Journal: Cancer Biology & Therapy

Article Title: Therapeutic enhancement of radiation and immunomodulation by gold nanoparticles in triple negative breast cancer

doi: 10.1080/15384047.2020.1861923

Figure Lengend Snippet: Effect of AuNPs and AuNP enhanced RT on tissue expression of cleaved Caspase 3, Calreticulin and macrophage infiltration

Article Snippet: The following primary antibodies and dilutions were used: anti-Calreticulin, 1:15000 for 20 min (Sino Biological, 13539-T60), anti-cleaved Caspase 3, 1:250 for 20 min (Cell Signaling, 9579S) and anti- F4/80 antibody, 1:500 dilution (Cell Signaling, 70076).

Techniques: Expressing

Journal: The Journal of Biological Chemistry

Article Title: Externalized Glycolytic Enzymes Are Novel, Conserved, and Early Biomarkers of Apoptosis *

doi: 10.1074/jbc.M111.314971

Figure Lengend Snippet: Cytofluorometric analysis of externalization of other molecules. Human transformed (Jurkat) T lymphocytes that had been induced to undergo apoptosis by treatment with actinomycin D or that had been left untreated were analyzed cytofluorometrically following staining with FITC-conjugated mouse anti-human annexin II monoclonal antibody (BD Biosciences) (A) or mouse anti-calreticulin monoclonal and FITC-conjugated goat anti-rabbit IgG secondary antibodies (Enzo Life Sciences) (B). Apoptotic (solid green histograms) and viable (dashed lines) cells were identified by scatter properties and gated electronically.

Article Snippet: Human transformed (Jurkat) T lymphocytes that had been induced to undergo apoptosis by treatment with actinomycin D or that had been left untreated were analyzed cytofluorometrically following staining with FITC-conjugated mouse anti-human annexin II monoclonal antibody (BD Biosciences) ( A ) or mouse anti-calreticulin monoclonal and FITC-conjugated goat anti-rabbit IgG secondary antibodies (Enzo Life Sciences) ( B ).

Techniques: Transformation Assay, Staining

Journal: Arthritis Research & Therapy

Article Title: Articular cartilage chondrocytes express aromatase and use enzymes involved in estrogen metabolism

doi: 10.1186/ar4539

Figure Lengend Snippet: Immunocytochemical and immunohistochemical staining of aromatase or aromatase combined with calreticulin in human articular cartilage tissue, in primary human articular chondrocytes and in immortalized chondrocytes of cell line C-28/I2. (A) Cultured primary articular chondrocytes react with aromatase antibody as revealed by red staining. (B) Cultured chondrocytes of cell line C-28/I2 react with the aromatase antibody. Control cells do not react. (C) Articular cartilage tissue shows immunoreactivity for aromatase, and the control remains unstained. (D) Localization of aromatase by immunofluorescence. The slides present staining of nuclei by 4′,6-diamidino-2-phenylindole (DAPI) and of cytoplasm with fluorescein isothiocyanate (FITC)–conjugated antibodies to aromatase (antibody) or combined staining of nuclei and aromatase (merged image). Cytoplasm of cultured C-28/I2 cells show positive reaction for aromatase. (E) Localization of aromatase combined with calreticulin by immunofluorescence. The slides present staining of nuclei by DAPI; staining of cytoplasm with FITC-conjugated secondary antibodies against aromatase or calreticulin; and combined staining of nuclei, aromatase and calreticulin (merged image). As shown by single-cell laser-scanning microscopy, staining for aromatase and calreticulin is observed at approximately identical cytoplasmic sites. ER: Endoplasmic reticulum. (F) Primary human articular chondrocytes from knee joint tissue with and without osteoarthritis (OA) show aromatase immunostaining equally in all chondrocytes. Scale bars: 50 μm (A and B) , 10 μm (C and E) and 20μm (F) .

Article Snippet: The second antibody used was goat anti-rabbit calreticulin (ABR-01176; Dianova, Hamburg, Germany) as a marker of endoplasmic reticulum.

Techniques: Immunohistochemical staining, Staining, Cell Culture, Immunofluorescence, Laser-Scanning Microscopy, Immunostaining

Journal: PLOS Biology

Article Title: Spatacsin regulates directionality of lysosome trafficking by promoting the degradation of its partner AP5Z1

doi: 10.1371/journal.pbio.3002337

Figure Lengend Snippet: ( A ) Western blot analysis of fractions obtained from Spg11 +/+ and Spg11 −/− mouse brains and separated by differential centrifugation. Immunoblots with antibodies raised against spatacsin, the ER proteins STIM1 and calreticulin, the lysosomal proteins cathepsin D (Cath D) and Lamp1, the mitochondrial protein VDAC. Light membrane fractions where spatacsin is enriched are encircled by the red rectangle. ( B ) Western blot analysis of ER- and lysosome-enriched fractions obtained from Spg11 +/+ and Spg11 −/− mouse brains. Immunoblots with antibodies raised against spatacsin, the ER proteins STIM1 and REEP5, lysosomal hydrolase cathepsin D and lysosomal membrane protein Lamp2. ER fractions are encircled by the red rectangle. The asterisk indicates a nonspecific signal. ( C ) Confocal images of MEFs expressing the ER marker GFP-Sec61β and V5-tagged spatacsin. Cells were immunostained with anti-V5 antibody and the lysosome marker Lamp1. Scale bar: 5 μm. ( D ) STED images of MEFs expressing the ER marker GFP-Sec61β and V5-tagged spatacsin. Cells were immunostained with anti-V5, anti Lamp1, and anti-GFP antibodies. Scale bar: 1 μm. Arrowheads point spatacsin-V5 colocalized with the ER marker GFP-Sec61β. Spatacsin V5 occasionnaly colocalized with the lysosome marker Lamp1 and the ER marker GFP-Sec61β (arrows). ( E ) Quantification of the proportion of V5-tagged spatacsin spots from STED images that are colocalizing either with Lamp1 staining or with GFP-Sec61β expressed in MEFs. Superplot: means and SEM, N = 18 cells from 3 independent experiments. Paired t test on the means. ( F ) Transmission electron microscopy images of ER-lysosomes contacts in Spg11 +/+ and Spg11 −/− MEFs. Arrows point toward areas where ER and lysosomes are in contact; asterisks indicate ER. Scale bar: 200 nm. ( G ) Proportion of lysosomes that are less than 30 nm from ER membrane analyzed on transmission electron microscopy images in Spg11 +/+ and Spg11 −/− MEFs. Superplot: means and SEM, N > 17 cells from 3 independent experiments. Paired t test on the means. ( H ) Quantification of the proportion of lysosomal membrane that is less than 30 nm from ER membrane analyzed on transmission electron microscopy images in Spg11 +/+ and Spg11 −/− MEFs. Superplot: means and SEM, N > 17 cells from 3 independent experiments. * P = 0.032, paired t test on the means. ( I ) Binarized image of fibroblasts expressing GFP-Sec61β and Lamp1-mCherry. Note the overlap (white) between the ER (magenta) and lysosome (green) masks. Scale bar: 5 μm. ( J ) Quantification of the proportion of lysosomes that have an area overlapping with the ER > 30% in Spg11 +/+ and Spg11 −/− MEFs. Superplot: means and SEM, N = 13 cells from 4 independent experiments. Paired t test on the means. The raw data underlying panels E, G, H, and J can be found in file.

Article Snippet: The antibodies used for immunofluorescence and immunoblotting were rat anti-Lamp1 (clone 1D4B, Development Studies Hybridoma Bank, University of Iowa, USA, deposited by JT August), rabbit anti-V5 (Cat#8137, Sigma), mouse anti-V5 (Cat#Ab27671, Abcam), rat anti-HA (clone 3F10, Cat#11867423001, Merck), rabbit anti-HA (Cat#ab9110, Abcam), rabbit anti-GFP (Cat#6556, Abcam), mouse anti-Myc (clone 9E10, Development Studies Hybridoma Bank), rabbit anti-Stim1 (Cat#5668, Cell Signaling Technology), rabbit anti-cathepsin D (Cat#Ab75852, Abcam), rabbit anti-spatacsin (Cat#16555-1-AP, ProteinTech), rabbit anti-spastizin (Cat#5023, ProSci), rabbit anti-AP5Z1 (Cat#HPA035693, Sigma), rabbit anti AP5M1 (provided by Dr. J Hirst; [ ]), mouse anti-clathrin heavy chain (clone 23, Cat#610500, BD Biosciences), rabbit anti-UBR4 (Cat#Ab86738, Abcam), mouse anti-p150 Glued (Cat#610474, BD Biosciences), rabbit anti-calreticulin (Cat#SPA600F, Enzo Life Sciences), mouse anti-VDAC1 (Cat#Ab16814, Abcam), rabbit anti-REEP5 (14643-1-AP, ProteinTech), mouse anti-p62 (Cat#Ab 56416, Abcam), rat anti Lamp2 (Cat#Ab13524, Abcam), mouse anti-α-tubulin (Clone DM1A, Cat#Ab7291, Abcam), mouse anti-actin (Clone C4, Cat#Ab3280, Abcam), and mouse anti-ubiquitine (Clone P4D1, Cat #3936S, Cell Signaling Technology).

Techniques: Western Blot, Centrifugation, Membrane, Expressing, Marker, Staining, Transmission Assay, Electron Microscopy

Journal: Journal of Clinical Medicine

Article Title: The Toll-like Receptor 7-Mediated Ro52 Antigen-Presenting Pathway in the Salivary Gland Epithelial Cells of Sjögren’s Syndrome

doi: 10.3390/jcm12134423

Figure Lengend Snippet: Increased expression of PLC and class I in SS patients’ LSGs. ( A ) The mean fluorescence intensity (MFI) of MHC class I (total 53 ducts from 10 SS, 19 ducts from 5 non-SS), TAP1 (53 ducts from 10 SS, 19 ducts from 5 non-SS), calreticulin (53 ducts from 10 SS, 20 ducts from 4 non-SS), ERp57 (54 ducts from 10 SS, 20 ducts from 5 non-SS) and tapasin (48 ducts from 10 SS, 28 ducts from 5 non-SS) on the ducts of labial salivary glands (LSGs) from patients with SS and non-SS subjects’ ducts. The MFI of immunostaining was captured and calculated with a hybrid cell count system. Representative samples of LSGs from SS patients ( B ) and non-SS subjects ( C ) stained with anti-class I, anti-TAP1, anti-calreticulin, anti-ERp57, anti-tapasin antibodies. mIgG1 (green), NRS (red), and NGS (red) were used as isotype controls. Hoechst was used for counterstaining the nuclei. White arrowheads: the identical ductal expression of staining for different proteins. Bar: 50 μM. Non-SS: these subjects were classified as non-SS sicca control subjects based on the AECG classification criteria. TAP1: transporter associated with antigen processing 1; ERp57: endoplasmic reticulum-resident protein 57; mIgG: mouse IgG; NRS: normal rabbit serum; NGS: normal goat serum. Significance was determined using Welch’s t -test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The primary antibodies ( ) used were rabbit anti-MHC class I polyclonal (Proteintech, Rosemont, IL, USA), mouse anti-MHC class I monoclonal (Novus Biologicals, Littleton, CO, USA), rabbit anti-Ro52 polyclonal (Cloud-Clone Corp.; Katy, TX, USA), mouse anti-Ro60 monoclonal (Santa Cruz, Dallas, TX, USA), mouse anti-ubiquitin monoclonal (Enzo Life Sciences, Farmingdale, NY, USA), rabbit anti-TAP1 monoclonal (Bioss Antibodies, Woburn, MA, USA), rabbit anti-tapasin polyclonal (GeneTex, Irvine, CA, USA), mouse anti-ERp57 monoclonal (Boster Bio, Pleasanton, CA, USA), and rabbit anti-calreticulin polyclonal antibody (LifeSpan BioSciences, Seattle, WA, USA).

Techniques: Expressing, Fluorescence, Immunostaining, Cell Counting, Staining

Journal: Journal of Clinical Medicine

Article Title: The Toll-like Receptor 7-Mediated Ro52 Antigen-Presenting Pathway in the Salivary Gland Epithelial Cells of Sjögren’s Syndrome

doi: 10.3390/jcm12134423

Figure Lengend Snippet: Increased expression of PLC and class I in SS patients’ TLR7-stimulated SGECs. Representative images in immunostaining showing the expressions of MHC class I, TAP1, calreticulin, ERp57, and tapasin in SGECs from SS patients ( n = 2) stimulated with 1 mM loxoribine for 6 h and 1000 U/mL of IFN-β for 12 h. mIgG1 (green), NRS (red), and NGS (green) were used as isotype controls. Deconvolution was performed for all images in . The insets show a magnified view of each panel. Bar: 10 μM. TAP1: transporter associated with antigen processing 1, ERp57: endoplasmic reticulum-resident protein 57, mIgG: mouse IgG, NRS: normal rabbit serum, NGS: normal goat serum.

Article Snippet: The primary antibodies ( ) used were rabbit anti-MHC class I polyclonal (Proteintech, Rosemont, IL, USA), mouse anti-MHC class I monoclonal (Novus Biologicals, Littleton, CO, USA), rabbit anti-Ro52 polyclonal (Cloud-Clone Corp.; Katy, TX, USA), mouse anti-Ro60 monoclonal (Santa Cruz, Dallas, TX, USA), mouse anti-ubiquitin monoclonal (Enzo Life Sciences, Farmingdale, NY, USA), rabbit anti-TAP1 monoclonal (Bioss Antibodies, Woburn, MA, USA), rabbit anti-tapasin polyclonal (GeneTex, Irvine, CA, USA), mouse anti-ERp57 monoclonal (Boster Bio, Pleasanton, CA, USA), and rabbit anti-calreticulin polyclonal antibody (LifeSpan BioSciences, Seattle, WA, USA).

Techniques: Expressing, Immunostaining