Journal: Cell

Article Title: Mechanism of orphan subunit recognition during assembly quality control

doi: 10.1016/j.cell.2023.06.016

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-CCT2 , Bethyl , Cat#A303-479A; RRID: AB_10950578.

Techniques: Recombinant, Transfection, Protease Inhibitor, Mutagenesis, In Vitro, Plasmid Preparation, Negative Control, Sequencing, Expressing, Software, Western Blot, Clear Native PAGE

Journal: Viruses

Article Title: TRiC/CCT Complex, a Binding Partner of NS1 Protein, Supports the Replication of Zika Virus in Both Mammalians and Mosquitoes

doi: 10.3390/v12050519

Figure Lengend Snippet: Identification of Zika virus (ZIKV) NS1 protein-binding partners by LC-MS/MS (Liquid chromatography–mass spectrometry).

Article Snippet: The rabbit anti-human CCT2 (One World Lab, San Diego, CA, USA), rabbit anti-ZIKV NS1 (Genetex, Irvine, CA, USA), rabbit anti-ZIKV Capsid (Cap) (Genetex, Irvine, CA, USA), mouse anti-HA (Abcam), mouse anti-c-Myc (Sigma-Aldrich, Burlington, MA, USA), mouse anti-actin (Abcam, Cambridge, MA, USA), HRP-linked rat anti-mouse IgG (Mouse TrueBlot ® ULTRA, ROCKLAND, Limerick, PA, USA), HRP-linked goat anti-rabbit IgG (Cell Signaling, Danvers, MA, USA), mouse anti-ZIKV NS1 monoclonal (GeneTex, Irvine, CA, USA), rabbit anti-CCT2 monoclonal (Abcam, Cambridge, MA, USA), goat anti-mouse IgG (H+L) cross-adsorbed secondary antibody-Alexa Fluor 555 (Invitrogen, Carlsbad, CA, USA), and F(ab’)2-goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody-Alexa Fluor 488 (Invitrogen, Carlsbad, CA, USA) antibodies were purchased.

Techniques: Virus, Protein Binding, Chromatography, Sequencing, Ubiquitin Proteomics

Journal: Viruses

Article Title: TRiC/CCT Complex, a Binding Partner of NS1 Protein, Supports the Replication of Zika Virus in Both Mammalians and Mosquitoes

doi: 10.3390/v12050519

Figure Lengend Snippet: Identification of the Zika virus NS1 region interacting with CCT2 in mammalian cells. ( a ) HeLa cells were infected with ZIKV Cam , and colocalization of ZIKV NS1 and CCT2 were examined using confocal microscopy (Leica SP5, 100× objective): blue, cell nucleus; red, ZIKV NS1 protein; green, CCT2 protein. Right panels show enlargement of area indicated by the white boxes. ( b ) Diagrams of NS1 deletion mutants. A series of NS1 deletion mutants was constructed by sequential deletion from the N- or C-terminus of NS1. A c-Myc-tag was added at the C-terminus. ( c ) Immunoprecipitation of wild-type and deletion mutant NS1 proteins with naïve CCT2. Wild-type and the indicated deletion mutant NS1 proteins were expressed in 293T cells. Twenty-four post transfection, immunoprecipitations were performed with anti-c-Myc beads, followed by immunoblotting.

Article Snippet: The rabbit anti-human CCT2 (One World Lab, San Diego, CA, USA), rabbit anti-ZIKV NS1 (Genetex, Irvine, CA, USA), rabbit anti-ZIKV Capsid (Cap) (Genetex, Irvine, CA, USA), mouse anti-HA (Abcam), mouse anti-c-Myc (Sigma-Aldrich, Burlington, MA, USA), mouse anti-actin (Abcam, Cambridge, MA, USA), HRP-linked rat anti-mouse IgG (Mouse TrueBlot ® ULTRA, ROCKLAND, Limerick, PA, USA), HRP-linked goat anti-rabbit IgG (Cell Signaling, Danvers, MA, USA), mouse anti-ZIKV NS1 monoclonal (GeneTex, Irvine, CA, USA), rabbit anti-CCT2 monoclonal (Abcam, Cambridge, MA, USA), goat anti-mouse IgG (H+L) cross-adsorbed secondary antibody-Alexa Fluor 555 (Invitrogen, Carlsbad, CA, USA), and F(ab’)2-goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody-Alexa Fluor 488 (Invitrogen, Carlsbad, CA, USA) antibodies were purchased.

