Journal: Nucleic Acids Research

Article Title: Tyrosine kinase c-Abl couples RNA polymerase II transcription to DNA double-strand breaks

doi: 10.1093/nar/gkz024

Figure Lengend Snippet: Formation and p-Dicer-dependent turnover of dsRNA at DSBs. ( A ) ChIP analysis of GFP-RNaseH1 occupancy at DS1 using site-specific primers. ( B ) Quantitative real-time PCR (qRT-PCT) of DNA immunopurified from DNA–RNA hybrids (DRIP) or upon incubation with recombinant RNaseH at DS1 using S9.6 hybridoma supernatant and region-specific primers. ( C ) qRT-PCR analysis of transcripts associated with CTD Y1P and immunopurification (mNET-IP). Values were normalized to data in absence of GFP-RNaseH1. (A–E) Asterisk, P -value < 0.05, two-tailed t -test. Error bar: mean ± SEM, n = 3. ( D, E ) Imaging and RGB quantitation of CTD Y1P and p-Dicer (p-DCR-1). White box, 2.5× zoom; n , number of cells with shown phenotype in %. ( F ) qRT-PCR of cDNA after J2 immunoselection with (J2 RIP) and reverse transcription with forward (upper panel)- and reverse (lower panel)-oriented primers spanning a region up to 1000 nts distant from DS1 in presence (J2+) or absence (J2-) of 4OHT, or upon preincubation of lysed material with recombinant RNaseIII (J2+ RNaseIII) prior to J2 RIP. J2+ values were set to 1. Asterisk, p-value <0.05, two-tailed t-test. Error bar: mean ±SEM, n = 3. ( G ) Autoradiograph detecting J2 immuno-selected (RNA IP) or total (IN) RNA or pBR322 MspI digest (M) upon end-labeling and PAGE separation. AU, arbitrary units. ( H ) as in (D), but preincubated with Leptomycin B (LMB) and stained with J2. Representative images are shown.

Article Snippet: Cells were permeabilized with PBS/0.3% Tween-20 (10 min, RT), washed 1× in PBS and incubated for 20 min at RT with either BSA (Sigma, 0.2 μg/ml final conc., diluted in PBS containing 0.02 mM NaOAc and 0.2 mM Tris), RNaseA (Sigma, 0.2 μg/ml final conc., diluted in PBS containing 0.02 mM NaOAc and 0.2 mM Tris) or RNaseIII (NEB, 2U final conc., diluted in RNase-free H 2 O containing 1× commercial reaction buffer (NEB) prior to fixation.

Techniques: Real-time Polymerase Chain Reaction, Incubation, Recombinant, Quantitative RT-PCR, Immu-Puri, Two Tailed Test, Imaging, Quantitation Assay, Autoradiography, End Labeling, Staining

Journal: Autophagy

Article Title: Impaired autophagy in macrophages promotes inflammatory eye disease

doi: 10.1080/15548627.2016.1207857

Figure Lengend Snippet: Blocking IL1B reverses severe uveitis. (A) The level of IL1B secreted by Atg5 CKO and floxed control peritoneal macrophages in response to LPS treatment can be reduced in both knockouts and controls by treatment with the pancaspase inhibitor, Z-VAD-FMK or CASP1and CASP4 inhibitor Z -YVAD-FMK. Two-way ANOVA (confidence level=95%) with the Bonferroni post-hoc test ****, P < 0.0001. (B) Pharmacological IL1B inhibition in Atg5 CKO mice using the IL1R (interleukin 1 receptor) antagonist Anakinra™. Representative retinal whole mounts from FITC-concavalin A lectin-perfused vehicle (n = 11) and Anakinra™ (n = 12) treated mice and quantification of adhered leukocytes per retina. Scale bar: 100 µm. Student t test **, P < 0.01 (C) Images of concavalin A lectin-perfused retinal wholemounts from control (Atg5fl/fl) (n = 6), Atg5 CKO (n = 15), casp1−/− casp4−/− knockout (n = 7) or Atg5 CKO casp1 casp4 triple-KO mice (n = 8) and quantification of adhered leukocytes in the retinal vasculature and IL1B serum levels from each group. (D). Representative histology from Atg5fl/fl control (n = 10), or Atg5 CKO (n = 14), casp1−/− casp4−/− double knockout (n = 14), and Atg5 CKO casp1−/− casp4−/− triple knockout mice (n = 14) treated with 200 ug RBP31-20 to induce EAU (top; scale bar: 200 µm) with corresponding fundus and OCT imaging, and EAU grading. Arrows indicate clusters of inflammatory cells in the vitreous and the circled areas highlight a region of retinal abnormality observed across imaging modalities. Kruskal-Wallis test with the Dunn post-hoc test *, P < 0.05; **, P < 0.01; ***, P < 0.001. All data are represented as mean ± SEM.

Article Snippet: After 24 h, mice were anesthetized with 86.9 mg/kg ketamine and 13.4 mg/kg xylazine and injected intravenously via the femoral vein with 100 µl of 5 mg/mL FITC-concanavalin A lectin (Vector Labs, FL-1001) to label both blood vessels and leukocytes.

Techniques: Blocking Assay, Inhibition, Knock-Out, Double Knockout, Triple Knockout, Imaging

Journal: Molecular Brain

Article Title: Human primary mixed brain cultures: preparation, differentiation, characterization and application to neuroscience research

doi: 10.1186/s13041-014-0063-0

Figure Lengend Snippet: Confocal imaging of Human mixed brain culture containing neurons, glia and neural progenitor cells. At each time point, cells were fixed using 4% Paraformaldehyde and dual-labeled with FITC-conjugated Neuronal antibody cocktail (Pan-N, 1:1000) and a Cy3-conjugate of either a glial marker (GFAP, 1:5000) or a neuronal progenitor (Nestin, 1:500). Results at DIV 12, and DIV 32 are shown. Antibodies against Pan-N and or Pan-N and GFAP were used with appropriate secondary antibodies (donkey-anti mouse; 1:500 or Goat-anti rabbit, 1:2000). Confocal fluorescence microscopy was performed on an inverted IX81microscope fitted with a FV1000-MPE and laser launch with confocal excitation with three diode lasers (405, 559 and 635 nm) and an Argon laser (458, 488, 514 nm), controlled by Fluoview v3.0 (Olympus). All imaging was performed with a 60x 1.2 NA water immersion objective. The scale bar is in the bottom right panel, and is applicable to all images.

Article Snippet: 5-Hydroxytryptamine (5-HT) (ICC) , 1:1000 , ImmunoStar , 20080 , Cy3-conj. Donkey anti Rabbit , 1:500 , Jackson Immuno Research , 711-165-152.

Techniques: Imaging, Labeling, Marker, Fluorescence, Microscopy

Journal: Molecular Brain

Article Title: Human primary mixed brain cultures: preparation, differentiation, characterization and application to neuroscience research

doi: 10.1186/s13041-014-0063-0

Figure Lengend Snippet: An increase in neuronal population over time. Human fetal brain (HFB) cells were grown as stated. At each time point, cells were fixed using 4% Paraformaldehyde and dual-labeled with FITC-conjugated Neuronal antibody cocktail (Pan-N, 1:1000) and a Cy3-conjugate of either a glial marker (GFAP, 1:5000) or a neuronal progenitor (Nestin, 1:500). Appropriate secondary antibodies were used. The values at the corners of the boxes refer to percent total of the cells gated for selection that were positive for the appropriate fluorescent-conjugated antibody. LL – Unlabeled cells. UL-Cells positive for Cy3-conjugated marker for either GFAP or Nestin. LR – Cells labeled with Pan-N. UR – Cells positive for either Pan-N and GFAP or Pan-N and Nestin. This data suggests that the stem-ness of the culture reduces over time and the neuronal phenotype beings to emerge.

Article Snippet: 5-Hydroxytryptamine (5-HT) (ICC) , 1:1000 , ImmunoStar , 20080 , Cy3-conj. Donkey anti Rabbit , 1:500 , Jackson Immuno Research , 711-165-152.

Techniques: Labeling, Marker, Selection

Journal: Molecular Brain

Article Title: Human primary mixed brain cultures: preparation, differentiation, characterization and application to neuroscience research

doi: 10.1186/s13041-014-0063-0

Figure Lengend Snippet: List of different primary and secondary antibodies used to characterize the culture

Article Snippet: 5-Hydroxytryptamine (5-HT) (ICC) , 1:1000 , ImmunoStar , 20080 , Cy3-conj. Donkey anti Rabbit , 1:500 , Jackson Immuno Research , 711-165-152.

Techniques: Marker

Journal: Infection and Immunity

Article Title: The Carbohydrate Lectin Receptor Dectin-1 Mediates the Immune Response to Exserohilum rostratum

doi: 10.1128/IAI.00903-16

Figure Lengend Snippet: Carbohydrate antigens differ between E. rostratum spores and hyphae. Spores were germinated in complete RPMI for 5 h (mannan and GAG) or 18 h (β-1,3-glucan and chitin). Cells were stained with calcofluor white (chitin stain), fluorescein-conjugated concanavalin A (mannan stain), anti-β-1,3-glucan with secondary anti-mouse IgG conjugated with Alexa Fluor 488 (β-1,3-glucan stain), and fluorescein-conjugated soybean agglutinin (GAG stain) and imaged via confocal microscopy using DIC and fluorescent imaging. All lectins and antibodies shown preferentially labeled the hyphae but not the spores of Exserohilum. White arrows pointing to spore bodies highlight the lack of staining. Bars = 5 μm.

Article Snippet: For mannan staining, cells were incubated with fluorescein-labeled concanavalin A (ConA; catalog number C827; Invitrogen, Carlsbad, CA) for 30 min and then washed with PBS–1% BSA three times prior to confocal imaging.

Techniques: Staining, Confocal Microscopy, Imaging, Labeling

Journal: Autophagy

Article Title: SNAPIN is critical for lysosomal acidification and autophagosome maturation in macrophages

doi: 10.1080/15548627.2016.1261238

Figure Lengend Snippet: Silencing SNAPIN by siRNA resulted in accumulation of late autophagic vacuoles in human macrophages. (A) Immunofluorescence microscopy of human macrophages following transfection with NS or SNAPIN siRNA, and staining with antibodies to SNAPIN (green) or LC3 (red). Arrows on the right identify cells in which SNAPIN was reduced. Scale bar: 20 μm. (B) Transmission electron microscopy of NS siRNA- (left) and SNAPIN siRNA-treated macrophages (middle and right). A normal autophagosome (red arrow, left panel) is identified in the NS siRNA-transfected cells. In the SNAPIN siRNA-treated macrophages, nondigested organelles (red arrows) or partially digested material were identified in autolysosomes (green arrows). Scale bar: 500 nm. Immunoblot analysis of macrophages following transfection with NS or SNAPIN siRNAs before (No) and after starvation for 2 h, (C) after chloroquine treatment for 2 h (D), or left untreated (E). Panel (A) is representative of 5 and panels (B-E) are representative of 3 independent experiments.

Article Snippet: In brief, macrophages were transfected with 200 nM of a nonspecific (NS) siRNA or SNAPIN siRNA (GACUGAGACGGCUAAACCA; Sigma, SASI-Hs01–00205757), SNAPIN -C siRNA (CAGUAGCAGUGUUGAUAGATT; Santa Cruz Biotechnology, sc-45545C), CTSD siRNA (Qiagen, SI00029813) with Lipofectamine 2000 (Life Technologies, 12566014), following the vender's protocol.

Techniques: Immunofluorescence, Microscopy, Transfection, Staining, Transmission Assay, Electron Microscopy, Western Blot

Journal: Autophagy

Article Title: SNAPIN is critical for lysosomal acidification and autophagosome maturation in macrophages

doi: 10.1080/15548627.2016.1261238

Figure Lengend Snippet: Silencing SNAPIN by siRNA reduced CTSD activation in human macrophages. Macrophages were transfected with NS or SNAPIN siRNAs or treated with bafilomycin A1 for 2 h and examined by immunofluorescence microscopy and immunoblot analysis for active CTSD. (A) Macrophages were stained with antibodies to SNAPIN (red) and BODIPY FL-pepstatin A (green), which binds to the active form of CTSD. Scale bar: 20 μm. Results are representative of 3 independent experiments. (B-D) proCTSD and mature CTSD (heavy chain, active form), and as a control proCTSB and mature CTSB were detected in whole cell lysates by immunoblot analysis (panel B) and quantified by densitometric analysis of immunoblots normalized to ACTB (n = 5) (panel D). Changes were presented as percentage of pro- and active forms of the CTSD with the results of the NS siRNA-treated cells set at 100%. The p value was determined by Student t test, comparing SNAPIN- to NS-siRNA reduced macrophages. Error bar: ± 1 SEM.

Article Snippet: In brief, macrophages were transfected with 200 nM of a nonspecific (NS) siRNA or SNAPIN siRNA (GACUGAGACGGCUAAACCA; Sigma, SASI-Hs01–00205757), SNAPIN -C siRNA (CAGUAGCAGUGUUGAUAGATT; Santa Cruz Biotechnology, sc-45545C), CTSD siRNA (Qiagen, SI00029813) with Lipofectamine 2000 (Life Technologies, 12566014), following the vender's protocol.

Techniques: Activation Assay, Transfection, Immunofluorescence, Microscopy, Western Blot, Staining, Control

Journal: Autophagy

Article Title: SNAPIN is critical for lysosomal acidification and autophagosome maturation in macrophages

doi: 10.1080/15548627.2016.1261238

Figure Lengend Snippet: Silencing of SNAPIN in macrophages induced the formation of swollen lysosomes. (A) Macrophages transfected with NS (left) or SNAPIN (middle and right) siRNAs were examined with antibodies to SNAPIN (green) and LAMP1 (red) by immunofluorescence microscopy. Large LAMP1-positive organelles are marked by arrowheads. The cell marked by a square in the middle panel was enlarged in the right panel. Macrophages transfected with (B) NS or (C) SNAPIN siRNAs were examined by immunofluorescence microscopy with antibodies to LAMP1 (magenta staining) and active CTSD (BODIPY FL-pepstatin A, green), with colocalization indicated in white. Arrows identify active CTSD within the LAMP1-positive lysosomes. Scale bar: 13.3 μm. The images were processed by deconvolution with NIS-element imaging software. The results are representative of 3 independent experiments.

Article Snippet: In brief, macrophages were transfected with 200 nM of a nonspecific (NS) siRNA or SNAPIN siRNA (GACUGAGACGGCUAAACCA; Sigma, SASI-Hs01–00205757), SNAPIN -C siRNA (CAGUAGCAGUGUUGAUAGATT; Santa Cruz Biotechnology, sc-45545C), CTSD siRNA (Qiagen, SI00029813) with Lipofectamine 2000 (Life Technologies, 12566014), following the vender's protocol.

Techniques: Transfection, Immunofluorescence, Microscopy, Staining, Imaging, Software

Journal: Autophagy

Article Title: SNAPIN is critical for lysosomal acidification and autophagosome maturation in macrophages

doi: 10.1080/15548627.2016.1261238

Figure Lengend Snippet: Silencing of SNAPIN in human macrophages did not affect fusion of late endosomes with lysosomes. Macrophages were transfected with NS (A) or SNAPIN siRNAs (B) and then examined by immunofluorescence microscopy employing antibodies to LAMP1 (green) and RAB7 (red). Both molecules exhibited perinuclear localization, however, colocalization was not apparent (left panel). The area marked in panel (B), was expanded in panel (C). Following SNAPIN siRNA transfection, a portion of the LAMP1-positive lysosomes incorporated RAB7 (arrow). (D) Human primary macrophages transfected with siRNAs were incubated with Alexa Fluor 488 dextran (green) for 2 h, chased for 16 h and stained with LAMP1. Swollen LAMP1-positive lysosomes containing dextran are marked by arrows. (E) Enlarged area indicated in panel (D). Scale bar: 13.3 μm. The images were processed by deconvolution with NIS-element imaging software. Results in this figure are representative of 3 independent experiments.

Article Snippet: In brief, macrophages were transfected with 200 nM of a nonspecific (NS) siRNA or SNAPIN siRNA (GACUGAGACGGCUAAACCA; Sigma, SASI-Hs01–00205757), SNAPIN -C siRNA (CAGUAGCAGUGUUGAUAGATT; Santa Cruz Biotechnology, sc-45545C), CTSD siRNA (Qiagen, SI00029813) with Lipofectamine 2000 (Life Technologies, 12566014), following the vender's protocol.

Techniques: Transfection, Immunofluorescence, Microscopy, Incubation, Staining, Imaging, Software

Journal: Autophagy

Article Title: SNAPIN is critical for lysosomal acidification and autophagosome maturation in macrophages

doi: 10.1080/15548627.2016.1261238

Figure Lengend Snippet: SNAPIN silencing in macrophages inhibited lysosome acidification. Human macrophages were transfected with NS (A) or SNAPIN siRNAs (B), then incubated for 2 h with dextran labeled with pH-sensitive pHrodo green dye and with dextran labeled with pH-insensitive Alexa Fluor 546 (red) and then chased for 6 h. (C) Bafilomycin A1 (100 nM) was added to cells that were not transfected with siRNA, for 2 h prior to harvesting the cells. Scale bar: 20 μm. (D) Green and red fluorescence mean intensity in each macrophage was measured with Nikon NIS element imaging software. The green:red ratios were calculated and the results summarized in the bar graph. The number of macrophages employed to measure fluorescence intensity was: NS = 160; SNAPIN = 166; bafilomycin A1 = 142. The ratio of green:red fluorescence was calculated from the data in panel (D). *** represents p<0.001, compare with NS siRNA-transfected macrophages following statistical analysis by AVONA. The results in this figure were obtained from 2 independent experiments.

Article Snippet: In brief, macrophages were transfected with 200 nM of a nonspecific (NS) siRNA or SNAPIN siRNA (GACUGAGACGGCUAAACCA; Sigma, SASI-Hs01–00205757), SNAPIN -C siRNA (CAGUAGCAGUGUUGAUAGATT; Santa Cruz Biotechnology, sc-45545C), CTSD siRNA (Qiagen, SI00029813) with Lipofectamine 2000 (Life Technologies, 12566014), following the vender's protocol.

Techniques: Transfection, Incubation, Labeling, Fluorescence, Imaging, Software

Journal: Autophagy

Article Title: SNAPIN is critical for lysosomal acidification and autophagosome maturation in macrophages

doi: 10.1080/15548627.2016.1261238

Figure Lengend Snippet: SNAPIN is important for autophagosome acidification. HEK293 cell lines stably transduced with scrambled (SCR)- (panel A) or SNAPIN shRNA (panel B)-expressing lenti-viruses, were transfected with the ptf-LC3 plasmid, which expresses LC3 tagged with tandem fluorescent RFP and GFP. The red fluorescence is expressed regardless of the pH, while the green fluorescence is quenched at low pH. (C) The average numbers of GFP- and RFP-positive puncta from SCR or SNAPIN shRNA-expressing cells are shown as a bar graph (n = 20; **p < 0.01, compared with the same colored puncta in SCR shRNA HEK293 cells). (D) The ratio of green:red puncta in cells was calculated and presented (n = 20 cells; **p < 0.01), comparing SCR and SNAPIN shRNA-expressing HEK293 cells. (E) Immunoblot analysis of the 2 cell lines. Scale bar: 13.3 μm. Results (panels A-D) were obtained from 4 independent experiments.

