hb 55 cell line  (ATCC)

Journal: bioRxiv

Article Title: Identification of druggable host dependency factors shared by multiple SARS-CoV-2 variants of concern

doi: 10.1101/2023.01.09.523209

Figure Lengend Snippet: Calu-3 cells were infected with the SARS-CoV-2 Wuhan, D614G, and Alpha variants (MOI 0.1). The viral intracellular RNA ( a ) and the viral particles released in the supernatant ( b ) were measured over time (24 and 48 h.p.i.). The RNA copies of the ORF1ab gene, specifically the region encoding for the RNA-dependent RNA polymerase, were calculated using a standard curve generated with a Rpdp-amplicon encoding plasmid. The viral titre was calculated by plaque reduction assay and expressed as PFU/ml. Data are mean ± s.d. of n=3 biological replicates in a , and of n=2 biological replicates, each tested in technical triplicate in b .

Article Snippet: Vero E6 (ATCC® CRL-1586™) were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Thermo Fisher Scientific), Calu-3 cells (ATCC®, HB-55) and Caco-2 cells (kind gift of Prof. Stefano Piccolo, University of Padua) were maintained in Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F-12, Thermo Fisher Scientific).

Techniques: Infection, Generated, Amplification, Plasmid Preparation

Journal: bioRxiv

Article Title: Identification of druggable host dependency factors shared by multiple SARS-CoV-2 variants of concern

doi: 10.1101/2023.01.09.523209

Figure Lengend Snippet: Relative expression of ( a ) viral genes ( ORF1ab and N ) and ( b ) cellular genes ( EGR1 and ATF3 ) in Calu-3 cells infected with the SARS-CoV-2 Wuhan, D614G, and Alpha variants (MOI 1). Intracellular RNA levels were measured at 6, 9, 12, 24 h.p.i. by qPCR. Data are mean-normalised pooled values from independent experiments (n=2 for Alpha; n=3 for Wuhan and D614G variants). Each condition was tested in 4 replicates per condition in each experiment. EGR1 and ATF3 are genes previously identified by Wyler and colleagues 22 as strongly induced at 12 h.p.i.

Article Snippet: Vero E6 (ATCC® CRL-1586™) were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Thermo Fisher Scientific), Calu-3 cells (ATCC®, HB-55) and Caco-2 cells (kind gift of Prof. Stefano Piccolo, University of Padua) were maintained in Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F-12, Thermo Fisher Scientific).

Techniques: Expressing, Infection

Journal: bioRxiv

Article Title: Identification of druggable host dependency factors shared by multiple SARS-CoV-2 variants of concern

doi: 10.1101/2023.01.09.523209

Figure Lengend Snippet: Relative expression of selected candidate hits after siRNA knockdown in ( a ) Calu-3 and ( b ) Caco-2 cell lines. RNA levels were measured by qPCR 48 hours post siRNA transfection. Data are mean ± s.d. of n=2 biological replicates.

Article Snippet: Vero E6 (ATCC® CRL-1586™) were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Thermo Fisher Scientific), Calu-3 cells (ATCC®, HB-55) and Caco-2 cells (kind gift of Prof. Stefano Piccolo, University of Padua) were maintained in Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F-12, Thermo Fisher Scientific).

Techniques: Expressing, Transfection

Journal: bioRxiv

Article Title: Identification of druggable host dependency factors shared by multiple SARS-CoV-2 variants of concern

doi: 10.1101/2023.01.09.523209

Figure Lengend Snippet: Calu-3 ( a ) and Caco-2 cells ( b ) treated with non-targeting (CTR) or indicated siRNAs and infected with SARS-CoV-2 (used variants are shown, MOI 0.1). Infectious SARS-CoV-2 particles in the supernatant were assessed by plaque reduction assay. Bars represent the mean of two independent replicates (± s.d.). “3, 2, 1 variants” indicate the number of variants against which the shown hit genes were found.

Article Snippet: Vero E6 (ATCC® CRL-1586™) were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Thermo Fisher Scientific), Calu-3 cells (ATCC®, HB-55) and Caco-2 cells (kind gift of Prof. Stefano Piccolo, University of Padua) were maintained in Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F-12, Thermo Fisher Scientific).

Techniques: Infection

Journal: bioRxiv

Article Title: Identification of druggable host dependency factors shared by multiple SARS-CoV-2 variants of concern

doi: 10.1101/2023.01.09.523209

Figure Lengend Snippet:

Article Snippet: Vero E6 (ATCC® CRL-1586™) were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Thermo Fisher Scientific), Calu-3 cells (ATCC®, HB-55) and Caco-2 cells (kind gift of Prof. Stefano Piccolo, University of Padua) were maintained in Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F-12, Thermo Fisher Scientific).

Techniques:

Journal: bioRxiv

Article Title: Identification of druggable host dependency factors shared by multiple SARS-CoV-2 variants of concern

doi: 10.1101/2023.01.09.523209

Figure Lengend Snippet: Calu-3 cells were pretreated for 24 h with the indicated compounds and infected with SARS-CoV-2 (strains Wuhan, D614G, Alpha and Delta, MOI 0.1). Two days post infection, cell medium was subjected to plaque reduction assay and the viral titre was calculated and expressed as PFU/ml. Data are mean ± s.d. of n=2 biological replicates. Each condition was tested in triplicate per replicate.

Article Snippet: Vero E6 (ATCC® CRL-1586™) were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Thermo Fisher Scientific), Calu-3 cells (ATCC®, HB-55) and Caco-2 cells (kind gift of Prof. Stefano Piccolo, University of Padua) were maintained in Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F-12, Thermo Fisher Scientific).

Techniques: Infection

Journal: bioRxiv

Article Title: Identification of druggable host dependency factors shared by multiple SARS-CoV-2 variants of concern

doi: 10.1101/2023.01.09.523209

Figure Lengend Snippet: a Intracellular ROS levels were measured in Calu-3 cells at different times after IKE administration (950 nM). IKE prompted a modest ROS increase from 2 to 6 h post administration, whereas longer times of exposure, induced ROS depletion. b Intracellular ROS levels were measured at different times post SARS-CoV-2 infection in Calu-3 cells, in the absence or presence of IKE (950 nM). In the latter case, cells were also pretreated with IKE 24 hours prior to infection. Data were normalized to the untreated control of each tested time point. The viral titre was assessed by PRA, each condition was assayed in = 3 samples. Data are mean ± s.d. of n = 2 biological replicates. c Intracellular ROS levels were measured in Calu-3 cells upon IKE (950 nM) or GSH (300 μM) administration (24 h treatment) and compared to untreated cells (-) as control. Both IKE and GSH induced ROS depletion in Calu-3 cells. IKE treatment diminished ROS levels up to the 70% with respect to the control, whereas GSH administration reduced intracellular ROS up to the 40%. Intracellular ROS levels were determined by H2dcfda assay, each conditions was evaluated in sextuplicate. Single values ± s.d. of n=2 biological replicates are shown. d Viral titre (Wuhan variant) upon 24 h pre-treatment of Calu-3 cells with IKE (950 nM) or GSH (300 μM) and testing after a single cycle of replication, i.e. 30 hpi. e Viral titre (Omicron variant) upon 24 h pre-treatment of Calu-3 cells with IKE (950 nM) or GSH (300 μM) and testing after a single cycle of replication, i.e. 30 hpi.

Article Snippet: Vero E6 (ATCC® CRL-1586™) were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Thermo Fisher Scientific), Calu-3 cells (ATCC®, HB-55) and Caco-2 cells (kind gift of Prof. Stefano Piccolo, University of Padua) were maintained in Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F-12, Thermo Fisher Scientific).

Techniques: Infection, Variant Assay

Journal: Journal of Translational Medicine

Article Title: Six-transmembrane epithelial antigen of the prostate and enhancer of zeste homolog 2 as immunotherapeutic targets for lung cancer

doi: 10.1186/1479-5876-9-191

Figure Lengend Snippet: HLA-DR restriction analysis of STEAP 281-296 - and EZH2 95-109 -reactive CD4 T-cell clones . (A) L243 but not W6/32 inhibited IFN-γ production from HLA-DR restricted, STEAP 281-296 -reactive CD4 T-cell clones. (B) HLA-DR restriction analysis of EZH2 95-109 -reactive CD4 T-cell clones. (C and D) STEAP 281-296 - and EZH2 95-109 -reactive CD4 T-cell clones were tested for their capacity to recognize autologous PBMCs as APCs in the presence of various concentrations of peptide. Points means of triplicate determinations, bars SD. Points without bars had SD < 10% the value of the mean. Results are representative of two experiments that were done with the same samples.

Article Snippet: To demonstrate antigen-specificity and HLA-DR restriction, blocking of antigen-induced responses were assessed by adding anti-HLA-DR mAb L243 (IgG2a prepared from supernatants of the hybridoma HB-55 obtained from the ATCC) or the anti-HLA-A/B/C mAb W6/32 (IgG2a, ATCC) at 10 μg/ml throughout the 48 h incubation period.

Techniques: Clone Assay

Journal: Journal of Translational Medicine

Article Title: Six-transmembrane epithelial antigen of the prostate and enhancer of zeste homolog 2 as immunotherapeutic targets for lung cancer

doi: 10.1186/1479-5876-9-191

Figure Lengend Snippet: HLA-DR expression in lung adenocarcinoma specimens . (A)(B) Adenocarcinoma cells and infiltrating APCs including lymphocytes or alveolar macrophages are positive for HLA-DR (×200).

Article Snippet: To demonstrate antigen-specificity and HLA-DR restriction, blocking of antigen-induced responses were assessed by adding anti-HLA-DR mAb L243 (IgG2a prepared from supernatants of the hybridoma HB-55 obtained from the ATCC) or the anti-HLA-A/B/C mAb W6/32 (IgG2a, ATCC) at 10 μg/ml throughout the 48 h incubation period.

Techniques: Expressing

Journal: Journal of Translational Medicine

Article Title: Six-transmembrane epithelial antigen of the prostate and enhancer of zeste homolog 2 as immunotherapeutic targets for lung cancer

doi: 10.1186/1479-5876-9-191

Figure Lengend Snippet: HLA-DR restriction analysis of STEAP 281-296 - and EZH2 95-109 -reactive CD4 T-cell clones . (A) L243 but not W6/32 inhibited IFN-γ production from HLA-DR restricted, STEAP 281-296 -reactive CD4 T-cell clones. (B) HLA-DR restriction analysis of EZH2 95-109 -reactive CD4 T-cell clones. (C and D) STEAP 281-296 - and EZH2 95-109 -reactive CD4 T-cell clones were tested for their capacity to recognize autologous PBMCs as APCs in the presence of various concentrations of peptide. Points means of triplicate determinations, bars SD. Points without bars had SD < 10% the value of the mean. Results are representative of two experiments that were done with the same samples.

Article Snippet: To demonstrate antigen-specificity and HLA-DR restriction, blocking of antigen-induced responses were assessed by adding anti-HLA-DR mAb L243 (IgG2a prepared from supernatants of the hybridoma HB-55 obtained from the ATCC) or the anti-HLA-A/B/C mAb W6/32 (IgG2a, ATCC) at 10 μg/ml throughout the 48 h incubation period.

Techniques: Clone Assay

Journal: Journal of Translational Medicine

Article Title: Six-transmembrane epithelial antigen of the prostate and enhancer of zeste homolog 2 as immunotherapeutic targets for lung cancer

doi: 10.1186/1479-5876-9-191

Figure Lengend Snippet: Definition of restricting HLA-DR alleles of the STEAP- or EZH2-reactive CD4 T-cell clones . IFN-γ production of the STEAP 281-296 -reactive CD4 T-cell clones (A) and EZH2 95-109 -reactive CD4 T-cell clones (B, C, and D) was evaluated using L-cells or semi-allogenic EBV-LCLs homozygous for HLA-DR molecules as APCs to define the restricting HLA-DR elements. Columns means of triplicate determinations, bars SD. Columns without bars had SD < 10% the values of the mean. Results are representative of at least two experiments.

Article Snippet: To demonstrate antigen-specificity and HLA-DR restriction, blocking of antigen-induced responses were assessed by adding anti-HLA-DR mAb L243 (IgG2a prepared from supernatants of the hybridoma HB-55 obtained from the ATCC) or the anti-HLA-A/B/C mAb W6/32 (IgG2a, ATCC) at 10 μg/ml throughout the 48 h incubation period.

Techniques: Clone Assay

Journal: Journal of Translational Medicine

Article Title: Six-transmembrane epithelial antigen of the prostate and enhancer of zeste homolog 2 as immunotherapeutic targets for lung cancer

doi: 10.1186/1479-5876-9-191

Figure Lengend Snippet: Direct recognition of STEAP-expressing lung tumor cells by STEAP 281- 296 - reactive CD4 T-cell clones . DR15-restricted CD4 T-cell clone SH31 (A) and DR53-restricted CD4 T-cell clones SH6 (B), HK7 (C), and Sa12 (D) were tested for their capacity to recognize antigen directly on STEAP positive LC cells that are either matched or mismatched for the restricting HLA-DR alleles. The HLA-DR-negative Jurkat was also used as negative control. The antigen specificity of these responses was demonstrated by blocking tumor recognition with L243. Columns without bars had SD < 10% the values of the mean. Results are representative of two separate experiments.

Article Snippet: To demonstrate antigen-specificity and HLA-DR restriction, blocking of antigen-induced responses were assessed by adding anti-HLA-DR mAb L243 (IgG2a prepared from supernatants of the hybridoma HB-55 obtained from the ATCC) or the anti-HLA-A/B/C mAb W6/32 (IgG2a, ATCC) at 10 μg/ml throughout the 48 h incubation period.

Techniques: Expressing, Clone Assay, Negative Control, Blocking Assay

Journal: Journal of Translational Medicine

Article Title: Six-transmembrane epithelial antigen of the prostate and enhancer of zeste homolog 2 as immunotherapeutic targets for lung cancer

doi: 10.1186/1479-5876-9-191

Figure Lengend Snippet: Granzyme B production from STEAP 281-296 - or EZH2 95-109 -reactive CD4 T-cell clones . STEAP 281-296 -reactive, DR15-restricted CD4 T-cell clone SH31 (A, left) and DR53-restricted CD4 T-cell clone Sa12 (A, right) were able to secrete granzyme B reacting with STEAP+/HLA-DR matched LC cells. EZH2 95-109 -reactive, DR15-restricted CD4 T-cell clone SK32 (B, left) and Sa361 (B, middle), and DR53-restricted CD4 T-cell clone Sa362 (B, right) were able to secrete granzyme B reacting with EZH2+/HLA-DR matched LC cells. The HLA-DR-negative Jurkat was also used as negative controls. The antigen specificity of these responses was demonstrated by blocking tumor recognition with L243. Columns without bars had SD < 10% the values of the mean. Results are representative of two separate experiments.

Article Snippet: To demonstrate antigen-specificity and HLA-DR restriction, blocking of antigen-induced responses were assessed by adding anti-HLA-DR mAb L243 (IgG2a prepared from supernatants of the hybridoma HB-55 obtained from the ATCC) or the anti-HLA-A/B/C mAb W6/32 (IgG2a, ATCC) at 10 μg/ml throughout the 48 h incubation period.

Techniques: Clone Assay, Blocking Assay

Journal: Journal of Translational Medicine

Article Title: Six-transmembrane epithelial antigen of the prostate and enhancer of zeste homolog 2 as immunotherapeutic targets for lung cancer

doi: 10.1186/1479-5876-9-191

Figure Lengend Snippet: Direct recognition of EZH2-expressing lung tumor cells by EZH2 95-109 -reactive CD4 T-cell clones . DR1-restricted CD4 T-cell clone SK31 (A), DR15-restricted CD4 T-cell clone SK32 (B), Sa362 (C), DR53-restricted CD4 T-cell clones Sa362 (D) and YM18 (E) were tested for their capacity to recognize antigen directly on EZH2 positive LC cells that are either matched or mismatched for the restricting HLA-DR alleles. The HLA-DR-negative Jurkat was also used as negative controls. The antigen specificity of these responses was demonstrated by blocking tumor recognition with L243. Columns without bars had SD < 10% the values of the mean. Results are representative of two separate experiments.

Article Snippet: To demonstrate antigen-specificity and HLA-DR restriction, blocking of antigen-induced responses were assessed by adding anti-HLA-DR mAb L243 (IgG2a prepared from supernatants of the hybridoma HB-55 obtained from the ATCC) or the anti-HLA-A/B/C mAb W6/32 (IgG2a, ATCC) at 10 μg/ml throughout the 48 h incubation period.

Techniques: Expressing, Clone Assay, Blocking Assay

Journal: Journal of Translational Medicine

Article Title: Six-transmembrane epithelial antigen of the prostate and enhancer of zeste homolog 2 as immunotherapeutic targets for lung cancer

doi: 10.1186/1479-5876-9-191

Figure Lengend Snippet: STEAP 281-296 -reactive CD4 T-cell recognize naturally processed exogenous antigen presented by autologous DCs . STEAP 281-296 -reactive CD4 T-cell clones SH31 (A), SH6 (B), and Sa12 (C) were tested for their reactivity against STEAP positive LC cell (LU65, A549, and RERF-LC-AI) lysates and STEAP negative Jurkat cell lysate when presented by autologous DCs. The capacity of L243 to inhibit these responses was assessed to demonstrate that T-cell reactivity was through TCR/HLA interactions. Columns means of triplicate determinations, bars SD. Columns without bars had SD < 10% the values of the mean. Results are representative of at two separate experiments.

Article Snippet: To demonstrate antigen-specificity and HLA-DR restriction, blocking of antigen-induced responses were assessed by adding anti-HLA-DR mAb L243 (IgG2a prepared from supernatants of the hybridoma HB-55 obtained from the ATCC) or the anti-HLA-A/B/C mAb W6/32 (IgG2a, ATCC) at 10 μg/ml throughout the 48 h incubation period.

Techniques: Clone Assay

Journal: PLoS ONE

Article Title: Limited Promiscuity of HLA-DRB1 Presented Peptides Derived of Blood Coagulation Factor VIII

doi: 10.1371/journal.pone.0080239

Figure Lengend Snippet: The presentation of FVIII peptides was compared between immature DCs and LPS-matured DCs. Cells were homogenized and PNS was fractionated on a sucrose density-gradient. Fractions from the sucrose gradient were analyzed by ELISA for MHC class II molecules and divided into a plasma membrane pool (PM) and an intracellular pool (IC) (see Figure S2 in File S1). Peptides were purified from the pools using L243-sepharose and analyzed by mass spectrometry. On the left y-axis the total number of FVIII peptides recovered from MHC class II is indicated (Peptide #). On the right y-axis number of “core peptides” (Core peptide #) is indicated. Core peptides are defined as a set of peptides with an overlapping sequence harboring an MHC class II binding motif. Heterogeneity in peptide length is due to amino-terminal and/or carboxy-terminal trimming of the presented peptides.

Article Snippet: Hybridomas L243 and IVA-12 (anti-HLA-DR) were obtained from ATCC (Wesel, Germany).

Techniques: Enzyme-linked Immunosorbent Assay, Purification, Mass Spectrometry, Sequencing, Binding Assay

Journal: PLoS ONE

Article Title: Limited Promiscuity of HLA-DRB1 Presented Peptides Derived of Blood Coagulation Factor VIII

doi: 10.1371/journal.pone.0080239

Figure Lengend Snippet: Endocytosis of FVIII by immature DCs was followed by maturation with LPS for either 12h, 24h, 48h, 72h or 96h. HLA-DRB1 bound peptides were purified from L243-sepharose and analyzed by mass spectrometry. Bar diagrams indicate the total amount of DRB1-bound peptides identified under each condition as well as the amount of FVIII peptides and core peptides. Core peptides are defined as a set of peptides with an overlapping sequence harboring an MHC class II binding motif. Heterogeneity in peptide length is due to amino-terminal and/or carboxy-terminal trimming of the presented peptides.

Article Snippet: Hybridomas L243 and IVA-12 (anti-HLA-DR) were obtained from ATCC (Wesel, Germany).

Techniques: Purification, Mass Spectrometry, Sequencing, Binding Assay

Journal: PLoS ONE

Article Title: Limited Promiscuity of HLA-DRB1 Presented Peptides Derived of Blood Coagulation Factor VIII

doi: 10.1371/journal.pone.0080239

Figure Lengend Snippet: A . Ovalbumin-derived peptides were identified by mass spectrometry after purification of HLA-DRB1 from lysates of ovalbumin-pulsed cells with either an anti-HLA-DR antibody or an isotype antibody. SIEVE was used to compare intensities of individual peptides and average intensities of each identified peptide are plotted. The diagonal line indicates an equal intensity under each conditions and the dotted lines indicate 5-fold differences in intensity. Peptide clusters with a fold change of 5 or higher were considered as true MHC class II-bound peptides. Only peptides that were sequenced by MS/MS are depicted. B . Ovalbumin peptides identified in this experiment are listed with sequence, charge, mass/charge ratio (m/z), retention time (RT), Xcorr value and HLA-DR/isotype ratio. This is the ratio of intensity with which the peptide was detected in the anti-MHC class II pulldown (L243) over the intensity with which it was detected in the pulldown with the isotype control antibody. Peptides excluded due to a ratio lower than 5 are marked in red.

Article Snippet: Hybridomas L243 and IVA-12 (anti-HLA-DR) were obtained from ATCC (Wesel, Germany).

Techniques: Derivative Assay, Mass Spectrometry, Purification, Tandem Mass Spectroscopy, Sequencing

Journal: PLoS ONE

Article Title: Activation of ERα Signaling Differentially Modulates IFN-γ Induced HLA-Class II Expression in Breast Cancer Cells

doi: 10.1371/journal.pone.0087377

Figure Lengend Snippet: MDA-MB-231, SK-BR-3, MCF-7, BT-474, and T47D were cultured in E 2 -depleted media, treated with vehicle (ethanol) or E 2 (10 −9 M) and stimulated or not with IFN-γ (100 U/ml) for 96 hours. (A & E) HLA-DR cell surface expression (L243) was analyzed by flow cytometry: grey line, isotype control; black line, constitutive expression; shaded histogram, IFN-γ induced expression. (B & F) Bar graphs represent the MFI (mean florescence intensity) ± SEM for HLA-DR expression of three independent experiments. (C & G) Western blot analysis was performed on cytoplasmic and nuclear extracts for ERα expression (HC-20) and on cytoplasmic extracts for HLA-DRα (TAL 1B5). Protein loading controls included α-tubulin (B-7) and P84 (5E10) for cytoplasmic and nuclear proteins, respectively. (D & H) Bar graphs show the ratio of band intensity for HLA-DRα, normalized to the α-tubulin band intensity and represent the mean ± SEM of three independent experiments (*p<0.05, **p<0.01, ***p<0.001).

Article Snippet: Expression of HLA-II and CIITA was determined as follows: HLA-DR conformers, clone L243 ATCC, purified IgG2a from supernatant diluted to 2.4 µg/ml for flow cytometry (FC) or 10 ng/ml for Western blot analysis; HLA-DRα, mouse IgG1 (clone Tal 1B5, Abcam, 40 ng/ml, IB); Ii, mouse IgG1 (clone LN2, BD Biosciences, 5 µg/ml, FC or 200 ng/ml, IB); HLA-DM, mouse IgG1 (clone MaP.DM1, BD Biosciences, 10 µg/ml, FC and clone TAL18.1, Abcam, 40 ng/ml, IB); CIITA (rabbit antiserum # 21, diluted 1/4000), prepared in Dr. Viktor Steimle's laboratory .

Techniques: Cell Culture, Expressing, Flow Cytometry, Western Blot

Journal: PLoS ONE

Article Title: Activation of ERα Signaling Differentially Modulates IFN-γ Induced HLA-Class II Expression in Breast Cancer Cells

doi: 10.1371/journal.pone.0087377

Figure Lengend Snippet: MDA-MB-231 clone 10A (MDA-231 c10A), VC5 (MDA-231 c10A, transfected with the empty plasmid vector) and MC2 (MDA-231 c10A, transfected with wild type ESR1 ) were cultured in E 2 -depleted medium and stimulated or not with IFN-γ (100 U/ml) for 96 hours. (A) HLA-DR cell surface expression (L243) was analyzed by flow cytometry: grey line, isotype control; black line, constitutive expression; shaded histogram, IFN-γ induced expression. (B) Bar graphs represent the MFI ± SEM for HLA-DR expression of three independent experiments (***p<0.001). (C) Western blot analysis was performed on whole cell lysates for HLA-DRα (TAL 1B5) and ERα (HC-20).

Article Snippet: Expression of HLA-II and CIITA was determined as follows: HLA-DR conformers, clone L243 ATCC, purified IgG2a from supernatant diluted to 2.4 µg/ml for flow cytometry (FC) or 10 ng/ml for Western blot analysis; HLA-DRα, mouse IgG1 (clone Tal 1B5, Abcam, 40 ng/ml, IB); Ii, mouse IgG1 (clone LN2, BD Biosciences, 5 µg/ml, FC or 200 ng/ml, IB); HLA-DM, mouse IgG1 (clone MaP.DM1, BD Biosciences, 10 µg/ml, FC and clone TAL18.1, Abcam, 40 ng/ml, IB); CIITA (rabbit antiserum # 21, diluted 1/4000), prepared in Dr. Viktor Steimle's laboratory .

Techniques: Transfection, Plasmid Preparation, Cell Culture, Expressing, Flow Cytometry, Western Blot

Journal: PLoS ONE

Article Title: Activation of ERα Signaling Differentially Modulates IFN-γ Induced HLA-Class II Expression in Breast Cancer Cells

doi: 10.1371/journal.pone.0087377

Figure Lengend Snippet: VC5 and MC2 cells were cultured in E 2 -depleted media, treated with vehicle (ethanol), E 2 (10 −9 M) or/and ICI (10 −6 M) followed by stimulation with IFN-γ (100 U/ml) for 96 hours. HLA-II expression was analyzed by surface flow cytometry using (A) anti-DR, (L243), and intracellular flow cytometry using (B) anti-DM (Map.DM1) and (C) anti-Ii (LN2). Bar graphs represent the MFI ± SEM of three independent experiments. (*p<0.05, **p<0.01). (D) Western blot analysis was performed on whole cell extracts using for HLA-DRα (TAL 1B5), HLA-DM (TAL18.1) and Ii (LN2); GAPDH (Ab8245) is the protein loading control. Bar graphs show the ratio of band intensities, normalized to GAPDH band intensities and represent the mean ± SEM ratio of three independent experiments: (E) HLA-DRα/GAPDH (F) HLA-DM/GAPDH, and (G) Ii/GAPDH (* p<0.05, ** p<0.01).

Article Snippet: Expression of HLA-II and CIITA was determined as follows: HLA-DR conformers, clone L243 ATCC, purified IgG2a from supernatant diluted to 2.4 µg/ml for flow cytometry (FC) or 10 ng/ml for Western blot analysis; HLA-DRα, mouse IgG1 (clone Tal 1B5, Abcam, 40 ng/ml, IB); Ii, mouse IgG1 (clone LN2, BD Biosciences, 5 µg/ml, FC or 200 ng/ml, IB); HLA-DM, mouse IgG1 (clone MaP.DM1, BD Biosciences, 10 µg/ml, FC and clone TAL18.1, Abcam, 40 ng/ml, IB); CIITA (rabbit antiserum # 21, diluted 1/4000), prepared in Dr. Viktor Steimle's laboratory .

Techniques: Cell Culture, Expressing, Flow Cytometry, Western Blot