/BD/15083/12 (H5N1), both original samples collected on the same day in January 2012 from the same live bird market in Dhaka city, fell within clade 2.3.2.1, but did not cluster together with the rest of the H5N1 isolates from Bangladesh.

Techniques: Generated

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: 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: