Journal: Nature communications

Article Title: Sleep disruption impairs hematopoietic stem cell transplantation in mice

doi: 10.1038/ncomms9516

Figure Lengend Snippet: HSCs from sleep-deprived mice have reduced homing capacity in vivo and in vitro (a) HSCs (2000 FACS-purified cells) derived from GFP-expressing, sleep-deprived mice were transplanted into lethally irradiated congenic mice that did not express GFP (control mice were allowed to sleep for the same duration). The donor HSCs were visualized 12 hours later in the recipient’s bone, under a fluorescence microscope. GFP expression was validated using anti-GFP staining and a representative image is shown in the insert. Scale 10µm (n=6–11 mice per group; mean±s.e.m). ( b ) The number of GFP-labeled cells in the recipient’s bone marrow was determined. Results are presented as the percentage of homing, assuming that two tibias and two femurs represent 20% of total mouse bone marrow (student’s t -test ; p=0.021; t=2.56; df=15 ). ( c ) Migration towards SDF-1α was determined in vitro using a transwell migration assay. KLS cells were placed in the upper chamber and the chemoattractant (SDF-1α, 50, 100, or 200ng/mL) was immersed in the medium of the lower chamber. Migration across the membrane in response to the chemoattractant was determined using flow cytometry, after four hours of incubation, and compared to baseline migration in the absence of the chemoattractant. Results are presented as a migration index (50ng/mL SDF-1α: 9.6 ± 1.7% in the sleep group compared to 2.06 ± 0.4% in the sleep-deprived group; 100ng/mL SDF-1α: 14.5 ± 3.4% in the sleep compared to 3.6 ± 1.4% in the sleep-deprived mice; Repeated measures ANOVA- sleep: F (2,15)=10.18, p

Article Snippet: Flow cytometry Mice were euthanized, using CO2, and bone marrow was harvested into PBS containing 2% fetal calf serum (FCS).

Techniques: Mouse Assay, In Vivo, In Vitro, FACS, Purification, Derivative Assay, Expressing, Irradiation, Fluorescence, Microscopy, Staining, Labeling, Migration, Transwell Migration Assay, Flow Cytometry, Incubation

Journal: Nature communications

Article Title: Sleep disruption impairs hematopoietic stem cell transplantation in mice

doi: 10.1038/ncomms9516

Figure Lengend Snippet: HSCs isolated from sleep-deprived mice have reduced mid-term and long-term reconstitution potential in lethally irradiated hosts ( a ) Mice were sleep-deprived, by gentle handling, for four hours, immediately after light onset (ZT0-ZT4; n=8 per group). Rapid eye movement (REM) and non-REM (NREM) duration were determined using electroencephalography (EEG) and electromyography (EMG), individually plotted for each hour (sleep deprived points in red; mean±s.e.m). ( b ) Plasma of the sleep and sleep-deprived mice was analyzed by ELISA for corticosterone levels (n=8 per group; mean±s.e.m). ( c ) We isolated HSCs from mice that were allowed to sleep or sleep-deprived mice. The flow cytometry gating scheme for HSCs isolation is shown on a representative mouse. (d) To test the mid-term and long-term transplantation potential of the isolated HSCs we intravenously injected 300 HSCs mice into lethally irradiated congenic recipients (to distinguish between the donor and recipient cells we used CD45.1 mice as donors and CD45.2 as recipients). Peripheral blood (PB) samples were collected from the recipient mice at ( e ) eight weeks (peripheral blood), ( f ) 16 weeks and ( g ) bone marrow (BM) post-transplantation. Cells were analyzed for myeloid chimerism, as determined by the percentage of myeloid cells derived from the donor compared to the total number of myeloid cells in the indicated tissue (mean±s.e.m; Student’s t -test; *** p

Article Snippet: Flow cytometry Mice were euthanized, using CO2, and bone marrow was harvested into PBS containing 2% fetal calf serum (FCS).

Techniques: Isolation, Mouse Assay, Irradiation, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Transplantation Assay, Injection, Derivative Assay

Journal: The Journal of Biological Chemistry

Article Title: The myosin-tail homology domain of centrosomal protein 290 is essential for protein confinement between the inner and outer segments in photoreceptors

doi: 10.1074/jbc.RA119.009712

Figure Lengend Snippet: Expression and localization of BBS proteins are not altered in Cep290 fl/fl ;Cre + retinas. A, immunoblot analysis of Cep290 +/ fl ;Cre ? and Cep290 fl/fl ;Cre + retinal protein extracts. Each lane represents individual animals, and three animals were analyzed per genotype. Thirty μg of proteins were loaded per lane. Locations of protein standards are shown on the right. Numbers in ratio column are relative band intensities of the indicated proteins in Cep290 fl/fl ;Cre + retinas compared with those in Cep290 +/ fl ;Cre ? retinas. B, BBSome assembly is not altered in Cep290 fl/fl ;Cre + retinas. Retinal protein extracts from Cep290 +/ fl ;Cre ? and Cep290 fl/fl ;Cre + retinas were subjected to immunoprecipitation ( IP ) with anti-BBS7 antibody. Normal rabbit IgG was used as a negative control. Co-precipitated BBS proteins were detected by immunoblotting. C, immunoblot analysis of outer segment fractions isolated from Cep290 +/ fl ;Cre ? and Cep290 fl/fl ;Cre + retinas. Each lane represents individual outer segment preparations, and the results from two independent preparations are shown. Five μg of proteins were loaded per lane. Others are same as in A. D, localization of BBS8 ( green ) in Cep290 +/ fl ;Cre + and Cep290 fl/fl ;Cre + photoreceptors. Connecting cilia were labeled with anti-CETN antibody ( red ). Scale bar represents 1 μm. E, localization of LZTFL1 ( green ) in control ( Lztfl1 + lgt ), Lztfl1 gt/gt , and Cep290 fl/fl ;Cre + photoreceptors. White arrowheads indicate binding of anti-LZTFL1 antibody to cross-reacting protein(s) around the basal body. Others are same as in D .

Article Snippet: AntibodiesThe following primary antibodies were used for immunofluorescence microscopy and immunoblotting: ABCA4 (EMD Millipore, MABN2439); β-actin (Sigma, A1978); ATP1A3 (abcam, ab2826); BBS2 (Proteintech Group, 66246-1-AP); BBS5 (Santa Cruz Biotechnology, sc-515331); BBS7 (Proteintech Group, 18961-1-AP); BBS8 (Sigma, HPA003310); CEP290 (EMD Millipore, ABN1710, for immunohistochemistry); CEP290 (Proteintech Group, 22490-1-AP, for immunoblotting); CETN (EMD Millipore, 04-1624); CNGA1 (EMD Millipore, MABN468); FLAG (Sigma, F1804 and A8592 (peroxidase-conjugated)); GRK1 (abcam, ab2776); GUCY2D (Proteintech Group, 55127-1-AP); HA (peroxidase-conjugated; Sigma, 12013819001); HCN1 (NeuroMab, 75-110); IMPG2 (Thermo Fisher Scientific, PA5-64926); LAMP1 (Sigma, L1418); LZTFL1 (custom-made ( )); PDE6A (Proteintech Group, 21200-1-AP); PDE6B (Proteintech Group, 22063-1-AP); polyglutamylation modification/GT335 (AdipoGen, AG-20B-0020-C100); PRPH2 (Proteintech Group, 18109-1-AP); RHO (EMD Millipore, MAB5356); ROM1 (Proteintech Group, 21984-1-AP); SNAP25 (abcam, ab24737); STX3 (EMD Millipore, MAB2258), STXBP1 (Proteintech Group, 11459-1-AP), SYP (Cell Signaling, 5461); and VAMP2 (Cell Signaling, 13508).

Techniques: Expressing, Immunoprecipitation, Negative Control, Isolation, Labeling, Binding Assay

Journal: Oncology Letters

Article Title: Comprehensive bioinformatics analysis of lncRNAs in gastric cancer

doi: 10.3892/ol.2018.9707

Figure Lengend Snippet: LINC00982 and LL22NC03-N14H11.1 have opposite roles in the proliferation of gastric cancer cells. (A) Reverse transcription-quantitative polymerase chain reaction revealed that the expression of LINC00982 in the LINC00982-overexpressed SGC-7901 cell line (left) and the expression of LL22NC03-N14H11.1 in the LL22NC03-N14H11.1-down-regulated SGC-7901 cell line (right). (B) The MTT assay revealed the proliferative status of the SGC-7901 cell line after LINC00982 being overexpressed or LL22NC03-N14H11.1 being downregulated. (C) Flow cytometric analysis of the expression of Ki-67 in the SGC-7901 cell line after LINC00982 was overexpressed or LL22NC03-N14H11.1 was downregulated. All the experiments were replicated a minimum of 3. The error bars represent the standard deviation. *P

Article Snippet: Anti-mouse-ki-67-PE flow cytometric antibody (eBioscience; Thermo Fisher Scientific, Inc.) was purchased from eBioscience.

Techniques: Real-time Polymerase Chain Reaction, Expressing, MTT Assay, Flow Cytometry, Standard Deviation

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: CK2 Controls Th17 and Regulatory T Cell Differentiation Through Inhibition of FoxO1

doi: 10.4049/jimmunol.1701592

Figure Lengend Snippet: CK2α Deletion Results in Impaired Th17 Cell and Enhanced Treg Differentiation During EAE (A) CK2α fl/fl and CK2α −/− were immunized with MOG 35-55 in CFA, and scored daily for signs of clinical disease; n=7 mice/group. Data is combined from 3 separate experiments. (B) At disease peak, between days 14 and 16 post immunization (P.I.), mononuclear cells (MNC) were enriched from the spinal cords, and CD4 was expression detected by flow cytometry. Total numbers of enriched MNCs, and numbers and frequencies of CD4 + cells are shown. (C) Cytokine production from CD4 + T cells in the spinal cords was detected by flow cytometry on MNCs. Cells are gated on live, CD4 + CD44 + cells. (D, E) Frequencies of total IL-17A + , IFN-γ + , and GM-CSF + cells (D) and single and double cytokine-producing cells (E) are quantified; CK2α fl/fl , n=8 mice; CK2α −/− , n=6 mice. Data is combined from 3 separate experiments. (F) During disease resolution, between days 22 and 24 P.I., MNCs were enriched from the spinal cords and Tregs detected by expression of CD25 and Foxp3 by flow cytometry. (G) Frequencies of Tregs in the spinal cord during resolution are quantified; n=7 mice/group. Data is combined from 3 separate experiments. *p

Article Snippet: Flow cytometry antibodies against mouse CD44, CD62L, CD69, Foxp3 and GM-CSF were purchased from eBioscience.

Techniques: Mouse Assay, Expressing, Flow Cytometry, Cytometry

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: CK2 Controls Th17 and Regulatory T Cell Differentiation Through Inhibition of FoxO1

doi: 10.4049/jimmunol.1701592

Figure Lengend Snippet: CK2α promotes Th17 Cell Pathogenicity in Adoptive Transfer EAE (A) CK2α fl/fl and CK2α −/− mice were immunized, and on days 10-14 P.I, lymph nodes cultured in Th17 conditions. Cytokine production was determined by flow cytometry. A representative plot from 3 separate experiments is shown. (B) Cells were adoptively transferred into Rag1 −/− recipients, and recipient mice scored daily; n=8 mice/group. (C) At disease peak, mononuclear cells were enriched from the spinal cords, counted, and CD4 expression detected by flow cytometry. (D) At disease peak, effector T cells from the spleens were stained for IL-17A and IFN-g. Representative flow plots and combined frequencies of total IL-17A + and IFN- g + CD4 + CD44 + T cells are shown. (E) Tregs in the spleens of recipient mice were identified by Foxp3. Representative flow plots and combined frequencies of Foxp3 + cells are shown; CK2a fl/fl , n = 5; CK2a −/− , n = 3. * p

Article Snippet: Flow cytometry antibodies against mouse CD44, CD62L, CD69, Foxp3 and GM-CSF were purchased from eBioscience.

Techniques: Adoptive Transfer Assay, Mouse Assay, Cell Culture, Flow Cytometry, Cytometry, Expressing, Staining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: CK2 Controls Th17 and Regulatory T Cell Differentiation Through Inhibition of FoxO1

doi: 10.4049/jimmunol.1701592

Figure Lengend Snippet: Characterization of CK2α −/− Mice (A) CD3 − and CD3 + cells were sorted from the spleens of CK2α fl/fl and CK2α −/− mice, genomic DNA isolated, and Csnk2a1 detected by PCR. (B) Cells from the thymus, spleen and lymph nodes of 12 week old CK2α fl/fl and CK2α −/− mice were counted; n=2. (C) Cells from the spleen and lymph nodes of 12-week old CK2α fl/fl and CK2α −/− mice were stained with anti-CD4 and anti-CD8 antibodies for detection by flow cytometry. (D) Cells from the spleen and lymph nodes of 12-week old CK2α fl/fl and CK2α −/− mice were stained with anti-CD62L and anti-CD44 antibodies for detection by flow cytometry. Plots are gated on CD4 + cells. (E) Cells from the spleens and lymph nodes of 12-week old CK2α fl/fl and CK2α −/− mice were stained with anti-CD25 and anti-Foxp3 antibodies for detection by flow cytometry. Plots are gated on CD4 + cells.

Article Snippet: Flow cytometry antibodies against mouse CD44, CD62L, CD69, Foxp3 and GM-CSF were purchased from eBioscience.

Techniques: Mouse Assay, Isolation, Polymerase Chain Reaction, Staining, Flow Cytometry, Cytometry

Journal: Cancer research

Article Title: Trastuzumab Increases HER2 Uptake and Cross-Presentation by Dendritic Cells

doi: 10.1158/0008-5472.CAN-16-2774

Figure Lengend Snippet: Trastuzumab increases cross-presentation of E75 by DCs. A, mature DCs were cultured with SKBR3 cells +/− trastzumab (10 µg/mL) for 24 hours after which the cell surface was stained for DC markers (CD11c + , HLA-DR + ) as well as an Fab targeting E75 peptide/HLA-A2. Cells were analyzed with flow cytometry and results are expressed as average E75/HLA-A2 Fab MFI. DCs cultured with SKBR3 cells + trastuzmab resulted in increased E75 cross-presentation compared to DCs cultured with SKBR3 cells alone. B, C mature DCs were cultured with SKBR3 cells +/− trastuzumab (10–40 µg/mL) to activate and expand E75-CTLs from HLA-A2 + PBMCs. After 1 week of co-culture, enumeration of E75-CTLs was performed using an E75/HLA-A2 dextramer. Results are expressed as percent CD8 + /E75 + cells from a parent gating of Live/CD16 − /CD19 − /CD56 − /CD14 − /CD3 + /CD8 + cells. DCs that had been cultured with SKBR3 cells + trastuzumab resulted in more efficient expansion of E75-CTLs from donor PBMCs. B, representative scatter plots. C, aggregate results of three experiments. * indicates P

Article Snippet: Anti-mouse flow cytometry antibodies include FITC-conjugated anti-CD3 (clone 145-2C11; Biolegend), Peridinin chlorophyll (PerCP)-Cy5.5-conjugated anti-CD8 (clone 53-6.7; Biolegend), and PE-conjugated HER2/neu-peptide (TYLPANASL)-loaded mouse H-2Kd tetramer produced by the Baylor College of Medicine MHC Tetramer Core (Houston, TX)( ).

Techniques: Cell Culture, Staining, Flow Cytometry, Cytometry, Co-Culture Assay

Journal: Cancer research

Article Title: Trastuzumab Increases HER2 Uptake and Cross-Presentation by Dendritic Cells

doi: 10.1158/0008-5472.CAN-16-2774

Figure Lengend Snippet: Trastuzumab enhances HER2 uptake by DCs. A, mature DCs were co-cultured with SKBR3 breast cancer cells. Cells were then stained for DC markers (CD11c + , HLA-DR + ) and intracellular HER2 and analyzed using Amnis® imaging flow cytometry which demonstrated uptake of HER2 by DCs. B, mature DCs were co-cultured with SKBR3, SKOV3, BT474, or MDA-MB-231 cells for 24 hours +/− trastuzumab (10 µg/ml). Cells were then stained for DC markers (CD11c + , HLA-DR + ) and intracellular HER2 and analyzed by flow cytometry. Results are expressed as average HER2 MFI for DCs cultured with each cancer cell line +/− trastuzumab. Our results show increased HER2 uptake when DCs were in culture with SKBR3 and BT474 cells (high HER2 shedding) when compared with SKOV3 and MDA-MB-231 cells (low HER2 shedding). *** indicates P

Article Snippet: Anti-mouse flow cytometry antibodies include FITC-conjugated anti-CD3 (clone 145-2C11; Biolegend), Peridinin chlorophyll (PerCP)-Cy5.5-conjugated anti-CD8 (clone 53-6.7; Biolegend), and PE-conjugated HER2/neu-peptide (TYLPANASL)-loaded mouse H-2Kd tetramer produced by the Baylor College of Medicine MHC Tetramer Core (Houston, TX)( ).

Techniques: Cell Culture, Staining, Imaging, Flow Cytometry, Cytometry, Multiple Displacement Amplification

Journal: Cancer research

Article Title: Trastuzumab Increases HER2 Uptake and Cross-Presentation by Dendritic Cells

doi: 10.1158/0008-5472.CAN-16-2774

Figure Lengend Snippet: Effect of treatment time and dose of trastuzumab on HER2 uptake by DCs. A, mature DCs were co-cultured with SKBR3 cells +/− trastuzumab at increasing durations up to 24 hours. Cells were then surface-stained for DC markers (CD11c + , HLA-DR + ) and intracellular HER2 and analyzed using flow cytometry. HER2 uptake by DCs is expressed using average HER2 MFI. Increasing HER2 uptake is observed over time with trastuzumab contributing significantly to uptake at 24 hours of treatment. B, mature DCs were co-cultured with SKBR3 cells at various doses of trastuzumab. Cells were stained for DC markers (CD11c + , HLA-DR + ) and intracellular HER2 and analyzed by flow cytometry. HER2 uptake by DCs is expressed using fold change in average HER2 MFI. All comparisons were made to DCs co-cultured with SKBR3 cells alone. A 50% increase in HER2 uptake is noted with trastuzumab treatment at all doses tested with little difference between low (10 µg/mL) and high (80 µg/mL) dose treatment. * indicates P

Article Snippet: Anti-mouse flow cytometry antibodies include FITC-conjugated anti-CD3 (clone 145-2C11; Biolegend), Peridinin chlorophyll (PerCP)-Cy5.5-conjugated anti-CD8 (clone 53-6.7; Biolegend), and PE-conjugated HER2/neu-peptide (TYLPANASL)-loaded mouse H-2Kd tetramer produced by the Baylor College of Medicine MHC Tetramer Core (Houston, TX)( ).

Techniques: Cell Culture, Staining, Flow Cytometry, Cytometry

Journal: Cancer research

Article Title: Trastuzumab Increases HER2 Uptake and Cross-Presentation by Dendritic Cells

doi: 10.1158/0008-5472.CAN-16-2774

Figure Lengend Snippet: Trastuzumab’s enhancing effect on HER2 uptake by DCs is specific to trastuzumab and is mediated by the Fc receptor. A, mature DCs were co-cultured with SKBR3 cells and trastuzumab (10 µg/mL) +/− human FcR binding inhibitor. Cells were stained for DC markers (CD11c + , HLA-DR + ) and intracellular HER2 and analyzed with flow cytometry. HER2 uptake by DCs is expressed as fold change in average HER2 MFI when compared to DCs alone. Data indicate that FcR inhibition abrogates the effect of trastuzumab on HER2 uptake by DCs. B, mature DCs were co-cultured with SKBR3 cells +/− trastuzumab or rituximab for 24 hours. Rituximab (10 µg/mL) was used as an antibody isotype control. Cells were stained for DC markers (CD11c + , HLA-DR + ) and intracellular HER2 and analyzed with flow cytometry. Results are represented by average HER2 MFI. No significant increase in HER2 uptake was noted in DCs cultured with SKBR3 cells + rituximab compared to SKBR3 cells alone. * indicates P

Article Snippet: Anti-mouse flow cytometry antibodies include FITC-conjugated anti-CD3 (clone 145-2C11; Biolegend), Peridinin chlorophyll (PerCP)-Cy5.5-conjugated anti-CD8 (clone 53-6.7; Biolegend), and PE-conjugated HER2/neu-peptide (TYLPANASL)-loaded mouse H-2Kd tetramer produced by the Baylor College of Medicine MHC Tetramer Core (Houston, TX)( ).

Techniques: Cell Culture, Binding Assay, Staining, Flow Cytometry, Cytometry, Inhibition

Journal: Cancer research

Article Title: Trastuzumab Increases HER2 Uptake and Cross-Presentation by Dendritic Cells

doi: 10.1158/0008-5472.CAN-16-2774

Figure Lengend Snippet: Surface expression and shedding of HER2 by cancer cell lines. A, surface expression of HER2 measured by flow cytometry in multiple breast cancer cell lines Results indicate high HER2 surface expression by the breast cancer cell lines SKBR3 and BT474, and the SKOV3 ovarian cancer cell line. MDA-MB-231 cells, express low levels of HER2. B, levels of soluble HER2 shedding by multiple cancer cell lines as determined by ELISA. SKBR3, BT474, SKOV3, and MDA-MB-231 cells were cultured for 48 hours in serum free media after which the supernatant was assayed for HER2 concentration by ELISA. SKBR3 and BT474 cells shed greater amounts of HER2 into the culture media compared with SKOV3 and MDA-MB-231 cells.

Article Snippet: Anti-mouse flow cytometry antibodies include FITC-conjugated anti-CD3 (clone 145-2C11; Biolegend), Peridinin chlorophyll (PerCP)-Cy5.5-conjugated anti-CD8 (clone 53-6.7; Biolegend), and PE-conjugated HER2/neu-peptide (TYLPANASL)-loaded mouse H-2Kd tetramer produced by the Baylor College of Medicine MHC Tetramer Core (Houston, TX)( ).

Techniques: Expressing, Flow Cytometry, Cytometry, Multiple Displacement Amplification, Enzyme-linked Immunosorbent Assay, Cell Culture, Concentration Assay

Journal: Journal of Neuroinflammation

Article Title: Mutant Huntingtin affects toll-like receptor 4 intracellular trafficking and cytokine production in mast cells

doi: 10.1186/s12974-020-01758-9

Figure Lengend Snippet: Dynamin activity mediates TLR4-dependent ERK 1/2 phosphorylation, c - fos and TNF mRNA accumulation and TNF secretion. WT BMMCs were pre-incubated with dynasore (80 μM) for 30 min before LPS addition (500 ng/mL). After stimulus, the cells were incubated at 37 °C for the indicated times and processed to obtain total protein or total RNA for RT-PCR. a ERK1/2 phosphorylation, b c - fos mRNA accumulation, c TNF mRNA synthesis, and d TNF secretion. Data are presented as the mean ± SD of 3–4 independent experiments. p

Article Snippet: Antibodies against TLR-4 receptor used for confocal microscopy were purchased from BioLegend (San Diego, CA, USA) (Cat. No. SA1521, recognizing TLR4/MD2), and those used for flow cytometry (Cat. No. SAB1300056, recognizing N-terminal region of TLR-4 receptor) were purchased from Sigma-Aldrich.

Techniques: Activity Assay, Incubation, Reverse Transcription Polymerase Chain Reaction