Techniques: Virus, Infection, Confocal Microscopy, Construct, Immunoprecipitation, Mutagenesis, Transfection, Western Blot

Journal: Viruses

Article Title: TRiC/CCT Complex, a Binding Partner of NS1 Protein, Supports the Replication of Zika Virus in Both Mammalians and Mosquitoes

doi: 10.3390/v12050519

Figure Lengend Snippet: ATP-dependent interaction between CCT2 and Zika virus NS1. c-Myc tagged NS1 was expressed in human 293T cells. Twenty-four post transfection, immunoprecipitation was performed with anti-c-Myc beads under several ATP concentrations (0, 10, 50, 100 mM), followed by immunoblotting.

Article Snippet: The rabbit anti-human CCT2 (One World Lab, San Diego, CA, USA), rabbit anti-ZIKV NS1 (Genetex, Irvine, CA, USA), rabbit anti-ZIKV Capsid (Cap) (Genetex, Irvine, CA, USA), mouse anti-HA (Abcam), mouse anti-c-Myc (Sigma-Aldrich, Burlington, MA, USA), mouse anti-actin (Abcam, Cambridge, MA, USA), HRP-linked rat anti-mouse IgG (Mouse TrueBlot ® ULTRA, ROCKLAND, Limerick, PA, USA), HRP-linked goat anti-rabbit IgG (Cell Signaling, Danvers, MA, USA), mouse anti-ZIKV NS1 monoclonal (GeneTex, Irvine, CA, USA), rabbit anti-CCT2 monoclonal (Abcam, Cambridge, MA, USA), goat anti-mouse IgG (H+L) cross-adsorbed secondary antibody-Alexa Fluor 555 (Invitrogen, Carlsbad, CA, USA), and F(ab’)2-goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody-Alexa Fluor 488 (Invitrogen, Carlsbad, CA, USA) antibodies were purchased.

Techniques: Virus, Transfection, Immunoprecipitation, Western Blot

Journal: Viruses

Article Title: TRiC/CCT Complex, a Binding Partner of NS1 Protein, Supports the Replication of Zika Virus in Both Mammalians and Mosquitoes

doi: 10.3390/v12050519

Figure Lengend Snippet: The effect of suppression of CCT2 on Zika virus replication. ( a ) CCT2 knockdown or control HeLa cells were infected with ZIKV at Multiplicity of Infection (MOI) of 1. At the indicated timepoints post infection (0, 6, 12, 18, 24, 30, and 36 h), samples were harvested and analyzed by immunoblotting with indicated antibodies. β-actin (ACTB) was used as a loading control. Red arrow shows detected NS1 protein. ( b ) CCT2 knockdown or control HeLa cells were infected with ZIKV at MOI of 1. At the indicated timepoints post transfection (6, 12, 24 and 36 h), samples were harvested and viral RNA levels were analyzed by qRT-PCR. Viral RNA levels were normalized to β-actin. ( c ) CCT2 knockdown and control cells were infected with ZIKV at MOI of 0.05. At the indicated timepoints post infection (12, 24, and 36 h), supernatants were collected, and virus titers were determined by plaque assay in Vero cells. **** p < 0.0001 by two-way ANOVA with multiple comparisons test. Data are plotted as the mean ± SEM.

Article Snippet: The rabbit anti-human CCT2 (One World Lab, San Diego, CA, USA), rabbit anti-ZIKV NS1 (Genetex, Irvine, CA, USA), rabbit anti-ZIKV Capsid (Cap) (Genetex, Irvine, CA, USA), mouse anti-HA (Abcam), mouse anti-c-Myc (Sigma-Aldrich, Burlington, MA, USA), mouse anti-actin (Abcam, Cambridge, MA, USA), HRP-linked rat anti-mouse IgG (Mouse TrueBlot ® ULTRA, ROCKLAND, Limerick, PA, USA), HRP-linked goat anti-rabbit IgG (Cell Signaling, Danvers, MA, USA), mouse anti-ZIKV NS1 monoclonal (GeneTex, Irvine, CA, USA), rabbit anti-CCT2 monoclonal (Abcam, Cambridge, MA, USA), goat anti-mouse IgG (H+L) cross-adsorbed secondary antibody-Alexa Fluor 555 (Invitrogen, Carlsbad, CA, USA), and F(ab’)2-goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody-Alexa Fluor 488 (Invitrogen, Carlsbad, CA, USA) antibodies were purchased.

Techniques: Virus, Knockdown, Control, Infection, Western Blot, Transfection, Quantitative RT-PCR, Plaque Assay

Journal: Viruses

Article Title: TRiC/CCT Complex, a Binding Partner of NS1 Protein, Supports the Replication of Zika Virus in Both Mammalians and Mosquitoes

doi: 10.3390/v12050519

Figure Lengend Snippet: The effect of suppression of CCT2 in Aedes aegypti mosquitoes on Zika virus replication. Aedes aegypti mosquitoes were intrathoracically injected with 500 ng dsRNA targeting green fluorescent protein (GFP ) (control) or CCT2 gene. After 3 days post dsRNA injection, Aedes aegypti mosquitoes were injected with 100 PFU of ZIKV. ( left panels ) At 3 and 7days post ZIKV injection, CCT2 expression levels in the whole Aedes aegypti mosquito were analyzed by qRT-PCR (Day 3: ds GFP -treated mosquitoes: n = 11, ds CCT2 -treated mosquitoes: n = 12, Day 7: ds GFP -treated mosquitoes: n = 13, ds CCT2 -treated mosquitoes: n = 12). CCT2 RNA levels were normalized to the levels of Rp49 . ( right panels ) At 3 and 7 days post ZIKV injection, viral RNA levels in the whole Aedes aegypti mosquito were analyzed by qRT-PCR (Day 3: ds GFP -treated mosquitoes: n = 11, ds CCT2 -treated mosquitoes: n = 12, Day 7: ds GFP -treated mosquitoes: n = 13, ds CCT2 -treated mosquitoes: n = 12). Viral RNA levels were normalized to the levels of Rp49 . Data are representative of three independent experiments with similar results. Significance is shown with asterisk ** p < 0.01 and *** p < 0.005 by Wilcoxon–Mann–Whitney test. Data are presented as the mean ± SEM.

Article Snippet: The rabbit anti-human CCT2 (One World Lab, San Diego, CA, USA), rabbit anti-ZIKV NS1 (Genetex, Irvine, CA, USA), rabbit anti-ZIKV Capsid (Cap) (Genetex, Irvine, CA, USA), mouse anti-HA (Abcam), mouse anti-c-Myc (Sigma-Aldrich, Burlington, MA, USA), mouse anti-actin (Abcam, Cambridge, MA, USA), HRP-linked rat anti-mouse IgG (Mouse TrueBlot ® ULTRA, ROCKLAND, Limerick, PA, USA), HRP-linked goat anti-rabbit IgG (Cell Signaling, Danvers, MA, USA), mouse anti-ZIKV NS1 monoclonal (GeneTex, Irvine, CA, USA), rabbit anti-CCT2 monoclonal (Abcam, Cambridge, MA, USA), goat anti-mouse IgG (H+L) cross-adsorbed secondary antibody-Alexa Fluor 555 (Invitrogen, Carlsbad, CA, USA), and F(ab’)2-goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody-Alexa Fluor 488 (Invitrogen, Carlsbad, CA, USA) antibodies were purchased.

Techniques: Virus, Injection, Control, Expressing, Quantitative RT-PCR, MANN-WHITNEY

Journal: PLoS ONE

Article Title: Chaperonin containing TCP1 as a marker for identification of circulating tumor cells in blood

doi: 10.1371/journal.pone.0264651

Figure Lengend Snippet: (A) UCSC Xena analysis from “TCGA, TARGET, GTEx” dataset (n = 17,200) comparing mRNA expression of CCT2 , KRT8 , KRT18 , and KRT19 in metastatic tissue (orange), normal tissues/GTEx (green), primary tumor (blue), and solid tissue normal/TCGA (purple). * = p<0.05, ** = p<0.005, *** = p<0.0001. (B) KMplotter analysis of overall survival (OS) with low vs. high CCT2 mRNA expression and low vs high mRNA expression of the mean of KRT8 , KRT18 , and KRT19 combined in breast cancer patients; n = 4,929. Hazard ratios (HR) and log-rank p-values, as calculated by kmplot.com software, are listed on the KmPlots.