Article Snippet: In brief, macrophages were transfected with 200 nM of a nonspecific (NS) siRNA or SNAPIN siRNA (GACUGAGACGGCUAAACCA; Sigma, SASI-Hs01–00205757), SNAPIN -C siRNA (CAGUAGCAGUGUUGAUAGATT; Santa Cruz Biotechnology, sc-45545C), CTSD siRNA (Qiagen, SI00029813) with Lipofectamine 2000 (Life Technologies, 12566014), following the vender's protocol.

Techniques: Stable Transfection, Transduction, shRNA, Expressing, Transfection, Plasmid Preparation, Fluorescence, Western Blot

Journal: Autophagy

Article Title: SNAPIN is critical for lysosomal acidification and autophagosome maturation in macrophages

doi: 10.1080/15548627.2016.1261238

Figure Lengend Snippet: SNAPIN silencing in macrophages did not affect lysosome-autophagosome fusion. (A) Human macrophages were transfected with NS or SNAPIN siRNAs, incubated with 2 μM of rapamycin or 100 nM bafilomycin A1 for 2 h. Cells were fixed with methanol for 20 min and stained with antibodies of LC3 (red) and LAMP1 (green). The highlighted areas in each panel were enlarged and shown in the inserted panels as indicated by the arrows. (B) Total LC3-positive puncta (red bars) and LC3 and LAMP1 colocalized puncta (yellow bars) were counted (numbers indicated in the bars) in 2 independent experiments. (C) The percentage of LC3+ LAMP1+ puncta to total LC3 puncta was calculated and presented. (D) Colocalization between LAMP1 and LC3 was measured by Pearson's correlation coefficient Rr employing NIS-element imaging software. *** represents p<0.001, following analysis by AVONA compare with NS-transfected cells. The presented images were processed by deconvolution.

Article Snippet: In brief, macrophages were transfected with 200 nM of a nonspecific (NS) siRNA or SNAPIN siRNA (GACUGAGACGGCUAAACCA; Sigma, SASI-Hs01–00205757), SNAPIN -C siRNA (CAGUAGCAGUGUUGAUAGATT; Santa Cruz Biotechnology, sc-45545C), CTSD siRNA (Qiagen, SI00029813) with Lipofectamine 2000 (Life Technologies, 12566014), following the vender's protocol.

Techniques: Transfection, Incubation, Staining, Imaging, Software

Journal: Autophagy

Article Title: SNAPIN is critical for lysosomal acidification and autophagosome maturation in macrophages

doi: 10.1080/15548627.2016.1261238

Figure Lengend Snippet: SNAPIN is important in maintaining lysosomal acidic pH. J774A1 macrophage cell line stably expressing a SCR shRNA or a Snapin shRNA was incubated overnight with a pH-sensitive pHrodo Red dextran and a pH-insensitive Alexa Fluor 488 green dextran. Then, dyes were replaced with fresh medium and the dextrans were chased for 8 h, allowing delivery to lysosomes. The H+-pump was inhibited by incubating cells in a Na+ buffer containing 2 μM concanamycin A (CcA). The leak of lysosomal H+ was assessed by the ratio change of red:green fluorescence in J774A1 living cells by photos taken automatically in half-minute intervals. (A) Representative photos of red:green color change in the J774A1 cell lines after CcA was added. (B) The ratio of red:green fluorescence intensity in J774A1 cells was calculated and presented. Data were obtained from 36 cells for the J774A1 SCR cell line and 42 cells for the J774A Snapin shRNA cell line. Results presented above are representative of 2 independent experiments. Every paired time point between SCR and Snapin shRNA cells was statistically different following ANOVA and Tukey's test (p < 0.05 at time 0 and p < 0.01–0.001 at all other time points).

Article Snippet: In brief, macrophages were transfected with 200 nM of a nonspecific (NS) siRNA or SNAPIN siRNA (GACUGAGACGGCUAAACCA; Sigma, SASI-Hs01–00205757), SNAPIN -C siRNA (CAGUAGCAGUGUUGAUAGATT; Santa Cruz Biotechnology, sc-45545C), CTSD siRNA (Qiagen, SI00029813) with Lipofectamine 2000 (Life Technologies, 12566014), following the vender's protocol.

Techniques: Stable Transfection, Expressing, shRNA, Incubation, Fluorescence

Journal: Autophagy

Article Title: SNAPIN is critical for lysosomal acidification and autophagosome maturation in macrophages

doi: 10.1080/15548627.2016.1261238

Figure Lengend Snippet: SNAPIN facilitates lysosomal acidification. J774A1 lines transformed with SCR or Snapin shRNAs were incubated overnight with a pH-sensitive pHrodo Red dextran and a pH-insensitive Alexa Fluor 488 green dextran. Afterwards, dextrans in J774A1 cells were chased for 8 h. J774A1 live-cell fluorescence imaging was taken automatically in half-minute intervals under a fluorescence microscope. (A) J774A1 cells were incubated with K+ buffer containing 450 μM carbonyl cyanide m-chlorophenyl hydrazone (CCCP), a protonophore capable of dissipating the lysosomal transmembrane ΔpH. The ratio of red:green fluorescence, which is inversely related to pH in lysosomes, was measured. Re-acidification of lysosomes started promptly after removing CCCP, replacing with Na+ buffer alone. The ratio of red:green was calculated and presented. (B) The rate of change of the red:green fluorescence per minute was calculated (ratiot2-ratiot1)/0.5 min) and the results plotted over time. (C) The rate of re-acidification in the first 2 min after CCCP removal was calculated and was summarized. *** represents p < 0.001 following ANOVA and Tukey's test. (D) The image demonstrates SNAPIN expression in the J774A1 cell lines by immunoblot analysis.

Article Snippet: In brief, macrophages were transfected with 200 nM of a nonspecific (NS) siRNA or SNAPIN siRNA (GACUGAGACGGCUAAACCA; Sigma, SASI-Hs01–00205757), SNAPIN -C siRNA (CAGUAGCAGUGUUGAUAGATT; Santa Cruz Biotechnology, sc-45545C), CTSD siRNA (Qiagen, SI00029813) with Lipofectamine 2000 (Life Technologies, 12566014), following the vender's protocol.

Techniques: Transformation Assay, Incubation, Fluorescence, Imaging, Microscopy, Expressing, Western Blot

Journal: Frontiers in Molecular Neuroscience

Article Title: N -Glycosylation Regulates the Trafficking and Surface Mobility of GluN3A-Containing NMDA Receptors

doi: 10.3389/fnmol.2018.00188

Figure Lengend Snippet: N -glycosylation regulates the mobility of GluN3A-containing NMDARs at the cell surface of hippocampal neurons. (A,C) Representative trajectories of quantum dot (QD)-labeled GFP-GluN3A (GluN3A) subunits in hippocampal neurons treated with 200 μg/ml DMM (A) or with the lectin AAL (20 μg/ml) (C) . (B,D) Comparison of the cumulative distribution of the diffusion coefficients measured for QD-labeled GluN3A subunits in hippocampal neurons treated for two days with 200 μg/ml DMM, 100 μg/ml swainsonine ( n = 208–314 trajectories per condition; B ), or incubated after QD labeling with 20 μg/ml of AAL, conA, or WGA ( n = 84–131 trajectories per condition; D ). The diffusion coefficients in fixed cells (shown in D ) were obtained from an independent experiment ( n = 49 trajectories). Pairwise comparisons of the distributions show the following statistically significant differences: DMM vs. control ( p < 0.003), AAL, conA, WGA vs. control ( p < 10 −23 ), AAL, conA, WGA vs. fixed ( p < 10 -13 ). The largest p-value obtained in Kolmogorov-Smirnov test and Mann-Whitney-Wilcoxon test is stated.

Article Snippet: Unconjugated conA, WGA and AAL (Vector Laboratories), which were used in QD tracking experiments, were dissolved in accordance with the manufacturer’s recommendations; the stock solutions were diluted to a final concentration of 20 μg/ml in extracellular recording solution prior to imaging.

Techniques: Labeling, Diffusion-based Assay, Incubation, MANN-WHITNEY

Journal: Methods in Molecular Biology

Article Title: Retinal Degeneration

doi: 10.1007/978-1-4939-8669-9

Figure Lengend Snippet: Fig. 2 Human (A, B) and mouse (C) retina whole mounts. (A) Fluorescence image of rhodopsin (Rho 1D4) label- ing of rods surrounding unlabeled cones in a human retinal whole mount. Cones are unlabeled and appear as holes surrounded by labeled rods. (B) Fluorescence image of ABCA4 (Rim 3F4) labeling of cones surrounded by labeled rods [10]. (C) Fluorescence image of cone opsin (blue and red/green opsin) labeling of cone photo- receptors in a mouse retinal whole mount

Article Snippet: Cryosections Table 2 Immunochemical markers for the identification of proteins and cellular compartments of mouse rod and cone photoreceptors Photoreceptor cell layer Antigen Antibody Source References Rod outer segment Rhodopsin Mab: Rho 1D4; Rho 4D2 Millipore [12, 13] Rod outer segment CNGA1 Mab: PMc1D1 Millipore [16, 24] Rod outer segment CNGB1 and GARP proteins Mab: Garp 4B1 Millipore [24, 25] Cone outer segments (M/L) Cone opsin Pab: Cone M/L Millipore [16] Rod and cone outer segments RetGC-1 Mab: GC1-8A5 Millipore [16] Rod and cone outer segments ABCA4 Mab: Rim 3F4; Rim 5B4 Millipore; Santa Cruz [12, 16] Rod and cone outer segments Peripherin-2 (peripherin/rds) Mab: Per 5H2 Millipore [16] Cilium/axoneme Retinitis pigmentosa 1 protein (RP1) Chicken anti-RP1 clone EAP15A Liu et al., 2004 [26] Photoreceptor inner segment Sodium/potassium ATPase α3 Mouse anti-Na/K ATPase clone XVIF9-G10 Abcam [10] Endoplasmic reticulum Calnexin Pab MilliporeSigma Endoplasmic reticulum KDEL Mab:10C3 Enzo, Stressgene [16] CNGA1 cyclic nucleotide-gated channel (Subunit A1), RetGC1 guanylate cyclase −1, Mab monoclonal antibody, Pab polyclonal antibody, GARP glutamic acid-rich proteins Retinal Cell Specific Markers for Immunofluorescence Microscopy 300 8.

Techniques: Fluorescence, Labeling

Journal: Methods in Molecular Biology

Article Title: Retinal Degeneration

doi: 10.1007/978-1-4939-8669-9

Figure Lengend Snippet: Fig. 5 Immunofluorescence of mouse retinal cryosections labeled with antibodies to various proteins localized in photoreceptors. (A) Labeling of wild-type (WT) mouse retina section with Rim 5B4 antibody to ABCA4, the ATP-binding cassette transporter found primarily in photoreceptor cells. (B) Labeling of ABCA4 knockout (KO) mouse section with Rim 5B4 antibody as a control. Labeling of outer segments is absent confirming the speci- ficity of the Rim 5B4 to ABCA4. Some autofluorescence is observed in the RPE cells arising from bisretinoid compounds such as A2E which accumulate in RPE cells of ABCA4 KO mice. (C) Labeling of WT mouse section with RetGC1 8A5 against guanylate cyclase 1 present in rod and cone outer segments. (D) Labeling of WT mouse section with calnexin antibody showing the location of the endoplasmic reticulum (ER) in photorecep- tors just above the ONL. Arrows in (A) and (C) show cone OS labeling. Labeling in (A–C) just below the ONL are blood cells with surface Ig labeled with the secondary antibody against mouse Ig. Bar = 20 μm

Article Snippet: Cryosections Table 2 Immunochemical markers for the identification of proteins and cellular compartments of mouse rod and cone photoreceptors Photoreceptor cell layer Antigen Antibody Source References Rod outer segment Rhodopsin Mab: Rho 1D4; Rho 4D2 Millipore [12, 13] Rod outer segment CNGA1 Mab: PMc1D1 Millipore [16, 24] Rod outer segment CNGB1 and GARP proteins Mab: Garp 4B1 Millipore [24, 25] Cone outer segments (M/L) Cone opsin Pab: Cone M/L Millipore [16] Rod and cone outer segments RetGC-1 Mab: GC1-8A5 Millipore [16] Rod and cone outer segments ABCA4 Mab: Rim 3F4; Rim 5B4 Millipore; Santa Cruz [12, 16] Rod and cone outer segments Peripherin-2 (peripherin/rds) Mab: Per 5H2 Millipore [16] Cilium/axoneme Retinitis pigmentosa 1 protein (RP1) Chicken anti-RP1 clone EAP15A Liu et al., 2004 [26] Photoreceptor inner segment Sodium/potassium ATPase α3 Mouse anti-Na/K ATPase clone XVIF9-G10 Abcam [10] Endoplasmic reticulum Calnexin Pab MilliporeSigma Endoplasmic reticulum KDEL Mab:10C3 Enzo, Stressgene [16] CNGA1 cyclic nucleotide-gated channel (Subunit A1), RetGC1 guanylate cyclase −1, Mab monoclonal antibody, Pab polyclonal antibody, GARP glutamic acid-rich proteins Retinal Cell Specific Markers for Immunofluorescence Microscopy 300 8.

Techniques: Immunofluorescence, Labeling, Binding Assay, Knock-Out, Control

Journal: Methods in Molecular Biology

Article Title: Retinal Degeneration

doi: 10.1007/978-1-4939-8669-9

Figure Lengend Snippet: Fig. 2 TPM imaging of the retina and RPE in mouse models of retinal pathologies in vivo. (a) TPM imaging based on endogenous fluorophores. Left, brightly fluorescent granules of retinal condensation products are visible in the retina and RPE of 3.5-month-old Mertk−/−Abca4−/−Rdh8−/− mice, with defective phagocytosis and retinoid cycle pathways [17]. Right, retinosomes outline the contours of RPE cells in 1-year-old albino Rpe65−/− mice, a mouse model of Leber congenital amaurosis. Both images were obtained with 800 nm laser light. (b) Visualization of retinal capillaries in vivo, in 1-year-old C57BL/6J-TyrC-2J mice, after tail vain injection with a fluorescein—BSA solution; this approach can be used to evaluate leakage from retinal capillaries in mice with induced diabetes. Both left and right images are from different sections of the retina in the same eye. Images were obtained with 820 nm laser light

Article Snippet: Cryosections Table 2 Immunochemical markers for the identification of proteins and cellular compartments of mouse rod and cone photoreceptors Photoreceptor cell layer Antigen Antibody Source References Rod outer segment Rhodopsin Mab: Rho 1D4; Rho 4D2 Millipore [12, 13] Rod outer segment CNGA1 Mab: PMc1D1 Millipore [16, 24] Rod outer segment CNGB1 and GARP proteins Mab: Garp 4B1 Millipore [24, 25] Cone outer segments (M/L) Cone opsin Pab: Cone M/L Millipore [16] Rod and cone outer segments RetGC-1 Mab: GC1-8A5 Millipore [16] Rod and cone outer segments ABCA4 Mab: Rim 3F4; Rim 5B4 Millipore; Santa Cruz [12, 16] Rod and cone outer segments Peripherin-2 (peripherin/rds) Mab: Per 5H2 Millipore [16] Cilium/axoneme Retinitis pigmentosa 1 protein (RP1) Chicken anti-RP1 clone EAP15A Liu et al., 2004 [26] Photoreceptor inner segment Sodium/potassium ATPase α3 Mouse anti-Na/K ATPase clone XVIF9-G10 Abcam [10] Endoplasmic reticulum Calnexin Pab MilliporeSigma Endoplasmic reticulum KDEL Mab:10C3 Enzo, Stressgene [16] CNGA1 cyclic nucleotide-gated channel (Subunit A1), RetGC1 guanylate cyclase −1, Mab monoclonal antibody, Pab polyclonal antibody, GARP glutamic acid-rich proteins Retinal Cell Specific Markers for Immunofluorescence Microscopy 300 8.

Techniques: Imaging, In Vivo, Injection

Journal: Methods in Molecular Biology

Article Title: Retinal Degeneration

doi: 10.1007/978-1-4939-8669-9

Figure Lengend Snippet: Fig. 3 Ex vivo TPM imaging of the retina after induction of photoreceptor damage in intact eyes of 1-month-old albino Abca4−/−Rdh8−/− mice. Reconstructed volumes obtained from a series of z-stack images obtained with 730 nm excitation light are shown in both (a) and (b). The RPE is at z = 0, and the section outlined in blue is 10 μm under the RPE (toward the ganglion cell layer). Red arrows indicate enlarged photoreceptors [12], yel- low arrows point to RPE nuclei. (a) Retinal 3D volume from a mouse treated with DMSO before induction of photoreceptor damage. (b) Retinal 3D volume from a mouse treated with an investigative pharmaceutical before induction of photoreceptor damage. Large quantities of enlarged photoreceptors present in mice treated with DMSO indicate severe damage to the retina

Article Snippet: Cryosections Table 2 Immunochemical markers for the identification of proteins and cellular compartments of mouse rod and cone photoreceptors Photoreceptor cell layer Antigen Antibody Source References Rod outer segment Rhodopsin Mab: Rho 1D4; Rho 4D2 Millipore [12, 13] Rod outer segment CNGA1 Mab: PMc1D1 Millipore [16, 24] Rod outer segment CNGB1 and GARP proteins Mab: Garp 4B1 Millipore [24, 25] Cone outer segments (M/L) Cone opsin Pab: Cone M/L Millipore [16] Rod and cone outer segments RetGC-1 Mab: GC1-8A5 Millipore [16] Rod and cone outer segments ABCA4 Mab: Rim 3F4; Rim 5B4 Millipore; Santa Cruz [12, 16] Rod and cone outer segments Peripherin-2 (peripherin/rds) Mab: Per 5H2 Millipore [16] Cilium/axoneme Retinitis pigmentosa 1 protein (RP1) Chicken anti-RP1 clone EAP15A Liu et al., 2004 [26] Photoreceptor inner segment Sodium/potassium ATPase α3 Mouse anti-Na/K ATPase clone XVIF9-G10 Abcam [10] Endoplasmic reticulum Calnexin Pab MilliporeSigma Endoplasmic reticulum KDEL Mab:10C3 Enzo, Stressgene [16] CNGA1 cyclic nucleotide-gated channel (Subunit A1), RetGC1 guanylate cyclase −1, Mab monoclonal antibody, Pab polyclonal antibody, GARP glutamic acid-rich proteins Retinal Cell Specific Markers for Immunofluorescence Microscopy 300 8.

Techniques: Ex Vivo, Imaging