Journal: Journal of cell science

Article Title: Increased levels of the cytoplasmic domain of Crumbs repolarise developing Drosophila photoreceptors.

doi: 10.1242/jcs.091223

Figure Lengend Snippet: Fig. 2. Overexpression of CrbFLAGintra during late larval and pupal development affects photoreceptor apico-basal polarity. (A–H) Immunostainings on cross-sections of adult wild-type (wt) eyes (A,C,E,G) and eyes overexpressing CrbFLAGintra using elavGal4 (elav.CrbFLAGintra) (B,D,F,H). The sections were stained for Drosophila E-Cad (DE-Cad) (A,B), Rh1 (C,D), Spam (Eys) (E,F) or the alpha subunit of Na+/K+-ATPase (G,H) (in red), and Sdt (A–C), Crb (D,E,G,H) or Drosophila PATJ (F) (in green). B-, D-, F- and H- represent higher magnifications of one single ommatidium shown in B0, D0, F0 and G0, respectively. The F-actin stain (blue) shows the rhabdomeres. Sdt (B9), Crb (D9,H9) and Drosophila PATJ (F0) exhibit expanded expression domains and often localise adjacent to ectopic rhabdomeres. Additionally, Sdt localises between split rhabdomeres (B9,B0, white arrowhead). Note ectopic Drosophila E-Cad flanking ectopic rhabdomeres (B, B0,B-, white arrows). Ectopic apical poles are positive for Rh1 (D,D0,D-, arrows) and Spam (Eys) (F, F0, F-, arrows). Na+/K+-ATPase is absent from ectopic rhabdomeres and diminished at membranes with high levels of ectopic Crb (H,H0,H-, the decrease is indicated by a cross). In addition, Na+/K+-ATPase is present at the stalk of cells with ectopic rhabdomeres, which are not R7 (H,H0,H-, some cells other than R7 are indicated by asterisks). Scale bars: 2 mm. (I,J) Schematic summary of the distribution of proteins (as shown in A–H) in wild-type ommatidia (I) and ommatidia overexpressing CrbFLAGintra (J).

Article Snippet: The following primary antibodies were used: rat anti-Crb2.8 antibody (1:1000) (Richard et al., 2006), rabbit anti-Sdt-PDZ antibody (1:500) (Berger et al., 2007), mouse anti-Sdt-PDZ antibody (B1-8, 1:200) (Bulgakova et al., 2010), rabbit antiDrosophila-PATJ antibody (1:500) (Richard et al., 2006), rat anti-DE-Cad antibody (DCAD2, 1:50, DSHB), mouse anti-Na+/K+-ATPase antibody (a5, alpha subunit, 1:500 concentrate, DSHB), rabbit anti-Rh1 antibody (1:1000) (Satoh et al., 2005), mouse anti-Rh1 (4C5, 1:100, DSHB), mouse anti-Spam antibody (21A6, 1:100, DSHB) (Zelhof et al., 2006), mouse anti-FLAG M2 antibody (1:1000, Sigma), rabbit anti-FLAG antibody (1:200, Sigma, F7425), rabbit anti-Baz antibody (1:400, kindly provided by Andreas Wodarz) (Wodarz et al., 1999), guinea pig anti-Par-6 antibody (1:1000, kindly provided by Andreas Wodarz) (Kim et al., 2009), mouse anti-Arm antibody (N2 7A1, 1:50, DSHB), rabbit anti-PKC f C20 (1:1000, Santa Cruz Biotechnology), rabbit anti-phosphorylated-Ezrin/Radixin/Moesin (1:100, Cell Signalling Technology), and rabbit anti-bH-spec (1:500, kindly provided by Graham Thomas) (Thomas and Kiehart, 1994).

Techniques: Over Expression, Staining, Expressing

Journal: Optometry and Vision Science

Article Title: Fibrous Catalyst–Enhanced Acanthamoeba Disinfection by Hydrogen Peroxide

doi: 10.1097/opx.0000000000001126

Figure Lengend Snippet: FIGURE 1. Killing efficacy of commercial peroxide systems against trophozoites of A. castellanii (ATCC 50370) (A) and A. polyphaga (ATCC 30461) (B). Fabric refers to the iron-containing fabric catalyst. Each data point represents the mean ± SD of three individual tests. In each case, the catalyst enhances reduction of the trophozoites 2 logs over the controls.

Article Snippet: According to US patents 8410011B2 and 8513303B2, creation of the iron-containing catalyst was carried out using a base material of 50:50 nonfunctional propylene:polyacryonitrile, with approximate density of 0.256 g/cm3 with the final catalytic iron content ranging from 0.92 to 1.71% wt./wt.11,12 Acanthamoeba castellanii (ATCC 50370) and Acanthamoeba polyphaga (ATCC 30461) were used in the study.

Techniques:

Journal: Optometry and Vision Science

Article Title: Fibrous Catalyst–Enhanced Acanthamoeba Disinfection by Hydrogen Peroxide

doi: 10.1097/opx.0000000000001126

Figure Lengend Snippet: FIGURE 2. Killing efficacy of commercial peroxide systems against cysts of A. castellanii (ATCC 50370) (A) and A. polyphaga (ATCC 30461) (B). Fabric refers to the iron-containing fabric catalyst. Each data point represents the mean ± SD of three individual tests. In each case, the catalyst enhances re- duction of the cysts 2 logs over the controls.

Article Snippet: According to US patents 8410011B2 and 8513303B2, creation of the iron-containing catalyst was carried out using a base material of 50:50 nonfunctional propylene:polyacryonitrile, with approximate density of 0.256 g/cm3 with the final catalytic iron content ranging from 0.92 to 1.71% wt./wt.11,12 Acanthamoeba castellanii (ATCC 50370) and Acanthamoeba polyphaga (ATCC 30461) were used in the study.

Techniques:

Journal: Cell reports

Article Title: APOE4-promoted gliosis and degeneration in tauopathy are ameliorated by pharmacological inhibition of HMGB1 release.

doi: 10.1016/j.celrep.2023.113252

Figure Lengend Snippet: Figure 1. APOE4 promotes the nucleocytoplasmic translocation of HMGB1 in hippocampal neurons in tauopathy mice (A) Representative images of immunostaining with anti-HMGB1, anti-NeuN, and DAPI in the dentate gyrus of the hippocampus (scale bar, 40 mm). (B) Representative high-magnification images of immunostaining with anti-HMGB1 and DAPI in the dentate gyrus of the hippocampus (scale bar, 10 mm).

Article Snippet: Measurement of HMGB1 levels with sandwich ELISA Biochemically extracted mouse hippocampal tissue lysates or collected hippocampal interstitial fluid (ISF) were run according to the provided manufacturer protocols (mouse HMGB1; (Novus Biologicals).

Techniques: Translocation Assay, Immunostaining

Journal: Cell reports

Article Title: APOE4-promoted gliosis and degeneration in tauopathy are ameliorated by pharmacological inhibition of HMGB1 release.

doi: 10.1016/j.celrep.2023.113252

Figure Lengend Snippet: Figure 3. APOE4 promotes acetylation of HMGB1 and decreases levels of SIRT1 deacetylase, and removal of neuronal APOE4 reduces HMGB1 acetylation and increases SIRT1 levels (A) Representative images of anti-acetyl-HMGB1 (green) and anti-GAPDH (red) western blots in hippocampal tissue lysates. (B) Quantification of acetyl-HMGB1 levels relative to GAPDH. (C and D) Quantification of acetyl-HMGB1 levels relative to GAPDH in the nuclear fraction (C) and cytoplasmic fraction (D).

Article Snippet: Measurement of HMGB1 levels with sandwich ELISA Biochemically extracted mouse hippocampal tissue lysates or collected hippocampal interstitial fluid (ISF) were run according to the provided manufacturer protocols (mouse HMGB1; (Novus Biologicals).

Techniques: Histone Deacetylase Assay, Western Blot

Journal: Cell reports

Article Title: APOE4-promoted gliosis and degeneration in tauopathy are ameliorated by pharmacological inhibition of HMGB1 release.

doi: 10.1016/j.celrep.2023.113252

Figure Lengend Snippet: Figure 4. Short-term treatment of PS19-E4 mice with HMGB1 inhibitors blocks the nucleocytoplasmic translocation of HMGB1 in hippo- campal neurons and its release into the ISF (A) Experimental design of the HMGB1 inhibitor study, with EP + GA treatment at three doses per week. (B) Representative high-magnification images of immunostaining with anti-HMGB1 and DAPI in hippocampal neurons (scale bar, 10 mm). (C and D) Quantification of the nuclear integrated density (C) and extranuclear integrated density (D) of HMGB1 immunostaining in hippocampal neurons. (E) HMGB1 protein levels measured by ELISA in the hippocampal interstitial fluid (ISF). (F) HMGB1 protein levels measured by ELISA in each collected ISF fraction. Fractions 1 and 2 were excluded from analyses in (E) and (F). (G) Representative images of microglia immunostaining with anti-Iba1 in the hippocampus (scale bar, 500 mm). (H) Quantification of the percentage of Iba1 coverage area in the hippocampus. (I) Representative images of astrocyte immunostaining with anti-GFAP in the hippocampus (scale bar, 500 mm). (J) Quantification of the percentage of GFAP coverage area in the hippocampus. (K) Representative images of activated microglia immunostaining with anti-CD68 in the hippocampus (scale bar, 500 mm). (L) Quantification of the percentage of CD68 coverage area in the hippocampus. (M) Representative images of activated astrocyte immunostaining with anti-S100b in the hippocampus (scale bar, 500 mm). (N) Quantification of the percentage of S100b coverage area in the hippocampus. (legend continued on next page)

Article Snippet: Measurement of HMGB1 levels with sandwich ELISA Biochemically extracted mouse hippocampal tissue lysates or collected hippocampal interstitial fluid (ISF) were run according to the provided manufacturer protocols (mouse HMGB1; (Novus Biologicals).

Techniques: Translocation Assay, Immunostaining, Enzyme-linked Immunosorbent Assay

Journal: Cell reports

Article Title: APOE4-promoted gliosis and degeneration in tauopathy are ameliorated by pharmacological inhibition of HMGB1 release.

doi: 10.1016/j.celrep.2023.113252

Figure Lengend Snippet: Figure 6. Long-term treatment of PS19-E4 mice with HMGB1 inhibitors drastically reduces Tau pathology, myelin deficits, and hippocampal degeneration (A) Representative images of p-Tau immunostaining with AT8 monoclonal antibody in the hippocampus (scale bar, 500 mm). (B) Quantification of the percentage of p-Tau (AT8+) coverage area in the hippocampus. (C) Representative images of myelin sheath staining with anti-MBP and DAPI in the stratum radiatum of CA1 in the hippocampus (scale bar, 100 mm). (D) Quantification of the percentage of MBP coverage area in the stratum radiatum of CA1. (E) Representative images of the ventral hippocampus after staining with Sudan black (scale bar, 1 mm). (F and G) Quantification of hippocampal volume (F) and posterior lateral ventricle volume (G). For all representative images and quantified data, mice were 9.5 months of age and belonged to either the PS19-E4 or the PS19-E3 group treated with saline or HMGB1 inhibitors. Quantified data in (B), (D), (F), and (G) (PS19-E4 saline, n = 16; PS19-E4 HMGB1-In, n = 18; PS19-E3 saline, n = 16, PS19-E3 HMGB1-In, n = 14) are represented as the mean ± SEM, one-way ANOVA with Tukey’s post hoc multiple comparisons test. HMGB1-In, HMGB1 inhibitors.

Article Snippet: Measurement of HMGB1 levels with sandwich ELISA Biochemically extracted mouse hippocampal tissue lysates or collected hippocampal interstitial fluid (ISF) were run according to the provided manufacturer protocols (mouse HMGB1; (Novus Biologicals).

Techniques: Immunostaining, Staining, Saline

Journal: International Journal of Molecular Medicine

Article Title: Eya2 overexpression promotes the invasion of human astrocytoma through the regulation of ERK/MMP9 signaling

doi: 10.3892/ijmm.2017.3132

Figure Lengend Snippet: Expression pattern of eyes absent (Eya) 2 in human astrocytoma. (A) Immunohistochemical staining of Eya2 protein in normal brain tissue was negative in glial cells and weak in neurons. The black arrows indicate neurons and the white arrows indicate glial cells. (B) Negative Eya2 staining in pilocytic astrocytoma (grade I). (C) Negative Eya2 staining in grade II diffuse astrocytoma. (D) Weak nuclear Eya2 staining in grade III anaplastic astrocytoma. (E) Strong nuclear Eya2 staining in grade III anaplastic astrocytoma. (F) Strong nuclear Eya2 staining in glioblastoma (grade IV) (magnification, ×200). Scale bar, 50 µ m.

Article Snippet: Eya2 rabbit polyclonal antibody (1:300 dilution; 11314-1-AP; Proteintech Inc., Chicago, IL, USA) was used to incubate the tissue sections at 4°C overnight.

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: International Journal of Molecular Medicine

Article Title: Eya2 overexpression promotes the invasion of human astrocytoma through the regulation of ERK/MMP9 signaling

doi: 10.3892/ijmm.2017.3132

Figure Lengend Snippet: The association between eyes absent 2 (Eya 2) and the clinical characteristics of astrocytoma.

Article Snippet: Eya2 rabbit polyclonal antibody (1:300 dilution; 11314-1-AP; Proteintech Inc., Chicago, IL, USA) was used to incubate the tissue sections at 4°C overnight.

Techniques: Expressing

Journal: International Journal of Molecular Medicine

Article Title: Eya2 overexpression promotes the invasion of human astrocytoma through the regulation of ERK/MMP9 signaling

doi: 10.3892/ijmm.2017.3132

Figure Lengend Snippet: Eyes absent (Eya) 2 expression in astrocytoma cell lines. (A) Western blot analysis indicated that Eya2 and Six1 protein expression was high in the U251 cell line and low in the U87 and A172 cell lines. (B) Eya2 siRNA treatment decreased its mRNA and protein expression in U251 cells. * P<0.05 Eya2 siRNA vs Neg siRNA in U251 cells. Eya2 plasmid transfection upregulated its mRNA and protein levels. * P<0.05 Eya2 plasmid vs control plasmid in A172 cells.

Article Snippet: Eya2 rabbit polyclonal antibody (1:300 dilution; 11314-1-AP; Proteintech Inc., Chicago, IL, USA) was used to incubate the tissue sections at 4°C overnight.

Techniques: Expressing, Western Blot, Plasmid Preparation, Transfection, Control

Journal: International Journal of Molecular Medicine

Article Title: Eya2 overexpression promotes the invasion of human astrocytoma through the regulation of ERK/MMP9 signaling

doi: 10.3892/ijmm.2017.3132

Figure Lengend Snippet: Eyes absent (Eya) 2 regulates the growth and invasion of astrocytoma cells. (A) Cell Counting kit-8 (CCK-8) assay demonstrated that Eya2 overexpression accelerated cell growth rate, while Eya2 knockdown inhibited cell proliferation. (B) Colony formation assay demonstrated that Eya2 overexpression upregulated the colony number, while Eya2 knockdown downregulated the colony number. * P<0.05 Eya2 siRNA vs Neg siRNA in U251 cells. * P<0.05 Eya2 plasmid vs control plasmid in A172 cells. (C) Matrigel invasion assay revealed that Eya2 knockdown decreased cell invasion in the U251 cell line. * P<0.05 Eya2 siRNA vs Neg siRNA in U251 cells. Transfection with Eya2 expession plasmid increased the A172 invading cell number. * P<0.05 Eya2 plasmid vs control plasmid in A172 cells.

Article Snippet: Eya2 rabbit polyclonal antibody (1:300 dilution; 11314-1-AP; Proteintech Inc., Chicago, IL, USA) was used to incubate the tissue sections at 4°C overnight.

Techniques: Cell Counting, CCK-8 Assay, Over Expression, Knockdown, Colony Assay, Plasmid Preparation, Control, Invasion Assay, Transfection

Journal: International Journal of Molecular Medicine

Article Title: Eya2 overexpression promotes the invasion of human astrocytoma through the regulation of ERK/MMP9 signaling

doi: 10.3892/ijmm.2017.3132

Figure Lengend Snippet: Eyes absent (Eya) 2 regulates cell cycle and related protein expression. (A) Eya2 overexpression in A172 cells increased the S phase percentage and decreased G1 phase percentage. Eya2 knockdown by siRNA in U251 cells had the opposite effect. * P<0.05 Eya2 siRNA vs Neg siRNA in U251 cells. * P<0.05 Eya2 plasmid vs control plasmid in A172 cells. (B) Eya2 overexpression upregulated cyclin D1, cyclin E, MMP9 mRNA and protein expression in the A172 cell line. * P<0.05 Eya2 plasmid vs control plasmid in A172 cells. Eya2 knockdown downregulated the expression of these proteins in the U251 cells. * P<0.05 Eya2 siRNA vs Neg siRNA in U251 cells. Eya2 overexpression activated ERK phosphorylation, while Eya2 knockdown downregulated ERK phosphorylation.

Article Snippet: Eya2 rabbit polyclonal antibody (1:300 dilution; 11314-1-AP; Proteintech Inc., Chicago, IL, USA) was used to incubate the tissue sections at 4°C overnight.

Techniques: Expressing, Over Expression, Knockdown, Plasmid Preparation, Control, Phospho-proteomics

Journal: International Journal of Molecular Medicine

Article Title: Eya2 overexpression promotes the invasion of human astrocytoma through the regulation of ERK/MMP9 signaling

doi: 10.3892/ijmm.2017.3132

Figure Lengend Snippet: Eyes absent (Eya) 2 interacts with Six1 and regulates ERK signaling. (A) ERK inhibitor, PD98059, blocked the promoting effect of Eya2 on A172 invasion. * P<0.05 Eya2 plasmid vs control plasmid in A172 cells. (B) Western blot analysis revealed that ERK inhibitor, PD98059, abolished the promoting effect of Eya2 on MMP9 mRNA and protein expression. * P<0.05 Eya2 plasmid vs control plasmid in A172 cells. (C) Eya2 co-immunoprecipitated with Six1 in U251 cells. (D) Transfection with Six1 siRNA significantly downregulated its protein in A172 cells. In Six1-depleted cells, transfection with Eya2 expression plasmid could not upregulate MMP9 expression at both the mRNA and protein level. * P<0.05 Eya2 plasmid vs control plasmid in A172 cells. Densitometry of western bands was measured using ImageJ software and relative intensity was calculated and indicated.

Article Snippet: Eya2 rabbit polyclonal antibody (1:300 dilution; 11314-1-AP; Proteintech Inc., Chicago, IL, USA) was used to incubate the tissue sections at 4°C overnight.

Techniques: Plasmid Preparation, Control, Western Blot, Expressing, Immunoprecipitation, Transfection, Software

Journal: Clinical kidney journal

Article Title: Mechanisms of pathogenicity and the quest for genetic modifiers of kidney disease in branchiootorenal syndrome.

doi: 10.1093/ckj/sfad260

Figure Lengend Snippet: Figure 2: Analysis of the EYA1 splice site affected by c.1698 + 1G > A. ( A) Gene structure and location of canonical splice site mutation c.1698 + 1G > A in EYA1 ( NM_000503.5) with transcription factor binding EYA domain formed by amino acids at positions 322–592. ( B) Three-dimensional structure of EYA1 protein ( PDB ID Q99502) with indicated N- and C-terminal ends of its EYA domain. The sequence preserved in the patients is depicted in light grey ( including three strands from the beta sheet structure ( black) ) , whereas the sequence part that is aberrant in the patients is shown in salmon ( including two strands from the beta sheet structure ( red) ) . ( C) Agarose gel image, showing separation of mutant from wild-type EYA1 cDNA fragments. The analysis is based on a 290 bp amplicon spanning the cDNA region from the transcribed exon 16 through the 3′ untranslated region. The transcript corresponding to the lower band in the middle lane lacks exon 17. That corresponding to the upper band seems to lack exon 17, too, while also containing additional nucleotides, probably as an artifact from PCR. The cDNA was derived from primary dermal fibroblasts. ( D) Chromatogram from Sanger sequencing the lower band evident in the middle lane in ( C) , showing the skip of exon 17.

Article Snippet: The following primary antibodies were sed: EYA1 rabbit polyclonal antibody ( #22658-1-AP, Proteintech, osemont, IL, USA) at 1:500 and recombinant anti-vinculin antiody ( #ab129002, Abcam, Cambridge, UK) produced in rabbit at :10 000.

Techniques: Mutagenesis, Binding Assay, Sequencing, Agarose Gel Electrophoresis, Amplification, Derivative Assay

Journal: Clinical kidney journal

Article Title: Mechanisms of pathogenicity and the quest for genetic modifiers of kidney disease in branchiootorenal syndrome.

doi: 10.1093/ckj/sfad260

Figure Lengend Snippet: Figure 3: Consequences of the EYA1 variant c.1698 + 1G > A for cellular EYA1 content and distribution. ( A) Immunofluorescence labeling of EYA1 ( red) in primary dermal fibroblasts. While control cells show consistent nuclear enrichment of EYA1 ( arrows) , patient cells are characterized by a heterogeneous cellular EYA1 distribution with many cells depleted of nuclear EYA1 ( arrowheads) . Nuclei stained with DAPI ( blue) . Scale bars, 50 μm. ( B) Quantification of nuclear fluorescence intensities exemplified in ( A) . Data from three biological replicates, each averaging signal intensities from 142–263 nuclei. Unpaired two-tailed t -test, * p < 0.05, mean ± SD. ( C) Immunoblot on whole cell lysates from primary dermal fibroblasts ( left, data from three biological replicates) and qPCR data ( right, mean of three technical replicates) . Together, immunoblot and qPCR data corroborate the results from immunofluorescence microscopy by showing increased cytosolic EYA1 at an overall reduced EYA1 expression level in patient cells.

Article Snippet: The following primary antibodies were sed: EYA1 rabbit polyclonal antibody ( #22658-1-AP, Proteintech, osemont, IL, USA) at 1:500 and recombinant anti-vinculin antiody ( #ab129002, Abcam, Cambridge, UK) produced in rabbit at :10 000.

Techniques: Variant Assay, Immunofluorescence, Labeling, Control, Staining, Fluorescence, Two Tailed Test, Western Blot, Microscopy, Expressing

Journal: Clinical kidney journal

Article Title: Mechanisms of pathogenicity and the quest for genetic modifiers of kidney disease in branchiootorenal syndrome.

doi: 10.1093/ckj/sfad260

Figure Lengend Snippet: Figure 4: CYP51A1 plays a role in SHH signaling and is expressed in the developing kidney. ( A) CYP51A1 gene structure and location of the c.770 + 1G > A splice site variant ( NM_000786.3) . ( B) Involvement of EYA1 and CYP51A1 in SHH signaling. GLI is activated into GLIA by Smoothened ( SMO) which is suppressed by Patched ( PTCH1) . PTCH1 is removed by SHH which in turn is regulated by CYP51A1 [11 ]. Together with the cofactor SIX1, EYA1 enhances GLI activity [18 ]. This figure was created with BioRender.com. ( C, D) Expression of CYP51A1 ( C) and EYA1 ( D) throughout nephron development based on single-cell RNA sequencing data from 14 weeks old human kidneys [17 ].

Article Snippet: The following primary antibodies were sed: EYA1 rabbit polyclonal antibody ( #22658-1-AP, Proteintech, osemont, IL, USA) at 1:500 and recombinant anti-vinculin antiody ( #ab129002, Abcam, Cambridge, UK) produced in rabbit at :10 000.

Techniques: Variant Assay, Activity Assay, Expressing, RNA Sequencing