Journal: Cellular and Molecular Life Sciences

Article Title: Cell-specific and lamin-dependent targeting of novel transmembrane proteins in the nuclear envelope

doi: 10.1007/s00018-010-0257-2

Figure Lengend Snippet: INM versus ONM targeting. NETs were imaged using high-resolution systems for localization in relation to the two sides of the NPC using Nup153 from the nuclear basket and Nup358 from the cytoplasmic filaments. ( a ) Schematic of expected patterns indicating INM or ONM localization. If a protein is in the INM, the NET and Nup153 signals should occur in the same plane, and the NET signal should appear internal to the Nup358 signal. If the protein is in the ONM, the Nup153 signal should be internal to the NET signal and the Nup358 signal should occur in the same plane as the NET signal. ( b ) Images using structured illumination show both characterized (LAP2ß) and many novel NETs in the same plane of the inner nuclear membrane with Nup153 and internal to Nup358. Only NET23 and controls Sec61ß and calreticulin yielded the pattern expected for ONM residence. ( c ) High-resolution deconvolved Deltavision images also can distinguish inner from outer nuclear membranes with Nup153 shown in red and Nup358 shown in green . ( d ) Many additional novel NETs appeared in the INM using again LAP2ß as a control and NET55 that had been separately tested with the OMX system. In contrast NETs 4, 24, and 31 together with the calreticulin control yielded ONM targeting. Scale bars for b - d 5 μm. ( e ) Immunogold-EM confirms the validity of OMX and Deltavision results as 5-nm gold particles recognizing GFP antibodies for expressed NET51 and NET55 proteins appeared in the INM, similarly to controls. C and N denote cytoplasmic and nucleoplasmic sides where NPCs are inserted in the membrane. Bars 100 nm

Article Snippet: Antibodies were: HA tag (mAb HA.11, Covance), lamin A and B1 (3262 and 3931; [ ]), Nup153 (Covance), Nup358 (raised against recombinant human protein aa 2595–2881, kind gift of F. Melchior), Calreticulin (2891S, Cell Signaling), Calnexin (SPA-860, Stressgen), or NETs: rabbit anti-peptide polyclonal generated for this study by Millipore NET4 (11780), NET23 (11815), NET29 (11796), NET30 (11827), NET31 (11830), NET33 (11835), NET34 (11231), NET39 (11668), NET50 (11850), NET51 (11856), NET55 (11862), NET59 (11866), and SUN2 (11905).

Techniques: Membrane, Control

Journal: Cellular and Molecular Life Sciences

Article Title: Cell-specific and lamin-dependent targeting of novel transmembrane proteins in the nuclear envelope

doi: 10.1007/s00018-010-0257-2

Figure Lengend Snippet: NE localization and detergent resistance of novel NETs. ( a ) NETs fused to mRFP at their carboxy-termini (except NET25 fused at its amino-terminus to an HA epitope tag) were transiently expressed in HT1080 cells also expressing lamin A-GFP. Cells were pre-extracted with Triton X-100 prior to fixation to remove membranes and soluble proteins, which typically also distorts morphology. NETs alone ( left ) and the merge between the NET ( red ) and lamin A ( green ) are shown. In some panels adjacent untransfected cells are shown, confirming that NE signal is not due to bleedthrough from the lamin channel. Like the emerin control ( top ), all NETs shown resisted detergent pre-extraction. Such resistance typically indicates association with the lamin polymer. Scale bars 7.5 μm. ( b ) Controls showing that the ER was fully removed by the detergent pre-extraction. Left calreticulin fused to GFP (but colored red ) was overexpressed in cells either directly fixed or pre-extracted with detergent. No colocalization with lamins ( green ) was observed, and no calreticulin remained after extraction, though lamins did remain. Both direct fixed and pre-extracted images were exposed for 2 s. Right untransfected cells were similarly treated, then stained with the ER lipid dye DiOC6. Endogenous ER staining did not exhibit notable accumulation at the NE and was completely removed by the pre-extraction. Both images were exposed for 500 ms. Scale bars 20 μm

Article Snippet: Antibodies were: HA tag (mAb HA.11, Covance), lamin A and B1 (3262 and 3931; [ ]), Nup153 (Covance), Nup358 (raised against recombinant human protein aa 2595–2881, kind gift of F. Melchior), Calreticulin (2891S, Cell Signaling), Calnexin (SPA-860, Stressgen), or NETs: rabbit anti-peptide polyclonal generated for this study by Millipore NET4 (11780), NET23 (11815), NET29 (11796), NET30 (11827), NET31 (11830), NET33 (11835), NET34 (11231), NET39 (11668), NET50 (11850), NET51 (11856), NET55 (11862), NET59 (11866), and SUN2 (11905).

Techniques: Expressing, Control, Extraction, Polymer, Staining

Journal: Cellular and Molecular Life Sciences

Article Title: Cell-specific and lamin-dependent targeting of novel transmembrane proteins in the nuclear envelope

doi: 10.1007/s00018-010-0257-2

Figure Lengend Snippet: NETs that did not resist detergent pre-extraction re-tested for NE accumulation in directly fixed cells. HA-tagged NET62 and mRFP-tagged NETs 20, 38, and 46 were transiently transfected into HT1080 cells expressing lamin A-GFP. Tagged emerin and calreticulin were separately overexpressed as controls. The NET alone ( black and white ) and the merge between the NET ( red ) and lamin A ( green ) are shown. The new NETs yielded clear nuclear rims against the high cytoplasmic accumulation, colocalizing with lamins ( yellow merge) similarly to emerin ( top left ). This staining pattern clearly differs from the ER localization of calreticulin ( top right ). Scale bars 20 μm

Article Snippet: Antibodies were: HA tag (mAb HA.11, Covance), lamin A and B1 (3262 and 3931; [ ]), Nup153 (Covance), Nup358 (raised against recombinant human protein aa 2595–2881, kind gift of F. Melchior), Calreticulin (2891S, Cell Signaling), Calnexin (SPA-860, Stressgen), or NETs: rabbit anti-peptide polyclonal generated for this study by Millipore NET4 (11780), NET23 (11815), NET29 (11796), NET30 (11827), NET31 (11830), NET33 (11835), NET34 (11231), NET39 (11668), NET50 (11850), NET51 (11856), NET55 (11862), NET59 (11866), and SUN2 (11905).

Techniques: Extraction, Transfection, Expressing, Staining

Journal: Cellular and Molecular Life Sciences

Article Title: Cell-specific and lamin-dependent targeting of novel transmembrane proteins in the nuclear envelope

doi: 10.1007/s00018-010-0257-2

Figure Lengend Snippet: Some NETs only accumulate at the NE in certain cell types. ( a ) NETs that failed to target to the NE in HT1080 (human fibrosarcoma) cells were re-tested in other cell lines derived from different tissues: HepG2 (human liver tumor), 293T (human embryonic kidney), C2C12 (mouse skeletal muscle), and 3T3-L1 (mouse pre-adipocyte). HA-tagged NET32 and mRFP-tagged NETs 11, 13, 45, 59, and emerin were transiently transfected into the different cells. To ensure that rim accumulation was not due to bleedthrough or cross-reactivity with NE markers, cells were not co-transfected with or stained for other NE proteins. Arrows mark cells in which different NETs yielded discernible rim staining by having a strong distinct rim as opposed to one that could be accounted by ER condensed against the nucleus. Scale bars 20 μm. ( b ) To further validate NE targeting, the relative pixel intensities in ER and NE were quantified compared to ER controls. Pixel intensity was measured at a point in the nuclear rim (based on DAPI staining) and at a point approximately 2 μm distant into the ER, and the NE/ER ratio was calculated. Eight such measurements were taken from each NET from 5 different cells, and Tukey’s boxplots for the 40 ratios for each NET in each cell line are shown with the median ( central line ), two quartiles above and below ( box ) and third quartile ( error bars ) shown. We compared each sample to each control (DiOC6 or calreticulin expressed in HT1080 cells) with the null hypothesis that ‘control to sample differences are by random chance.’ After analysis we reject the null hypothesis for each sample at P < 0.001 by Mann-Whitney (Wilcoxon) U test (Table ). The ER dye DiOC6 and calreticulin-GFP both were very similar in intensity between the ER and NE, yielding a ratio of ~1. Though NET ratios tended to be in the 1.3–1.5 range, they were highly statistically significant, even for NET11 in C2C12 cells where a strong rim was not visually evident

Article Snippet: Antibodies were: HA tag (mAb HA.11, Covance), lamin A and B1 (3262 and 3931; [ ]), Nup153 (Covance), Nup358 (raised against recombinant human protein aa 2595–2881, kind gift of F. Melchior), Calreticulin (2891S, Cell Signaling), Calnexin (SPA-860, Stressgen), or NETs: rabbit anti-peptide polyclonal generated for this study by Millipore NET4 (11780), NET23 (11815), NET29 (11796), NET30 (11827), NET31 (11830), NET33 (11835), NET34 (11231), NET39 (11668), NET50 (11850), NET51 (11856), NET55 (11862), NET59 (11866), and SUN2 (11905).

Techniques: Derivative Assay, Transfection, Staining, Control, MANN-WHITNEY

Journal: Cellular and Molecular Life Sciences

Article Title: Cell-specific and lamin-dependent targeting of novel transmembrane proteins in the nuclear envelope

doi: 10.1007/s00018-010-0257-2

Figure Lengend Snippet: Mann-Whitney U-test (Wilcoxon) P -values for comparison of each protein to the DiOC6 control or the calreticulin control for Tukey’s boxplot shown in Fig. 6 b (all are significant)

Article Snippet: Antibodies were: HA tag (mAb HA.11, Covance), lamin A and B1 (3262 and 3931; [ ]), Nup153 (Covance), Nup358 (raised against recombinant human protein aa 2595–2881, kind gift of F. Melchior), Calreticulin (2891S, Cell Signaling), Calnexin (SPA-860, Stressgen), or NETs: rabbit anti-peptide polyclonal generated for this study by Millipore NET4 (11780), NET23 (11815), NET29 (11796), NET30 (11827), NET31 (11830), NET33 (11835), NET34 (11231), NET39 (11668), NET50 (11850), NET51 (11856), NET55 (11862), NET59 (11866), and SUN2 (11905).

Techniques: Mann-Whitney U-Test, Comparison, Control, Significance Assay

Journal: Cureus

Article Title: Study of Clinical Outcome and Healthcare Modalities of COVID-19 Patients Treated With Remdesivir at a Tertiary Care Teaching Hospital

doi: 10.7759/cureus.21535

Figure Lengend Snippet: Gender-wise patient characteristics.

Article Snippet: Comorbidity analysis revealed that remdesivir is likely to affect the comorbidity-driven outcome in COVID-19 patients.

Techniques:

Journal: Cureus

Article Title: Study of Clinical Outcome and Healthcare Modalities of COVID-19 Patients Treated With Remdesivir at a Tertiary Care Teaching Hospital

doi: 10.7759/cureus.21535

Figure Lengend Snippet: Age-wise patient characteristics.

Article Snippet: Comorbidity analysis revealed that remdesivir is likely to affect the comorbidity-driven outcome in COVID-19 patients.

Techniques:

Journal: Cureus

Article Title: Study of Clinical Outcome and Healthcare Modalities of COVID-19 Patients Treated With Remdesivir at a Tertiary Care Teaching Hospital

doi: 10.7759/cureus.21535

Figure Lengend Snippet: Comorbidity-wise patient characteristics.

Article Snippet: Comorbidity analysis revealed that remdesivir is likely to affect the comorbidity-driven outcome in COVID-19 patients.

Techniques:

Journal: Cureus

Article Title: Study of Clinical Outcome and Healthcare Modalities of COVID-19 Patients Treated With Remdesivir at a Tertiary Care Teaching Hospital

doi: 10.7759/cureus.21535

Figure Lengend Snippet: Comorbidity association with health care modalities. *u value denotes value of Mann-Whitney U test, **p <0.05 is kept at a statistically significant level, †duration is in days and mean is with 95% CI, OR: odds ratio.

Article Snippet: Comorbidity analysis revealed that remdesivir is likely to affect the comorbidity-driven outcome in COVID-19 patients.

Techniques: Mann-Whitney U-Test, Significance Assay

Journal: Protein Science : A Publication of the Protein Society

Article Title: Linking structural features of protein complexes and biological function

doi: 10.1002/pro.2736

Figure Lengend Snippet: Discriminatory PPI Features Among Functional Groups (Using Kruskal-Wallis Rank Sum Test)

Article Snippet: The discriminatory PPI features among functional groups were thus tested for statistical significance with P < 0.05 (for the Kruskal-Wallis rank sum test) in RStudio.

Techniques: Functional Assay, Solvent, Binding Assay

Journal: eNeuro

Article Title: Heterozygous Dab1 Null Mutation Disrupts Neocortical and Hippocampal Development

doi: 10.1523/ENEURO.0433-22.2023

Figure Lengend Snippet: Half reduction of the Dab1 gene dosage does not cause a significant positional change of neocortical layer 2–6 neurons. A , TBR1-positive, RORB-positive, or BRN2-positive cells in the somatosensory (Rostral), parietal (Middle), and visual (Caudal) cortices of P7 wild-type (+/+) and heterozygous yotari mice (+/ yot ). Representative images of coronal neocortical sections in the middle brain region are shown. B , Schematic illustration showing a region of the cerebral neocortex. Distances from the ventricular surface (VS) to the target cells (D1) and cortical wall thickness (D2) were measured, and the relative positions of cells were calculated by dividing D1 by D2 and multiplying by 10. C , D , Combination of dot, violin, and scatter plots showing the relative positions of layer marker-positive cells ( C ) and distances of layer marker-positive cells from the ventricular surface ( D ) within three brain regions along the rostro-caudal axis. The black dot and bar in the leftmost dot plot indicate the mean and SD, respectively, and each gray dot indicates the mean of one brain. Each black circle in the scatter plot is a raw datapoint. Measurements were performed on three brains from different litters ( n = 3) per group. The total number of analyzed cells (pooled from three mice per group) are shown below each graph. After calculation of mean values for each brain, most group differences were analyzed by independent samples Student’s t test; however, some experiments were analyzed by Mann–Whitney U test [ C , BRN2 (Middle)] or unpaired Welch’s t test [ C , TBR1 (Rostral); D , TBR1 (Rostral)]. The p -values are labeled on the graph. ns (not significant) p > 0.05. Scale bar: 100 μm.

Article Snippet: For layer marker staining, sections were washed three times (5 min/wash) in PBS containing 0.1% Tween 20 (PBStw) at room temperature (RT), autoclaved at 105°C for 5 min in 0.01 m citrate buffer (pH 6.0) for antigen retrieval, then incubated sequentially with 5% bovine serum albumin (BSA)/PBStw at RT for 5 min and primary antibodies against BRN2 (Santa Cruz Biotechnology, sc-6029; RRID: AB_2167385 ), RORB (Perseus Proteomics, PP-N7927-00; RRID: AB_1964364 ), or TBR1 (abcam, ab31940; RRID: AB_2200219 ) in 5% BSA/PBStw at RT for 1 h. For visualization of immunolabeling, sections were washed three times in PBStw and incubated first with Alexa 488-labeled secondary antibody against mouse IgG (Thermo Fisher Scientific, A-21202; RRID: AB_141607 ) or rabbit mouse IgG (Thermo Fisher Scientific, A-11008; RRID: AB_143165 ) with 4′,6-diamidino-2-phenylindole (DAPI; Life Technologies) at RT for 1 h. After three washes in PBS, sections were mounted with PermaFluor Aqueous Mounting Medium (Thermo Fisher Scientific).

Techniques: Marker, MANN-WHITNEY, Labeling

Journal: eNeuro

Article Title: Heterozygous Dab1 Null Mutation Disrupts Neocortical and Hippocampal Development

doi: 10.1523/ENEURO.0433-22.2023

Figure Lengend Snippet: There were no significant positional changes in late-born, BRN2-positive, superficial neocortical neurons in yotari heterozygous mice. A , Coronal sections of P9 parietal neocortex from wild-type (+/+) and heterozygous yotari (+/ yot ) mice injected with BrdU at E16.5. Sections were co-stained with BrdU (green), the layer 2 marker BRN2 (magenta), and the nuclear stain DAPI (blue). B , C , Combination of dot, violin, and scatter plots showing relative positions of BrdU/BRN2 double-positive cells between the ventricular surface and pial surface ( B ) and distances of BrdU/BRN2 double-positive cells from the ventricular surface ( C ) in somatosensory (Rostral), parietal (Middle), and visual (Caudal) cortices of wild-type (W) and heterozygous yotari (He) mice. The black dot and bar in the leftmost dot plot indicate the mean and SD, respectively, each gray dot indicates the mean from one brain, and each black circle in the scatter plot is a raw datapoint. Data were obtained from three brains per group, each from different litters ( n = 3). Total analyzed cell numbers pooled from three mice per group are shown below each graph. After calculation of the mean values for each brain, group differences were analyzed by independent samples Student’s t test. The p -values are labeled on the graph. ns (not significant). Scale bar: 100 μm.

Article Snippet: For layer marker staining, sections were washed three times (5 min/wash) in PBS containing 0.1% Tween 20 (PBStw) at room temperature (RT), autoclaved at 105°C for 5 min in 0.01 m citrate buffer (pH 6.0) for antigen retrieval, then incubated sequentially with 5% bovine serum albumin (BSA)/PBStw at RT for 5 min and primary antibodies against BRN2 (Santa Cruz Biotechnology, sc-6029; RRID: AB_2167385 ), RORB (Perseus Proteomics, PP-N7927-00; RRID: AB_1964364 ), or TBR1 (abcam, ab31940; RRID: AB_2200219 ) in 5% BSA/PBStw at RT for 1 h. For visualization of immunolabeling, sections were washed three times in PBStw and incubated first with Alexa 488-labeled secondary antibody against mouse IgG (Thermo Fisher Scientific, A-21202; RRID: AB_141607 ) or rabbit mouse IgG (Thermo Fisher Scientific, A-11008; RRID: AB_143165 ) with 4′,6-diamidino-2-phenylindole (DAPI; Life Technologies) at RT for 1 h. After three washes in PBS, sections were mounted with PermaFluor Aqueous Mounting Medium (Thermo Fisher Scientific).

Techniques: Injection, Staining, Marker, Labeling

Journal: eNeuro

Article Title: Heterozygous Dab1 Null Mutation Disrupts Neocortical and Hippocampal Development

doi: 10.1523/ENEURO.0433-22.2023

Figure Lengend Snippet: Summary of statistical analyses

Article Snippet: For layer marker staining, sections were washed three times (5 min/wash) in PBS containing 0.1% Tween 20 (PBStw) at room temperature (RT), autoclaved at 105°C for 5 min in 0.01 m citrate buffer (pH 6.0) for antigen retrieval, then incubated sequentially with 5% bovine serum albumin (BSA)/PBStw at RT for 5 min and primary antibodies against BRN2 (Santa Cruz Biotechnology, sc-6029; RRID: AB_2167385 ), RORB (Perseus Proteomics, PP-N7927-00; RRID: AB_1964364 ), or TBR1 (abcam, ab31940; RRID: AB_2200219 ) in 5% BSA/PBStw at RT for 1 h. For visualization of immunolabeling, sections were washed three times in PBStw and incubated first with Alexa 488-labeled secondary antibody against mouse IgG (Thermo Fisher Scientific, A-21202; RRID: AB_141607 ) or rabbit mouse IgG (Thermo Fisher Scientific, A-11008; RRID: AB_143165 ) with 4′,6-diamidino-2-phenylindole (DAPI; Life Technologies) at RT for 1 h. After three washes in PBS, sections were mounted with PermaFluor Aqueous Mounting Medium (Thermo Fisher Scientific).

Techniques: Comparison, Significance Assay, Mann-Whitney U-Test