Article Snippet: To incorporate the anti-CCT2-PE antibody into the CSS protocol, we used the Veridex PDF “Guidelines for user-defined markers in CSS” [ ].

Techniques: Expressing, Software

Journal: PLoS ONE

Article Title: Chaperonin containing TCP1 as a marker for identification of circulating tumor cells in blood

doi: 10.1371/journal.pone.0264651

Figure Lengend Snippet: (A) Representative images from tissue microarray of normal breast tissue stained for CCT2. (B) Representative images from a cohort of MBC patient tissues (breast and metastatic sites; ) stained for CCT2. The images are—Left column: cancer adjacent tissue (CAT) from locations indicated in the figure, which had minimal staining. Middle column: CCT2 lo tissue, which was classified as a score of 1 or 2. Right column: CCT2 hi tissue, which was classified as a score of 3 or 4.

Article Snippet: To incorporate the anti-CCT2-PE antibody into the CSS protocol, we used the Veridex PDF “Guidelines for user-defined markers in CSS” [ ].

Techniques: Microarray, Staining

Journal: PLoS ONE

Article Title: Chaperonin containing TCP1 as a marker for identification of circulating tumor cells in blood

doi: 10.1371/journal.pone.0264651

Figure Lengend Snippet: Analysis of different prognostic markers from metastatic breast cancer (MBC) patients.

Article Snippet: To incorporate the anti-CCT2-PE antibody into the CSS protocol, we used the Veridex PDF “Guidelines for user-defined markers in CSS” [ ].

Techniques:

Journal: PLoS ONE

Article Title: Chaperonin containing TCP1 as a marker for identification of circulating tumor cells in blood

doi: 10.1371/journal.pone.0264651

Figure Lengend Snippet: Spearman correlation for CCT2 tumor stain score, circulating tumor cell (CTC) count, and time gap.

Article Snippet: To incorporate the anti-CCT2-PE antibody into the CSS protocol, we used the Veridex PDF “Guidelines for user-defined markers in CSS” [ ].

Techniques: Staining

Journal: PLoS ONE

Article Title: Chaperonin containing TCP1 as a marker for identification of circulating tumor cells in blood

doi: 10.1371/journal.pone.0264651

Figure Lengend Snippet: Multiple linear regression for CCT2 tumor stain score and time gap, accounting for various variables.

Article Snippet: To incorporate the anti-CCT2-PE antibody into the CSS protocol, we used the Veridex PDF “Guidelines for user-defined markers in CSS” [ ].

Techniques: Staining

Journal: PLoS ONE

Article Title: Chaperonin containing TCP1 as a marker for identification of circulating tumor cells in blood

doi: 10.1371/journal.pone.0264651

Figure Lengend Snippet: (A) RT-PCR data comparing lentiviral control (T47D-GFP) (black) with T47D-CCT2 (grey) for expression of EMT genetic markers: SNAIL and TWIST , epithelial markers: EpCAM and E-cadherin , and mesenchymal markers: vimentin and N-cadherin . p-values are shown on the graph. (B) Flow cytometry data detecting surface expression of EpCAM, E-cadherin, and N-cadherin protein in T47D-CCT2 cells (green) compared to isotype controls (grey). (C) Flow cytometry data detecting surface expression of EpCAM, E-cadherin, and N-cadherin proteins in MDA-MB-231 cells (red) compared to isotype controls (grey). All experiments were performed in duplicate.

Article Snippet: To incorporate the anti-CCT2-PE antibody into the CSS protocol, we used the Veridex PDF “Guidelines for user-defined markers in CSS” [ ].

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Flow Cytometry

Journal: PLoS ONE

Article Title: Chaperonin containing TCP1 as a marker for identification of circulating tumor cells in blood

doi: 10.1371/journal.pone.0264651

Figure Lengend Snippet: Representative images of (A) whole healthy human blood without spiked cancer cells processed through the CSS and stained for CCT2, and (B-C) Representative images of MDA-MB-231 (B) or T47D-CCT2 (C) cells spiked into human blood, processed through the CSS, and stained for CCT2. Light blue arrows: leukocyte that is CCT2 positive. Red arrows: cells with dim CK signal and CCT2 positive signal. Yellow arrows: leukocytes that have dim CCT2 signal. Dark blue arrow: doublet of spiked cancer cells with different CCT2 staining intensities. Grey arrows: cells with dim CCT2 signal. CK-FLU. This data is representative of ten experiments.

Article Snippet: To incorporate the anti-CCT2-PE antibody into the CSS protocol, we used the Veridex PDF “Guidelines for user-defined markers in CSS” [ ].

Techniques: Staining

Journal: PLoS ONE

Article Title: Chaperonin containing TCP1 as a marker for identification of circulating tumor cells in blood

doi: 10.1371/journal.pone.0264651

Figure Lengend Snippet: Recovery of breast cancer cells spiked in blood and analyzed with the CSS Analyzer II.

Article Snippet: To incorporate the anti-CCT2-PE antibody into the CSS protocol, we used the Veridex PDF “Guidelines for user-defined markers in CSS” [ ].

Techniques: Cell Counting

Journal: PLoS ONE

Article Title: Chaperonin containing TCP1 as a marker for identification of circulating tumor cells in blood

doi: 10.1371/journal.pone.0264651

Figure Lengend Snippet: MDA-MB-231 cells recovered from the spike in blood were stained and measured for CD44 levels in gated CCT2 positive cells. Alexa-405 served as the isotype control (black). The left panel shows the negative control which was healthy blood cells only stained for CD44 (dark green). The middle panel shows the CCT2 positive spiked cancer cells stained for CD44 (yellow). The right panel shows the positive control which was MDA-MB-231 cells (not spiked in blood) stained for CD44 (light green).

Article Snippet: To incorporate the anti-CCT2-PE antibody into the CSS protocol, we used the Veridex PDF “Guidelines for user-defined markers in CSS” [ ].

Techniques: Staining, Negative Control, Positive Control

Journal: PLoS ONE

Article Title: Chaperonin containing TCP1 as a marker for identification of circulating tumor cells in blood

doi: 10.1371/journal.pone.0264651

Figure Lengend Snippet: (A-B) Representative images from CSS Analyzer II show SCLC cells spiked into healthy human blood and processed with the CSS Autoprep using the anti-CCT2-PE antibody. (A) CRL 5853 and (B) CRL 5903. Red arrows: cells that have dim CK and are CCT2 positive. Blue arrows: doublet of spiked cancer cells with different CCT2 staining intensities. The experiment was performed in duplicate. (C-E) Representative images of lung cancer cells (C) CRL 5903 and (D-E) CRL 5853, stained with reduced anti-CCT2 antibody as indicated in Methods. Red arrows: cells with dim CK staining and CCT2 positive staining. CK-FLU.

Article Snippet: To incorporate the anti-CCT2-PE antibody into the CSS protocol, we used the Veridex PDF “Guidelines for user-defined markers in CSS” [ ].

Techniques: Staining

Journal: PLoS ONE

Article Title: Chaperonin containing TCP1 as a marker for identification of circulating tumor cells in blood

doi: 10.1371/journal.pone.0264651

Figure Lengend Snippet: (A) Representative images of CTCs, based on standard CTC criteria that were CCT2 positive at varying concentrations of the anti-CCT2-PE antibody. (B) Representative images of CTCs from each SCLC patient. Each image was taken from collections of relevant events that were analyzed using standard CTC criteria for the CSS CXC kits as described above. (C) Representative images from CTCs collected using the CTC analysis algorithm (instead of CXC analysis algorithm) where the DAPI signal overlaps with CCT2-PE instead of CK-FLU as in (A, B). Note that images that contain faint CD45 expression are a result of bleed-over from the signal in the PE channel.

Article Snippet: To incorporate the anti-CCT2-PE antibody into the CSS protocol, we used the Veridex PDF “Guidelines for user-defined markers in CSS” [ ].

Techniques: Expressing

Journal: PLoS ONE

Article Title: Chaperonin containing TCP1 as a marker for identification of circulating tumor cells in blood

doi: 10.1371/journal.pone.0264651

Figure Lengend Snippet: Venn diagram showing possible CTC subsets that are CK + /CCT2 - , CK + /CCT2 + , and CK - /CCT2 + .

Article Snippet: To incorporate the anti-CCT2-PE antibody into the CSS protocol, we used the Veridex PDF “Guidelines for user-defined markers in CSS” [ ].

Techniques: