Journal: Biochemical pharmacology

Article Title: Identification of membrane palmitoylated protein 1 (MPP1) as a heart-failure-promoting protein triggered by cardiovascular risk factors and aging.

doi: 10.1016/j.bcp.2023.115789

Figure Lengend Snippet: Fig. 3. Generation of Tg-MPP1 mice. A, Upper panel: scheme of the plasmid used for the generation of Tg-MPP1 mice. Lower panel: PCR genotyping of ear- punch biopsies from 11 Tg-MPP1-positive mice with stable integration of the transgenic MPP1 cDNA into the genomic DNA. The negative control (-) did not contain genomic DNA, and the linearized MPP1 plasmid DNA (P) was used as a positive control. The lane marked with M, is the DNA marker. B, Immunoblot detection of the MPP1 protein in heart protein extracts from Tg-MPP1 mice and non-transgenic B6 mice. The left panel is a representative immunoblot, and the right panel shows quantitative data (mean ± s.d., n = 4 mice per group). The p- value is indicated and was determined by the unpaired, two-tailed, t-test. The lower panel is a control immunoblot detecting α-tubulin. C, As a specificity control of the monoclonal anti-MPP1 antibody, immunoblot detection of MPP1 in MPP1-transfected HEK cells was performed in comparison to mock- transfected HEK cells. The lower blot shows a loading control detecting GAPDH.

Article Snippet: Antibodies used for immunoblot detection, immunohistology and immunofluorescence The study used the following antibodies: rabbit monoclonal antiMPP1 antibody was raised against a synthetic peptide derived from the sequence of MPP1 ([EPR5865], ab108528; Abcam, Cambridge, UK); mouse monoclonal anti-MPP1 antibody was raised against an epitope within amino acids 320–376 of human MPP1 (A-7 HRP, sc-374506 HRP; Santa Cruz Biotechnology Inc., Dallas, TX, USA); mouse monoclonal anti-α-Tubulin antibody, clone DM1A (T6199; Merck KGaA, Darmstadt, Germany); rabbit polyclonal anti-GFP antibodies were raised against full length GFP protein (ab290, Abcam, Cambridge, UK); mouse monoclonal anti-GAPDH antibody (0411) was raised against recombinant human GAPDH (sc-47724, Santa Cruz Biotechnology Inc., Dallas, TX, USA); peroxidase-conjugated AffiniPure F(ab’)2 Fragment Goat anti-rabbit IgG (Fc fragment-specific produced in goat; Cat. No. 111–036-046; Jackson ImmunoResearch Europe Ltd, Ely, UK); peroxidase-conjugated AffiniPure F(ab’)2 fragment goat anti-mouse IgG (Fcγ fragment-specific; Cat. No. 115–036-071; Jackson ImmunoResearch Europe Ltd, Ely, UK); goat anti-mouse IgG (H + L) cross-adsorbed secondary antibody, Alexa FluorTM 568 (A11004; Invitrogen by ThermoFisher Scientific, Waltham, MA USA); goat anti-rabbit IgG (H + L) cross-adsorbed secondary antibody, Alexa FluorTM 488 (A11008; Invitrogen by ThermoFisher Scientific, Waltham, MA USA).

Techniques: Plasmid Preparation, Transgenic Assay, Negative Control, Positive Control, Marker, Western Blot, Two Tailed Test, Control, Transfection, Comparison

Journal: Biochemical pharmacology

Article Title: Identification of membrane palmitoylated protein 1 (MPP1) as a heart-failure-promoting protein triggered by cardiovascular risk factors and aging.

doi: 10.1016/j.bcp.2023.115789

Figure Lengend Snippet: Fig. 2. Upregulation of the MAGUK family protein, MPP1, in three different heart failure models. A,B, Probe set intensities of cardiac Mpp iso forms were determined by whole genome microarray gene expression profiling of the AAC-induced heart failure model in comparison to sham-operated con trols (A), and of Apoe−/− mice with long-term atherosclerosis-induced heart failure in comparison to age-matched non-transgenic B6 mice (B). Affymetrix IDs of probe sets detecting Mpp1, Mpp2, Mpp3, Mpp4, Mpp5, Mpp6, and Mpp7 are indicated. Data are mean values ± s.d. (four hearts per microarray chip with two microarray chips per group). Probe set intensities are taken from NCBI GEO dataset GSE25765. C, Cardiac transcript levels of Mpp isoforms in 8-month-old, male Tg-RKIP mice were determined by NGS in comparison to age- and sex- matched, non-transgenic FVB controls (NCBI GEO dataset GSE191316) (mean ± s.d., n = 3 mice per group). Statistically significant differences between transcript levels of the heart failure groups and the respective control group were determined by Tukey’s test, and are indicated for each individual MAGUK gene (A,B,C). P-values for statistically different MAGUK genes are indicated. All other MAGUK genes were not significantly different (n.s.) between the heart failure and control groups.

Article Snippet: Antibodies used for immunoblot detection, immunohistology and immunofluorescence The study used the following antibodies: rabbit monoclonal antiMPP1 antibody was raised against a synthetic peptide derived from the sequence of MPP1 ([EPR5865], ab108528; Abcam, Cambridge, UK); mouse monoclonal anti-MPP1 antibody was raised against an epitope within amino acids 320–376 of human MPP1 (A-7 HRP, sc-374506 HRP; Santa Cruz Biotechnology Inc., Dallas, TX, USA); mouse monoclonal anti-α-Tubulin antibody, clone DM1A (T6199; Merck KGaA, Darmstadt, Germany); rabbit polyclonal anti-GFP antibodies were raised against full length GFP protein (ab290, Abcam, Cambridge, UK); mouse monoclonal anti-GAPDH antibody (0411) was raised against recombinant human GAPDH (sc-47724, Santa Cruz Biotechnology Inc., Dallas, TX, USA); peroxidase-conjugated AffiniPure F(ab’)2 Fragment Goat anti-rabbit IgG (Fc fragment-specific produced in goat; Cat. No. 111–036-046; Jackson ImmunoResearch Europe Ltd, Ely, UK); peroxidase-conjugated AffiniPure F(ab’)2 fragment goat anti-mouse IgG (Fcγ fragment-specific; Cat. No. 115–036-071; Jackson ImmunoResearch Europe Ltd, Ely, UK); goat anti-mouse IgG (H + L) cross-adsorbed secondary antibody, Alexa FluorTM 568 (A11004; Invitrogen by ThermoFisher Scientific, Waltham, MA USA); goat anti-rabbit IgG (H + L) cross-adsorbed secondary antibody, Alexa FluorTM 488 (A11008; Invitrogen by ThermoFisher Scientific, Waltham, MA USA).

Techniques: Microarray, Gene Expression, Comparison, Transgenic Assay, Control

Journal: Biochemical pharmacology

Article Title: Identification of membrane palmitoylated protein 1 (MPP1) as a heart-failure-promoting protein triggered by cardiovascular risk factors and aging.

doi: 10.1016/j.bcp.2023.115789

Figure Lengend Snippet: Fig. 4. Tg-MPP1 mice develop features of heart failure with cardiac enlarge ment at an age of 8 months. A, Echo cardiographic measurement of the left ventricular ejection fraction (LVEF, %), the fractional shortening (FS, %), the left ventricular internal diameter in diastole (LVIDd), and the left ventricular internal diameter in systole (LVIDs) of 8-month- old, male Tg-MPP1 mice, and sex- and age-matched, non-transgenic B6 mice. Echocardiographic measurements were performed under anesthesia. B, Determi nation of the body weights (BW), heart weights (HW), and the heart weight to body weight ratios (HW/BW) of 8-month- old, male Tg-MPP1 mice, and of sex- and age-matched, non-transgenic B6 mice. Data (A,B) are the mean ± s.d., n = 6 mice per group. P-values were determined by the unpaired, two-tailed t-test. C, Immu nohistological detection of MPP1 on heart sections of Tg-MPP1 mice in comparison to those of non-transgenic B6 mice (n = 4 mice/group; bar: 2 mm). Sections were stained with the anti-MPP1 antibody (MPP1) and counterstained with hema toxylin (HE). The right panels show higher magnification images of representative sections from a Tg-MPP1 mouse and a non- transgenic B6 control (bar: 20 μm).

Article Snippet: Antibodies used for immunoblot detection, immunohistology and immunofluorescence The study used the following antibodies: rabbit monoclonal antiMPP1 antibody was raised against a synthetic peptide derived from the sequence of MPP1 ([EPR5865], ab108528; Abcam, Cambridge, UK); mouse monoclonal anti-MPP1 antibody was raised against an epitope within amino acids 320–376 of human MPP1 (A-7 HRP, sc-374506 HRP; Santa Cruz Biotechnology Inc., Dallas, TX, USA); mouse monoclonal anti-α-Tubulin antibody, clone DM1A (T6199; Merck KGaA, Darmstadt, Germany); rabbit polyclonal anti-GFP antibodies were raised against full length GFP protein (ab290, Abcam, Cambridge, UK); mouse monoclonal anti-GAPDH antibody (0411) was raised against recombinant human GAPDH (sc-47724, Santa Cruz Biotechnology Inc., Dallas, TX, USA); peroxidase-conjugated AffiniPure F(ab’)2 Fragment Goat anti-rabbit IgG (Fc fragment-specific produced in goat; Cat. No. 111–036-046; Jackson ImmunoResearch Europe Ltd, Ely, UK); peroxidase-conjugated AffiniPure F(ab’)2 fragment goat anti-mouse IgG (Fcγ fragment-specific; Cat. No. 115–036-071; Jackson ImmunoResearch Europe Ltd, Ely, UK); goat anti-mouse IgG (H + L) cross-adsorbed secondary antibody, Alexa FluorTM 568 (A11004; Invitrogen by ThermoFisher Scientific, Waltham, MA USA); goat anti-rabbit IgG (H + L) cross-adsorbed secondary antibody, Alexa FluorTM 488 (A11008; Invitrogen by ThermoFisher Scientific, Waltham, MA USA).

Techniques: Transgenic Assay, Two Tailed Test, Comparison, Staining, Control

Journal: Biochemical pharmacology

Article Title: Identification of membrane palmitoylated protein 1 (MPP1) as a heart-failure-promoting protein triggered by cardiovascular risk factors and aging.

doi: 10.1016/j.bcp.2023.115789

Figure Lengend Snippet: Fig. 5. Co-localization of AGTR1 with MPP1 in vivo, and increased cardiac AGTR1 protein levels in Tg- MPP1 mice. A, Immunofluorescence detection of MPP1 and AGTR1 on cardiac cryosections from Tg-CMV- AGTR1-Cerulean mice shows co-localization of AGTR1 with MPP1 on sarcolemmal membranes (yellow). MPP1 was stained with mouse monoclonal anti-MPP1 antibody (red), AGTR1-Cerulean was stained with rabbit poly clonal anti-GFP antibodies (green), and nuclei were stained with DAPI (blue). The immunofluorescence co- localization study shows cryosections from four different mice (bar: 40 μm). B, Cardiac AGTR1-specific binding sites were determined on sarcolemmal mem branes of Tg-MPP1 mice and non-transgenic B6 mice by radioligand binding with Sar1,[125I]Tyr4,Ile8-angiotensin II. Data are shown as mean values ± s.d., n = 6 mice per group. The p-value was determined by the unpaired, two- tailed t-test. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Antibodies used for immunoblot detection, immunohistology and immunofluorescence The study used the following antibodies: rabbit monoclonal antiMPP1 antibody was raised against a synthetic peptide derived from the sequence of MPP1 ([EPR5865], ab108528; Abcam, Cambridge, UK); mouse monoclonal anti-MPP1 antibody was raised against an epitope within amino acids 320–376 of human MPP1 (A-7 HRP, sc-374506 HRP; Santa Cruz Biotechnology Inc., Dallas, TX, USA); mouse monoclonal anti-α-Tubulin antibody, clone DM1A (T6199; Merck KGaA, Darmstadt, Germany); rabbit polyclonal anti-GFP antibodies were raised against full length GFP protein (ab290, Abcam, Cambridge, UK); mouse monoclonal anti-GAPDH antibody (0411) was raised against recombinant human GAPDH (sc-47724, Santa Cruz Biotechnology Inc., Dallas, TX, USA); peroxidase-conjugated AffiniPure F(ab’)2 Fragment Goat anti-rabbit IgG (Fc fragment-specific produced in goat; Cat. No. 111–036-046; Jackson ImmunoResearch Europe Ltd, Ely, UK); peroxidase-conjugated AffiniPure F(ab’)2 fragment goat anti-mouse IgG (Fcγ fragment-specific; Cat. No. 115–036-071; Jackson ImmunoResearch Europe Ltd, Ely, UK); goat anti-mouse IgG (H + L) cross-adsorbed secondary antibody, Alexa FluorTM 568 (A11004; Invitrogen by ThermoFisher Scientific, Waltham, MA USA); goat anti-rabbit IgG (H + L) cross-adsorbed secondary antibody, Alexa FluorTM 488 (A11008; Invitrogen by ThermoFisher Scientific, Waltham, MA USA).

Techniques: In Vivo, Immunofluorescence, Staining, Binding Assay, Transgenic Assay, Two Tailed Test

Journal: Biochemical pharmacology

Article Title: Identification of membrane palmitoylated protein 1 (MPP1) as a heart-failure-promoting protein triggered by cardiovascular risk factors and aging.

doi: 10.1016/j.bcp.2023.115789

Figure Lengend Snippet: Fig. 6. MPP1 increased the cellular contents of AGTR1eYFP in HEK cells. A,B, Cellular AGTR1eYFP levels were increased by co-transfection of HEK293 cells with an MPP1-encoding pcDNA3 expression plasmid (+). Control cells were transfected with the pcDNA3 plasmid without insert (-). Panel (A) shows cellular AGTR1eYFP fluorescence peak intensities at an emission wavelength of 527 nm, and panel (B) shows representative AGTR1eYFP fluorescence emis sion spectra without (grey) and with MPP1-encoding plasmid co-transfection (red). The black line shows a spectrum of control cells transfected with pcDNA3 without insert (Cont.). C,D, Co-transfection of the MPP1-encoding plasmid did not significantly alter cellular ADRB1eYFP levels. Control cells were trans fected with the pcDNA3 plasmid without insert (-). Panel (C) shows cellular ADRB1eYFP fluorescence peak intensities at an emission wavelength of 527 nm, and panel (D) shows representative fluorescence emission spectra of ADRB1eYFP-expressing cells without and with MPP1-encoding plasmid co- transfection. Data (A,C) show mean values ± s.d. (n = 8 biological replicates). P-values were determined by Tukey’s test. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Antibodies used for immunoblot detection, immunohistology and immunofluorescence The study used the following antibodies: rabbit monoclonal antiMPP1 antibody was raised against a synthetic peptide derived from the sequence of MPP1 ([EPR5865], ab108528; Abcam, Cambridge, UK); mouse monoclonal anti-MPP1 antibody was raised against an epitope within amino acids 320–376 of human MPP1 (A-7 HRP, sc-374506 HRP; Santa Cruz Biotechnology Inc., Dallas, TX, USA); mouse monoclonal anti-α-Tubulin antibody, clone DM1A (T6199; Merck KGaA, Darmstadt, Germany); rabbit polyclonal anti-GFP antibodies were raised against full length GFP protein (ab290, Abcam, Cambridge, UK); mouse monoclonal anti-GAPDH antibody (0411) was raised against recombinant human GAPDH (sc-47724, Santa Cruz Biotechnology Inc., Dallas, TX, USA); peroxidase-conjugated AffiniPure F(ab’)2 Fragment Goat anti-rabbit IgG (Fc fragment-specific produced in goat; Cat. No. 111–036-046; Jackson ImmunoResearch Europe Ltd, Ely, UK); peroxidase-conjugated AffiniPure F(ab’)2 fragment goat anti-mouse IgG (Fcγ fragment-specific; Cat. No. 115–036-071; Jackson ImmunoResearch Europe Ltd, Ely, UK); goat anti-mouse IgG (H + L) cross-adsorbed secondary antibody, Alexa FluorTM 568 (A11004; Invitrogen by ThermoFisher Scientific, Waltham, MA USA); goat anti-rabbit IgG (H + L) cross-adsorbed secondary antibody, Alexa FluorTM 488 (A11008; Invitrogen by ThermoFisher Scientific, Waltham, MA USA).

Techniques: Cotransfection, Expressing, Plasmid Preparation, Control, Transfection, Fluorescence

Journal: Biochemical pharmacology

Article Title: Identification of membrane palmitoylated protein 1 (MPP1) as a heart-failure-promoting protein triggered by cardiovascular risk factors and aging.

doi: 10.1016/j.bcp.2023.115789

Figure Lengend Snippet: Fig. 7. AGTR1-(1–319)-eYFP with deletion of the carboxyl terminal tail is also enhanced by MPP1 in HEK cells. A, Cellular fluorescence peak intensities at an emission wavelength of 527 nm were deter mined of HEK cells with expression of the full-length AGTR1-(1–359)-eYFP without (-) and with (+) co- transfection of the MPP1-encoding plasmid, and of HEK cells with expression of the truncated AGTR1- (1–319)-eYFP without (-), and with (+) co- transfection of MPP1. Data are mean values ± s.d. (n = 10 biological replicates). P-values were deter mined by Tukey’s test. B, Topological scheme of the full-length AGTR1-(1–359) protein sequence. Trun cated residues of AGTR1-(1–319) are marked in red. The AGTR1 topology was derived from Uniprot (P30556 AGTR1_Human), and the scheme was drawn with Protter, version 1.0. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Antibodies used for immunoblot detection, immunohistology and immunofluorescence The study used the following antibodies: rabbit monoclonal antiMPP1 antibody was raised against a synthetic peptide derived from the sequence of MPP1 ([EPR5865], ab108528; Abcam, Cambridge, UK); mouse monoclonal anti-MPP1 antibody was raised against an epitope within amino acids 320–376 of human MPP1 (A-7 HRP, sc-374506 HRP; Santa Cruz Biotechnology Inc., Dallas, TX, USA); mouse monoclonal anti-α-Tubulin antibody, clone DM1A (T6199; Merck KGaA, Darmstadt, Germany); rabbit polyclonal anti-GFP antibodies were raised against full length GFP protein (ab290, Abcam, Cambridge, UK); mouse monoclonal anti-GAPDH antibody (0411) was raised against recombinant human GAPDH (sc-47724, Santa Cruz Biotechnology Inc., Dallas, TX, USA); peroxidase-conjugated AffiniPure F(ab’)2 Fragment Goat anti-rabbit IgG (Fc fragment-specific produced in goat; Cat. No. 111–036-046; Jackson ImmunoResearch Europe Ltd, Ely, UK); peroxidase-conjugated AffiniPure F(ab’)2 fragment goat anti-mouse IgG (Fcγ fragment-specific; Cat. No. 115–036-071; Jackson ImmunoResearch Europe Ltd, Ely, UK); goat anti-mouse IgG (H + L) cross-adsorbed secondary antibody, Alexa FluorTM 568 (A11004; Invitrogen by ThermoFisher Scientific, Waltham, MA USA); goat anti-rabbit IgG (H + L) cross-adsorbed secondary antibody, Alexa FluorTM 488 (A11008; Invitrogen by ThermoFisher Scientific, Waltham, MA USA).

Techniques: Fluorescence, Expressing, Cotransfection, Plasmid Preparation, Sequencing, Derivative Assay

Journal: Biochemical pharmacology

Article Title: Identification of membrane palmitoylated protein 1 (MPP1) as a heart-failure-promoting protein triggered by cardiovascular risk factors and aging.

doi: 10.1016/j.bcp.2023.115789

Figure Lengend Snippet: Fig. 9. The AGTR1-enhancing effect mediated by MPP1 requires all functional domains of MPP1. A, Scheme of MPP1 functional domains, and of the two MPP1 fragments 1–267 and 268–466, which were tested. B, Cellular fluorescence peak intensities at an emission wavelength of 527 nm were determined of AGTR1eYFP-expressing HEK cells without (-) and with (+) co- transfection of MPP1-encoding plasmid, MPP1-(1–267)-encoding plasmid, MPP1-(268–466)-encoding plasmid, or MPP1-(1–267) and MPP1-(268–466)- encoding plasmids together. Data are presented as mean values ± s.d. (n = 4 biological replicates). P-values were determined by Tukey’s test.

Article Snippet: Antibodies used for immunoblot detection, immunohistology and immunofluorescence The study used the following antibodies: rabbit monoclonal antiMPP1 antibody was raised against a synthetic peptide derived from the sequence of MPP1 ([EPR5865], ab108528; Abcam, Cambridge, UK); mouse monoclonal anti-MPP1 antibody was raised against an epitope within amino acids 320–376 of human MPP1 (A-7 HRP, sc-374506 HRP; Santa Cruz Biotechnology Inc., Dallas, TX, USA); mouse monoclonal anti-α-Tubulin antibody, clone DM1A (T6199; Merck KGaA, Darmstadt, Germany); rabbit polyclonal anti-GFP antibodies were raised against full length GFP protein (ab290, Abcam, Cambridge, UK); mouse monoclonal anti-GAPDH antibody (0411) was raised against recombinant human GAPDH (sc-47724, Santa Cruz Biotechnology Inc., Dallas, TX, USA); peroxidase-conjugated AffiniPure F(ab’)2 Fragment Goat anti-rabbit IgG (Fc fragment-specific produced in goat; Cat. No. 111–036-046; Jackson ImmunoResearch Europe Ltd, Ely, UK); peroxidase-conjugated AffiniPure F(ab’)2 fragment goat anti-mouse IgG (Fcγ fragment-specific; Cat. No. 115–036-071; Jackson ImmunoResearch Europe Ltd, Ely, UK); goat anti-mouse IgG (H + L) cross-adsorbed secondary antibody, Alexa FluorTM 568 (A11004; Invitrogen by ThermoFisher Scientific, Waltham, MA USA); goat anti-rabbit IgG (H + L) cross-adsorbed secondary antibody, Alexa FluorTM 488 (A11008; Invitrogen by ThermoFisher Scientific, Waltham, MA USA).

Techniques: Functional Assay, Fluorescence, Expressing, Cotransfection, Plasmid Preparation

Journal: Biochemical pharmacology

Article Title: Identification of membrane palmitoylated protein 1 (MPP1) as a heart-failure-promoting protein triggered by cardiovascular risk factors and aging.

doi: 10.1016/j.bcp.2023.115789

Figure Lengend Snippet: Fig. 8. Deletion of a putative internal PDZ domain-binding motif in AGTR1-(1–319)-(Δ213-220)-eYFP abolishes the AGTR1-enhancing effect by MPP1 in HEK cells. A, Topological scheme of the AGTR1-(1–359) protein sequence, in which deletions made in construct AGTR1-(1–319)-(Δ213-220) are marked in red. The scheme was drawn with Protter, version 1.0. Residues 213–220 at the beginning of the third intracellular loop of AGTR1 include the sequence “Y-T-L-I”, which could be an internal PDZ domain-binding motif, which is defined by “X-S/T-X-ϕ“ where “X” can be any amino acid, and “ϕ“ is a hydrophobic amino acid. B, Cellular fluorescence peak intensities at an emis sion wavelength of 527 nm were determined of HEK cells without (-) and with stable MPP1 (+) expression, and transfection of AGTR1-(1–319)-eYFP, or AGTR1-(1–319)-(Δ213-220)-eYFP with deletion of a putative internal PDZ domain-binding motif (Δ213-220). Data are presented as mean values ± s.d. (n = 3 biological replicates). P-values were determined by Tukey’s test. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Antibodies used for immunoblot detection, immunohistology and immunofluorescence The study used the following antibodies: rabbit monoclonal antiMPP1 antibody was raised against a synthetic peptide derived from the sequence of MPP1 ([EPR5865], ab108528; Abcam, Cambridge, UK); mouse monoclonal anti-MPP1 antibody was raised against an epitope within amino acids 320–376 of human MPP1 (A-7 HRP, sc-374506 HRP; Santa Cruz Biotechnology Inc., Dallas, TX, USA); mouse monoclonal anti-α-Tubulin antibody, clone DM1A (T6199; Merck KGaA, Darmstadt, Germany); rabbit polyclonal anti-GFP antibodies were raised against full length GFP protein (ab290, Abcam, Cambridge, UK); mouse monoclonal anti-GAPDH antibody (0411) was raised against recombinant human GAPDH (sc-47724, Santa Cruz Biotechnology Inc., Dallas, TX, USA); peroxidase-conjugated AffiniPure F(ab’)2 Fragment Goat anti-rabbit IgG (Fc fragment-specific produced in goat; Cat. No. 111–036-046; Jackson ImmunoResearch Europe Ltd, Ely, UK); peroxidase-conjugated AffiniPure F(ab’)2 fragment goat anti-mouse IgG (Fcγ fragment-specific; Cat. No. 115–036-071; Jackson ImmunoResearch Europe Ltd, Ely, UK); goat anti-mouse IgG (H + L) cross-adsorbed secondary antibody, Alexa FluorTM 568 (A11004; Invitrogen by ThermoFisher Scientific, Waltham, MA USA); goat anti-rabbit IgG (H + L) cross-adsorbed secondary antibody, Alexa FluorTM 488 (A11008; Invitrogen by ThermoFisher Scientific, Waltham, MA USA).

Techniques: Binding Assay, Sequencing, Construct, Fluorescence, Expressing, Transfection

Journal: Biochemical pharmacology

Article Title: Identification of membrane palmitoylated protein 1 (MPP1) as a heart-failure-promoting protein triggered by cardiovascular risk factors and aging.

doi: 10.1016/j.bcp.2023.115789

Figure Lengend Snippet: Fig. 10. Upregulation of cardiac Mpp1 transcript levels by diabetes- induced cardiac dysfunction and by Hdac3 deficiency in rodents. A, Car diac Mpp1 transcript levels were up-regulated in rats with diabetes-induced cardiac dysfunction. Data were retrieved from the GEO profile GDS3153 (31), probe set ID 1389963_at of the Affymetrix Rat Expression 230A Array. Hearts were obtained from 12-week-old rats with four weeks of streptozotocin-induced diabetes and from control rats (mean ± s.d., n = 3 hearts per group). B, Upregulation of cardiac Mpp1 in hearts from 6-week-old mice with Hdac3- deficiency (Hdac3 KO) in heart and skeletal muscle (HDAC3fl/fl/MCK-Cre), which develop a severe hypertrophic cardiomyopathy on a high fat diet (32). Control hearts were isolated from wild-type mice (HDAC3fl/fl). Data were taken from the GEO profile GDS4886, probe set ID 106447481 of the Affymetrix Mouse Gene 1.0 ST Array (mean ± s.d., n = 4 male mice per group). P-values were determined by the unpaired, two-tailed t-test.

Article Snippet: Antibodies used for immunoblot detection, immunohistology and immunofluorescence The study used the following antibodies: rabbit monoclonal antiMPP1 antibody was raised against a synthetic peptide derived from the sequence of MPP1 ([EPR5865], ab108528; Abcam, Cambridge, UK); mouse monoclonal anti-MPP1 antibody was raised against an epitope within amino acids 320–376 of human MPP1 (A-7 HRP, sc-374506 HRP; Santa Cruz Biotechnology Inc., Dallas, TX, USA); mouse monoclonal anti-α-Tubulin antibody, clone DM1A (T6199; Merck KGaA, Darmstadt, Germany); rabbit polyclonal anti-GFP antibodies were raised against full length GFP protein (ab290, Abcam, Cambridge, UK); mouse monoclonal anti-GAPDH antibody (0411) was raised against recombinant human GAPDH (sc-47724, Santa Cruz Biotechnology Inc., Dallas, TX, USA); peroxidase-conjugated AffiniPure F(ab’)2 Fragment Goat anti-rabbit IgG (Fc fragment-specific produced in goat; Cat. No. 111–036-046; Jackson ImmunoResearch Europe Ltd, Ely, UK); peroxidase-conjugated AffiniPure F(ab’)2 fragment goat anti-mouse IgG (Fcγ fragment-specific; Cat. No. 115–036-071; Jackson ImmunoResearch Europe Ltd, Ely, UK); goat anti-mouse IgG (H + L) cross-adsorbed secondary antibody, Alexa FluorTM 568 (A11004; Invitrogen by ThermoFisher Scientific, Waltham, MA USA); goat anti-rabbit IgG (H + L) cross-adsorbed secondary antibody, Alexa FluorTM 488 (A11008; Invitrogen by ThermoFisher Scientific, Waltham, MA USA).

Techniques: Expressing, Control, Isolation, Two Tailed Test

Journal: Biochemical pharmacology

Article Title: Identification of membrane palmitoylated protein 1 (MPP1) as a heart-failure-promoting protein triggered by cardiovascular risk factors and aging.

doi: 10.1016/j.bcp.2023.115789

Figure Lengend Snippet: Fig. 11. Detection of increased MPP1 transcript levels in peripheral blood mononuclear cells of old human research participants. A-F, Transcript levels of MPP1 (A), GRK2 (B), GRK3 (C), DUSP3 (D), LRRN3 (E), and CD27 (F) in PBMC from old (age: 75–89 years, y; n = 5) human research participants were determined by whole genome microarray gene expression profiling. PBMC isolated from middle-aged research participants (age: 35–50 years, y; n = 4) served as the control group. Data are shown as mean values ± s.d. P-values were determined by the two- tailed (A,B,D,E,F), or one-tailed (C), unpaired t-test.

Article Snippet: Antibodies used for immunoblot detection, immunohistology and immunofluorescence The study used the following antibodies: rabbit monoclonal antiMPP1 antibody was raised against a synthetic peptide derived from the sequence of MPP1 ([EPR5865], ab108528; Abcam, Cambridge, UK); mouse monoclonal anti-MPP1 antibody was raised against an epitope within amino acids 320–376 of human MPP1 (A-7 HRP, sc-374506 HRP; Santa Cruz Biotechnology Inc., Dallas, TX, USA); mouse monoclonal anti-α-Tubulin antibody, clone DM1A (T6199; Merck KGaA, Darmstadt, Germany); rabbit polyclonal anti-GFP antibodies were raised against full length GFP protein (ab290, Abcam, Cambridge, UK); mouse monoclonal anti-GAPDH antibody (0411) was raised against recombinant human GAPDH (sc-47724, Santa Cruz Biotechnology Inc., Dallas, TX, USA); peroxidase-conjugated AffiniPure F(ab’)2 Fragment Goat anti-rabbit IgG (Fc fragment-specific produced in goat; Cat. No. 111–036-046; Jackson ImmunoResearch Europe Ltd, Ely, UK); peroxidase-conjugated AffiniPure F(ab’)2 fragment goat anti-mouse IgG (Fcγ fragment-specific; Cat. No. 115–036-071; Jackson ImmunoResearch Europe Ltd, Ely, UK); goat anti-mouse IgG (H + L) cross-adsorbed secondary antibody, Alexa FluorTM 568 (A11004; Invitrogen by ThermoFisher Scientific, Waltham, MA USA); goat anti-rabbit IgG (H + L) cross-adsorbed secondary antibody, Alexa FluorTM 488 (A11008; Invitrogen by ThermoFisher Scientific, Waltham, MA USA).

Techniques: Microarray, Gene Expression, Isolation, Control, Two Tailed Test, One-tailed Test

Journal: Scientific Reports

Article Title: IER5 generates a novel hypo-phosphorylated active form of HSF1 and contributes to tumorigenesis

doi: 10.1038/srep19174

Figure Lengend Snippet: ( A ) H1299 or 293T cells were transfected with control vector or an IER5 expression vector. Cells were harvested 21 hrs or 27 hrs post-transfection and microarray expression analysis was performed. The table shows the HSP family genes, among the genes induced by IER5. ( B ) H1299 cells were transfected with control, IER5-Flag or mutant IER5-Flag expression vectors (representative image of mut 1 is shown in ). Cells were harvested 27 hrs post-transfection, and mRNA expressions of the HSP family genes were analyzed by Northern blotting. ( C ) H1299 and 293T cells were transfected with control vector or IER5-Flag expression vector, and cells were harvested 24 hrs post-transfection. Expressions of the HSP family proteins were analyzed by Western blotting. ( D–F ) Control or IER5-targeting siRNAs were introduced into OE33 cells. Cells were harvested 52 hrs post-transfection. Expression of IER5 ( D,F ) and HSPA1A ( E,F ) were analyzed by quantitative RT-PCR ( D,E ) and Western blotting ( F ). (**p < 0.01). ( G ) The promoter regions of HSPA1A , HSPA1B and HSPA6 were inserted into the luciferase reporter plasmid containing a minimal promoter, and assayed 24 hrs post-transfection. Experiments were run in triplicate, and data are represented as the mean-fold activation ±SD. ( H ) Serially deleted regions of the HSPA1A promoter were analyzed as in ( G ). Numbers indicate the position of the 5′ most nucleotide relative to the transcription initiation site. A heat shock element (HSE), to which HSF1 binds, was found between positions −132 and −109.

Article Snippet: Antibodies used in this study: anti-IER5 rabbit polyclonal antibody (HPA029894), anti-Flag M2 mouse monoclonal antibody (F1804), anti-β-actin mouse monoclonal antibody (A2228) from SIGMA, anti-p53 goat polyclonal antibody (sc-6243-G), anti-p21 rabbit polyclonal antibody (C-19), anti-HSF1 rabbit polyclonal antibody (sc-9144), anti-phospho-HSF1 (Ser230) rabbit polyclonal antibody (sc-30443-R), anti-PP2A-B55 (sc-18330) from Santa Cruz Biotechnology, anti-phospho-HSF1 (Ser320) rabbit monoclonal antibody (#2446-1) and anti-phospho-HSF1 (Ser326) rabbit monoclonal antibody (#2092-1) from Epitomics, anti-phospho HSF1 (Ser121) rabbit polyclonal antibody from Assay bio Tech, anti-phospho-HSF1 (Ser303/Ser307) rabbit monoclonal antibody (ab81281) from abcam, anti-HSPB1 mouse monoclonal antibody (ADI-SPA-800), anti-DNAJB1 rabbit polyclonal antibody (ADI-SPA-400), anti-HSPA6 mouse monoclonal antibody (ADI-SPA-754), anti-HSPA1A/1B mouse monoclonal antibody (ADI-SPA-810) from Enzo Life Sciences, anti-HA mouse monoclonal antibody (12CA5) from Roche, and anti-PP2A C subunit (clone 1D6) from Merck Millipore.

Techniques: Transfection, Control, Plasmid Preparation, Expressing, Microarray, Mutagenesis, Northern Blot, Western Blot, Quantitative RT-PCR, Luciferase, Activation Assay

Journal: Scientific Reports

Article Title: IER5 generates a novel hypo-phosphorylated active form of HSF1 and contributes to tumorigenesis

doi: 10.1038/srep19174

Figure Lengend Snippet: ( A–C ) Control or HSF1-targeting siRNAs were introduced into H1299 cells. Subsequently, cells were transfected with control vector or an IER5 expression vector 24 hrs post-siRNA transfection. Cells were harvested 24 hrs post-DNA transfection. Expression of HSPA1A ( A ), HSPA6 ( B ) and HSF1 ( C ) mRNAs were analyzed by quantitative RT-PCR. ( # p < 0.0001). ( D ) 293T cells were transfected with HA-HSF1 and control vector or IER5-Flag. Cells were harvested 24 hrs post-transfection. Cell lysates were crosslinked by DSP (1 mg/ml), and immunoprecipitated using anti-HA antibody. HSP90 binding to HA-HSF1 was detected by Western blotting. ( E ) H1299 cells were transfected with control vector or IER5-Flag expression vector. Cells were harvested 25 hrs post-transfection. Whole cell lysates were crosslinked with EGS. HSF1 and IER-Flag expression was analyzed by Western blotting. ( F ) H1299 cells were transfected with control vector, IER5-Flag, mut 1-Flag expression vector. Cells were harvested 24 hrs post-transfection. Subcellular localizations of endogenous HSF1 (detected using anti-HSF1 antibody), IER5-Flag and mut 1 (detected using anti-Flag antibody) were analyzed. HSF1 and IER5 expression levels were quantitated and shown at the bottom. ( G ) HSF1 binding to Heat Shock Elements (HSEs) was analyzed by streptavidin pull-down assay. Biotinylated control or HSE-containing DNA probes were bound to streptavidin-coated beads and mixed with control or IER5-expressing cell lysates. Bead-bound HSF1 was denatured in Laemmli buffer and analyzed by Western blotting.

Article Snippet: Antibodies used in this study: anti-IER5 rabbit polyclonal antibody (HPA029894), anti-Flag M2 mouse monoclonal antibody (F1804), anti-β-actin mouse monoclonal antibody (A2228) from SIGMA, anti-p53 goat polyclonal antibody (sc-6243-G), anti-p21 rabbit polyclonal antibody (C-19), anti-HSF1 rabbit polyclonal antibody (sc-9144), anti-phospho-HSF1 (Ser230) rabbit polyclonal antibody (sc-30443-R), anti-PP2A-B55 (sc-18330) from Santa Cruz Biotechnology, anti-phospho-HSF1 (Ser320) rabbit monoclonal antibody (#2446-1) and anti-phospho-HSF1 (Ser326) rabbit monoclonal antibody (#2092-1) from Epitomics, anti-phospho HSF1 (Ser121) rabbit polyclonal antibody from Assay bio Tech, anti-phospho-HSF1 (Ser303/Ser307) rabbit monoclonal antibody (ab81281) from abcam, anti-HSPB1 mouse monoclonal antibody (ADI-SPA-800), anti-DNAJB1 rabbit polyclonal antibody (ADI-SPA-400), anti-HSPA6 mouse monoclonal antibody (ADI-SPA-754), anti-HSPA1A/1B mouse monoclonal antibody (ADI-SPA-810) from Enzo Life Sciences, anti-HA mouse monoclonal antibody (12CA5) from Roche, and anti-PP2A C subunit (clone 1D6) from Merck Millipore.

Techniques: Control, Transfection, Plasmid Preparation, Expressing, Quantitative RT-PCR, Immunoprecipitation, Binding Assay, Western Blot, Pull Down Assay

Journal: Scientific Reports

Article Title: IER5 generates a novel hypo-phosphorylated active form of HSF1 and contributes to tumorigenesis

doi: 10.1038/srep19174

Figure Lengend Snippet: ( A–E ) Whole cell extracts and immunoprecipitated samples were analyzed by Western blotting. ( A ) 293T cells were transfected with HSF1-Flag together with control vector or an IER5 expression vector. Cells were harvested 21 hrs post-transfection. ( B ) 293T cells were transfected with control vector or HSF1-Flag expression vector, together with control or IER5-targetting siRNAs. Cells were harvested 49 hrs post-transfection. ( C ) 293T cells were transfected with HSF1-Flag and control vector or IER5. Cells were harvested 27 hrs post-transfection. Cell lysates were immunoprecipitated using anti-Flag antibody, and incubated with or without CIAP for 30 min. Total HSF1 and p-Ser320 were detected. ( D ) 293T cells were transfected with control vector or HA-HSF1 expression vector. Cells were treated with or without heat shock at 42 °C for 3 hrs, and harvested 24 hrs post-DNA transfection. ( E ) 293T cells were transfected with HA-HSF1 and control or IER5-Flag expression vector. Cells were harvested 24 hrs post-DNA transfection. ( F ) HSF1 modification was analyzed by LC-MS/MS. The experiment was performed nine times, and the numbers of analysis that detected each phosphorylation are shown. Significant reductions in phosphorylation at 5 residues (S121, S307, S314, T3232 and T367) were detected. Phosphorylation sites previously reported to be involved in the repression of HSF1 activity (S121 and S307) are shown in blue. ( G ) 293T cells were transfected with the indicated plasmids and harvested 24 hrs post-transfection. HSPA1A mRNA expression was analyzed by quantitative RT-PCR and other proteins were detected by Western Blotting. (***p < 0.001, # p < 0.0001). ( H,I ) 293T cells were transfected with control vector or IER5-Flag expression vector. Cells were treated with or without heat shock at 42 °C for 3 hrs, and harvested 24 hrs post-DNA transfection. Endogenous HSF1, IER5-Flag protein expression was analyzed by Western blotting ( H ), and HSPA1A , HSPA6 and HSPA1B mRNA expression was analyzed by quantitative RT-PCR ( I ). (*p < 0.05, **p < 0.01, ***p < 0.001, # p < 0.0001).

Article Snippet: Antibodies used in this study: anti-IER5 rabbit polyclonal antibody (HPA029894), anti-Flag M2 mouse monoclonal antibody (F1804), anti-β-actin mouse monoclonal antibody (A2228) from SIGMA, anti-p53 goat polyclonal antibody (sc-6243-G), anti-p21 rabbit polyclonal antibody (C-19), anti-HSF1 rabbit polyclonal antibody (sc-9144), anti-phospho-HSF1 (Ser230) rabbit polyclonal antibody (sc-30443-R), anti-PP2A-B55 (sc-18330) from Santa Cruz Biotechnology, anti-phospho-HSF1 (Ser320) rabbit monoclonal antibody (#2446-1) and anti-phospho-HSF1 (Ser326) rabbit monoclonal antibody (#2092-1) from Epitomics, anti-phospho HSF1 (Ser121) rabbit polyclonal antibody from Assay bio Tech, anti-phospho-HSF1 (Ser303/Ser307) rabbit monoclonal antibody (ab81281) from abcam, anti-HSPB1 mouse monoclonal antibody (ADI-SPA-800), anti-DNAJB1 rabbit polyclonal antibody (ADI-SPA-400), anti-HSPA6 mouse monoclonal antibody (ADI-SPA-754), anti-HSPA1A/1B mouse monoclonal antibody (ADI-SPA-810) from Enzo Life Sciences, anti-HA mouse monoclonal antibody (12CA5) from Roche, and anti-PP2A C subunit (clone 1D6) from Merck Millipore.

Techniques: Immunoprecipitation, Western Blot, Transfection, Control, Plasmid Preparation, Expressing, Incubation, Modification, Liquid Chromatography with Mass Spectroscopy, Phospho-proteomics, Activity Assay, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: IER5 generates a novel hypo-phosphorylated active form of HSF1 and contributes to tumorigenesis

doi: 10.1038/srep19174

Figure Lengend Snippet: ( A–C ) 293T cells were transfected with HA-HSF1 together with control vector or IER5-Flag. Cells were treated with 20 mM NaF acid for 4 hrs and harvested 24 hrs post-transfection. Expression of HSPA1A and HSPA6 were analyzed by quantitative RT-PCR ( A,B ) and expression of IER5-Flag and HA-HSF1 were detected by Western Blotting ( C ). (**p < 0.01, ***p < 0.001). ( D ) 293T cells were transfected with HA-HSF1 together with control vector or IER5-Flag. Cells were treated with 500 nM okadaic acid for 8 hrs and harvested 24 hrs post-transfection. Expression of HSPA1A was analyzed by quantitative RT-PCR and expression of IER5-Flag and HA-HSF1 were detected by Western Blotting. (***p < 0.001). ( E ) 293T cells were transfected as in ( D ). Cells were treated with 250 nM okadaic acid for 4 hrs and harvested 24 hrs post-transfection. Cell lysates were immunoprecipitated using anti-HA antibody. Total HSF1 and HSF1 phosphorylated at Ser121 or p-Ser303, 307 were detected by Western Blotting. ( F–H ) Control or PPP2CA (PP2A catalytic subunit)-targeting siRNAs were introduced into 293T cells. Subsequently, cells were transfected with HA-HSF1 and control vector or IER5-Flag expression vector 72 hrs post-siRNA transfection. Cells were harvested 24 hrs post-DNA transfection. IER5, HSF1 and PPP2CA protein levels were analyzed by Western blotting, and HSPA1A ( G ) and HSPA6 ( H ) mRNAs were analyzed by quantitative RT-PCR. (**p < 0.01). ( I,J ) 293T cells were transfected with HA-HSF1 together with control vector, IER5-Flag or IER5-Flag mut 1-Flag, and harvested 24 hrs post-transfection. IER5-Flag ( I ) or HA-HSF1 ( J ) was immunoprecipitated using anti-Flag or anti-HA antibodies. Association of HSF1, PP2A catalytic subunit PPP2CA and PP2A regulatory subunit B55 with IER5 was analyzed by Western blotting in ( I ), and association of IER5, PPP2CA and B55 with HSF1 was analyzed in ( J ). ( K ) Endogenous IER5 in OE33 cells was immunoprecipitated using anti-IER5 antibody. Association of HSF1 and PPP2CA with IER5 was analyzed. ( L ) PP2A phosphatase activities were analyzed using the indicated amounts of cell lysates. 293T cells were treated with or without Okadaic acid (500 nM) for 8 hrs. Control or IER5-Flag expression vector were transfected and harvested 24 hrs post-transfection.

Article Snippet: Antibodies used in this study: anti-IER5 rabbit polyclonal antibody (HPA029894), anti-Flag M2 mouse monoclonal antibody (F1804), anti-β-actin mouse monoclonal antibody (A2228) from SIGMA, anti-p53 goat polyclonal antibody (sc-6243-G), anti-p21 rabbit polyclonal antibody (C-19), anti-HSF1 rabbit polyclonal antibody (sc-9144), anti-phospho-HSF1 (Ser230) rabbit polyclonal antibody (sc-30443-R), anti-PP2A-B55 (sc-18330) from Santa Cruz Biotechnology, anti-phospho-HSF1 (Ser320) rabbit monoclonal antibody (#2446-1) and anti-phospho-HSF1 (Ser326) rabbit monoclonal antibody (#2092-1) from Epitomics, anti-phospho HSF1 (Ser121) rabbit polyclonal antibody from Assay bio Tech, anti-phospho-HSF1 (Ser303/Ser307) rabbit monoclonal antibody (ab81281) from abcam, anti-HSPB1 mouse monoclonal antibody (ADI-SPA-800), anti-DNAJB1 rabbit polyclonal antibody (ADI-SPA-400), anti-HSPA6 mouse monoclonal antibody (ADI-SPA-754), anti-HSPA1A/1B mouse monoclonal antibody (ADI-SPA-810) from Enzo Life Sciences, anti-HA mouse monoclonal antibody (12CA5) from Roche, and anti-PP2A C subunit (clone 1D6) from Merck Millipore.

Techniques: Transfection, Control, Plasmid Preparation, Expressing, Quantitative RT-PCR, Western Blot, Immunoprecipitation

Journal: Scientific Reports

Article Title: IER5 generates a novel hypo-phosphorylated active form of HSF1 and contributes to tumorigenesis

doi: 10.1038/srep19174

Figure Lengend Snippet: ( A–D ) OE33 cells (2 × 10 3 cells) were plated in adherent ( A ) or suspension ( B ) 96 well culture plates, and control, IER5-targeting or HSF1-targeting siRNAs were introduced. Cell growth assays were performed on the indicated days. Relative cell numbers were analyzed with CellTiter-Glo reagents from four wells and the mean cell numbers ±SD are shown. IER5 and HSF1 mRNA expression was analyzed by quantitative RT-PCR 48 hrs post-transfection ( C,D ). (**p < 0.01, # p < 0.0001). ( E ) OE33 cells were stably transfected with caHSF1, and the indicated siRNAs were introduced. Cell lysates were prepared 48 hrs post-transfection, and expression of caHSF1-Flag, IER5, HSPA1A/1B were analyzed by Western blotting. ( F ) Cell growth assays were performed as in ( B ) using OE33 cells expressing caHSF1. (**p < 0.01). ( G,H ) Expression of IER5 ( G ) and HSPA6 ( H ) and prognosis in cancer patients. Disease-specific survival of patients with bladder cancer (Transitional cell carcinoma, dataset GSE13507) was analyzed using the PrognoScan database. ( I–K ) Expression of IER5 ( I ), HSPA6 ( J ) and HSPA1A ( K ) mRNA in cells treated with Adriamycin. Cell lines carrying wild-type p53 were treated with Adriamycin (1 μM) for 24 hrs (MRC5, MCF7, U2OS) or 19 hrs (A549). (*p < 0.05, **p < 0.01). ( L ) Expression of IER5, p53 and HSPA1A protein level in U2OS cells. Control, p53 and IER5 targeting siRNAs were introduced. Cells were treated with Adriamycin (1 μM) for 24 hrs and harvested 48 hrs post siRNA-transfection. ( M ) IER5 is transiently induced downstream of p53 and activates HSF1 in stressed cells, while IER5 is overexpressed and constitutively activates HSF1 in cancer cells.

Article Snippet: Antibodies used in this study: anti-IER5 rabbit polyclonal antibody (HPA029894), anti-Flag M2 mouse monoclonal antibody (F1804), anti-β-actin mouse monoclonal antibody (A2228) from SIGMA, anti-p53 goat polyclonal antibody (sc-6243-G), anti-p21 rabbit polyclonal antibody (C-19), anti-HSF1 rabbit polyclonal antibody (sc-9144), anti-phospho-HSF1 (Ser230) rabbit polyclonal antibody (sc-30443-R), anti-PP2A-B55 (sc-18330) from Santa Cruz Biotechnology, anti-phospho-HSF1 (Ser320) rabbit monoclonal antibody (#2446-1) and anti-phospho-HSF1 (Ser326) rabbit monoclonal antibody (#2092-1) from Epitomics, anti-phospho HSF1 (Ser121) rabbit polyclonal antibody from Assay bio Tech, anti-phospho-HSF1 (Ser303/Ser307) rabbit monoclonal antibody (ab81281) from abcam, anti-HSPB1 mouse monoclonal antibody (ADI-SPA-800), anti-DNAJB1 rabbit polyclonal antibody (ADI-SPA-400), anti-HSPA6 mouse monoclonal antibody (ADI-SPA-754), anti-HSPA1A/1B mouse monoclonal antibody (ADI-SPA-810) from Enzo Life Sciences, anti-HA mouse monoclonal antibody (12CA5) from Roche, and anti-PP2A C subunit (clone 1D6) from Merck Millipore.

Techniques: Suspension, Control, Expressing, Quantitative RT-PCR, Transfection, Stable Transfection, Western Blot

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Additive Effect of CD73 Inhibitor in Colorectal Cancer Treatment With CDK4/6 Inhibitor Through Regulation of PD-L1

doi: 10.1016/j.jcmgh.2022.07.005

Figure Lengend Snippet: Effects of extracellular adenosine on CCND1 levels and PD-L1 expression in human macrophages. ( A ) Experimental scheme for human macrophage differentiation from PB and adenosine treatment ( upper panel ). Flow cytometric analysis for 56 immune-related molecules (n = 7). Relative amounts of immune-related molecules on macrophages treated with or without 200 μM adenosine for 48 hours, as analyzed using shown through flow cytometry. ( B ) PD-L1 expression on CD68 + human macrophages after treatment with 200 μM adenosine for 48 hours. Mean fluorescence intensity (MFI) of PD-L1 relative to an isotype control (n = 11). ( C ) Relative amount of PD-L1 protein on macrophages treated with or without 200 μM adenosine for 48 h (n = 18) ( D ) Representative images of 3D-cultured macrophages treated with 200 μM ATP or 200 μM adenosine for 48 hours; the results are from 4 independent experiments. Scale bars have been presented. Pooled data indicating ATP- or adenosine-induced sphere-forming capability in macrophages, as measured using ( E ) sphere number and ( F ) sphere diameter (the longest sphere diameters). Experiments were performed in triplicate. ( G ) Dose-dependent increase in PD-L1 levels following ATP and adenosine treatment in the 3D-culture system (n = 5). Data have been presented as mean ± standard error of the mean. ns, not significant. ∗ P < .05, ∗∗ P < .01, ∗∗∗ P < .001, and ++ P < .01 (Student t test).

Article Snippet: The membrane were blocked with 5% skim milk and probed with a rabbit anti-PD-L1 antibody (1:1,000; Cell Signaling Technology, Danvers, MA), rabbit anti-CDK4 antibody (1:500; Cell Signaling Technology), rabbit anti-SPOP antibody (1:1,000; Proteintech, Tokyo, Japan), anti-cyclin D1 antibody (1:1,000, Cell Signaling Technology), or mouse anti-β-actin antibody (1:5,000, Cell Signaling Technology) at 4 °C overnight.

Techniques: Expressing, Flow Cytometry, Fluorescence, Control, Cell Culture

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Additive Effect of CD73 Inhibitor in Colorectal Cancer Treatment With CDK4/6 Inhibitor Through Regulation of PD-L1

doi: 10.1016/j.jcmgh.2022.07.005

Figure Lengend Snippet: Adenosine-induced decrease in CCND1 levels drives PD-L1 protein upregulation. ( A ) Comparative RNA-seq analysis of untreated and adenosine (200 μM)-treated human macrophages. In the network, each node represents a GO biological pathway, and the edges indicate the relationships among biological pathways, based on kappa values. ( B ) Genes most significantly influenced by adenosine treatment: cell cycle-related (50 genes) and DNA damage repair-related (38 genes). Cyclin D1 ( CCND1 ) was selectively decreased upon adenosine treatment, at the mRNA (n = 4) ( C ) and protein levels (n = 7) ( D, E ). ( F ) Representative histogram of 5 independent experiments and ( G ) quantitative measurements of PD-L1 expression following treatment with anti- CCND1 shRNA or negative control shRNA in the presence or absence of adenosine (200 μM) treatment, as analyzed using fluorescence-activated cell sorting analysis. Data have been presented as mean ± standard error of the mean. ns, not significant. ∗∗∗ P < .001 (Student t test).

Article Snippet: The membrane were blocked with 5% skim milk and probed with a rabbit anti-PD-L1 antibody (1:1,000; Cell Signaling Technology, Danvers, MA), rabbit anti-CDK4 antibody (1:500; Cell Signaling Technology), rabbit anti-SPOP antibody (1:1,000; Proteintech, Tokyo, Japan), anti-cyclin D1 antibody (1:1,000, Cell Signaling Technology), or mouse anti-β-actin antibody (1:5,000, Cell Signaling Technology) at 4 °C overnight.

Techniques: RNA Sequencing, Expressing, shRNA, Negative Control, Fluorescence, FACS

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Additive Effect of CD73 Inhibitor in Colorectal Cancer Treatment With CDK4/6 Inhibitor Through Regulation of PD-L1

doi: 10.1016/j.jcmgh.2022.07.005

Figure Lengend Snippet: DEGs of CCND1 -related pathway from extracellular adenosine treated macrophages. ( A ) Comparative RNA-seq analysis of untreated and adenosine (200 μM)-treated human macrophages. Genes most significantly influenced by adenosine treatment: CCND1-related (54 genes). ( B ) Results of the GO enrichment analysis of total DEGs from extracellular adenosine-treated human macrophages compared with untreated macrophages. Data have been presented as mean ± standard error of the mean. ∗∗∗ P < .001 (Student t test).

Article Snippet: The membrane were blocked with 5% skim milk and probed with a rabbit anti-PD-L1 antibody (1:1,000; Cell Signaling Technology, Danvers, MA), rabbit anti-CDK4 antibody (1:500; Cell Signaling Technology), rabbit anti-SPOP antibody (1:1,000; Proteintech, Tokyo, Japan), anti-cyclin D1 antibody (1:1,000, Cell Signaling Technology), or mouse anti-β-actin antibody (1:5,000, Cell Signaling Technology) at 4 °C overnight.

Techniques: RNA Sequencing

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Additive Effect of CD73 Inhibitor in Colorectal Cancer Treatment With CDK4/6 Inhibitor Through Regulation of PD-L1

doi: 10.1016/j.jcmgh.2022.07.005

Figure Lengend Snippet: Negative correlation between ecto-enzymes and CCND1 expression in CRC. ( A ) IHC staining of human normal colon (n = 40) and colon adenocarcinoma (n = 64) specimens in a tissue microarray for CCND1 and CD163 (a macrophage marker). ( B ) Relative intensities of CCND1 staining in the lamina propria of a normal and cancerous colon. ∗∗ P < .01 (unpaired t test). ( C ) Correlation analysis of NT5E and CCND1 expression in the TCGA colorectal adenocarcinoma dataset. ∗∗∗ P < .001 (Student t test).

Article Snippet: The membrane were blocked with 5% skim milk and probed with a rabbit anti-PD-L1 antibody (1:1,000; Cell Signaling Technology, Danvers, MA), rabbit anti-CDK4 antibody (1:500; Cell Signaling Technology), rabbit anti-SPOP antibody (1:1,000; Proteintech, Tokyo, Japan), anti-cyclin D1 antibody (1:1,000, Cell Signaling Technology), or mouse anti-β-actin antibody (1:5,000, Cell Signaling Technology) at 4 °C overnight.

Techniques: Expressing, Immunohistochemistry, Microarray, Marker, Staining

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Additive Effect of CD73 Inhibitor in Colorectal Cancer Treatment With CDK4/6 Inhibitor Through Regulation of PD-L1

doi: 10.1016/j.jcmgh.2022.07.005

Figure Lengend Snippet: Network analysis of DEGs from Ccnd1 high and Ccnd1 low intra-tumoral myeloid cells. ( A ) Distinct transcriptional profiles of Ccnd1 high and Ccnd1 low intra-tumoral myeloid cells obtained from the CT26 tumor cell-bearing mouse model. Uniform manifold approximation and projection (UMAP) plot for myeloid cell subclusters. ( B ) Proportion of myeloid cluster 13 between the control and AB680-treated tumors. ( C ) UMAP plot for the expression of Ccnd1 and Entpd1 in myeloid cells. ( D ) Heat map representing cluster-specific gene expressions. ( E ) Volcano blot for Ccnd1 low intra-tumoral myeloid cells, compared to Ccnd1 high intra-tumoral myeloid cells. ( F ) UMAP plot for the expression of inflammatory response- and leukocyte migration-related genes. ( G ) Comparative scRNA-seq analysis of Ccnd1 low intratumoral myeloid cells, as compared with Ccnd1 high intratumoral myeloid cells. In the functional network, each node represents a GO biological pathway, and the edges indicate the relationships among biological pathways, based on kappa values.

Article Snippet: The membrane were blocked with 5% skim milk and probed with a rabbit anti-PD-L1 antibody (1:1,000; Cell Signaling Technology, Danvers, MA), rabbit anti-CDK4 antibody (1:500; Cell Signaling Technology), rabbit anti-SPOP antibody (1:1,000; Proteintech, Tokyo, Japan), anti-cyclin D1 antibody (1:1,000, Cell Signaling Technology), or mouse anti-β-actin antibody (1:5,000, Cell Signaling Technology) at 4 °C overnight.

Techniques: Control, Expressing, Migration, Functional Assay

Journal: PLOS ONE

Article Title: Investigation of the anti-tumor mechanism of tirabrutinib, a highly selective Bruton’s tyrosine kinase inhibitor, by phosphoproteomics and transcriptomics

doi: 10.1371/journal.pone.0282166

Figure Lengend Snippet: Microarray analysis was conducted in the TMD8 xenograft model after 21 days of tirabrutinib administration. (A) Volcano plot for differential gene expression between the vehicle and tirabrutinib 10 mg/kg group with Welch’s t test. (B) Heat map of top altered gene group including 59 upregulated probes and 87 downregulated probes (fold change, >4 or <0.25; P < 0.00001) in the vehicle, tirabrutinib 3 mg/kg, and 10 mg/kg groups. (C) Gene set enrichment analysis (GSEA) plots involved in IRF4, MYC, and mTORC1 signaling in the tirabrutinib 10 mg/kg group versus vehicle group.

Article Snippet: Rabbit monoclonal anti- phosphorylated BTK (Tyr-223) antibody (p-BTK; Novus Biologicals, #NB100-79907), rabbit monoclonal BTK antibody (Cell Signaling Technology, Inc., #3533), rabbit polyclonal phosphorylated AKT (Ser-473) antibody (p-AKT; Cell Signaling Technology, Inc., #9271), rabbit monoclonal AKT (pan) (C67E7) antibody (Cell Signaling Technology, Inc., #4691), rabbit polyclonal phosphorylated p44/42 MAPK (ERK1/2) (Thr-202/Tyr-204) antibody (p-ERK1/2; Cell Signaling Technology, Inc., #9101), rabbit monoclonal p44/42 MAPK (ERK1/2) (137F5) antibody (Cell Signaling Technology, Inc., #4695), rabbit monoclonal phosphorylated PLCγ2 (Tyr-759) (E9E9Y) antibody (p-PLCγ2; Cell Signaling Technology, Inc., #50535), rabbit monoclonal PLCγ2 (E5U4T) antibody (Cell Signaling Technology, Inc., #55512), mouse monoclonal phosphorylated IκBα (Ser-32/36) (5A5) antibody (p-IκBα; Cell Signaling Technology, Inc., #9246), rabbit monoclonal IκBα (44D4) antibody (Cell Signaling Technology, Inc., #4812), rabbit polyclonal phosphorylated PKCβ (phospho-Thr-641) antibody (p-PKCβ; Signalway Antibody LLC, #11172), rabbit monoclonal IRF4 (D9P5H) antibody (Cell Signaling Technology, Inc., #15106), rabbit monoclonal BCL6 (D65C10) antibody (Cell Signaling Technology, Inc., #5650), rabbit monoclonal c-MYC (D84C12) antibody (Cell Signaling Technology, Inc., #5605), and rabbit monoclonal GAPDH (14C10) antibody (Cell Signaling Technology, Inc., #2118) were used.

Techniques: Microarray, Gene Expression

Journal: Cancer immunology research

Article Title: Mammary tumor cells with high metastatic potential are hypersensitive to macrophage-derived HGF

doi: 10.1158/2326-6066.CIR-19-0234

Figure Lengend Snippet: (A) Genes encoding trans-membrane receptors in microarray data sets that were significantly upregulated (logFC >1, dotted red line, P<0.01) in HML2 compared with parental cells. The scale is exponential. (B) A Kaplan-Meier plot of metastasis-free survival of grade III basal type breast cancer patients with high and low expression of MET. In a database established using multiple microarray data sets downloaded from GEO, 146 patient data fitting to the selected parameters (Survival: DMFS, Intrinsic subtype: basal, and Grade: 3) were analyzed using KM Plotter. (C) Relative Met mRNA expression in parental and HML2 cells (n=3). *P<0.05 versus parental, Student’s t test. (D) Representative images of western blots and (E) total and phosphorylated MET and GAB1 relative to β-actin in parental (Prnt) and HML2 cells stimulated with HGF (n=5) at the indicated time in hours (hr) after HGF stimulation. *P<0.05 versus time 0; #P<0.05 versus parental, Student’s t test. Results are mean±SEM.

Article Snippet: The following antibodies from Cell Signaling Technology were used: Phospho-Met (#3077P), Met (#4560S), Phospho-Gab1 (#3234T), Gab1 (#3232T), β-actin (#3700S), anti-Rabbit IgG (#7074P2), and anti-mouse IgG (#7076P2).

Techniques: Microarray, Expressing, Western Blot

Journal: Cancer immunology research

Article Title: Mammary tumor cells with high metastatic potential are hypersensitive to macrophage-derived HGF

doi: 10.1158/2326-6066.CIR-19-0234

Figure Lengend Snippet: (A) Phospho-GAB1 relative to β-actin in parental and HML2 cells stimulated with normal medium or BMM-conditioned medium (BMM-CM) for 4 hours with MET inhibitor crizotinib (Crz) or vehicle (n=5). *P<0.05 versus parental; §P<0.05 versus normal; #P<0.01 versus vehicle, Student’s t test. (B) Average number of invading cells in E0771-HML2 spheroids cultured with normal medium or BMM-CM for 48 hours in the presence of Crizotinib, vehicle, blocking antibody to MET (αMet), or control IgG (n=9). *P<0.01 versus normal; #P<0.01 versus vehicle/IgG controls, Student’s t test. (C) Number of extravasated E0771-HML2 cells in vitro. Cancer cells were cultured for 36 hours in the presence or absence of BMMs with or without HGF and crizotinib (n=6). *P<0.01 versus PBS:BMM–; #P<0.01 versus HGF, Student’s t test. (D) Relative Hgf mRNA expression in BMMs, E0771-HML2, and 3B11 endothelial cells (n=6). *P<0.01 versus BMMs, Student’s t test. (E,F) Expression of HGF and markers for macrophages (F4/80), neutrophils (Ly6G), T cells (CD3), or B cells (B220) in the lungs of animals with metastatic E0771 tumors (T), and macrophages in normal adjacent tissue (20x magnification). Dotted line indicates a border of metastatic foci (n=3). Scale bar: 100 μm; Square indicates enlarged area at 60x magnification.

Article Snippet: The following antibodies from Cell Signaling Technology were used: Phospho-Met (#3077P), Met (#4560S), Phospho-Gab1 (#3234T), Gab1 (#3232T), β-actin (#3700S), anti-Rabbit IgG (#7074P2), and anti-mouse IgG (#7076P2).

Techniques: Cell Culture, Blocking Assay, In Vitro, Expressing

Journal: Cancer cell

Article Title: Tinagl1 Suppresses Triple-Negative Breast Cancer Progression and Metastasis by Simultaneously Inhibiting Integrin/FAK and EGFR Signaling.

doi: 10.1016/j.ccell.2018.11.016

Figure Lengend Snippet: Figure 4. Identification of Tinagl1-Interacting Proteins (A–C) LM2 cells expressing the C-terminal HA-tagged Tinagl1 (Tinagl1-HA) were lysed and immunoprecipitated (IP) with immunoglobulin G (IgG) (control) or anti- HA antibody. The IP samples were subjected to silver staining and WB (A) before mass spectrometry analysis. Tinagl1-interacting partners were clustered with KEGG pathway analysis, and the three top pathways are shown in (B). The members of the top three pathways have overlaps. The EGFR and integrin b1 subunits are the core members of each pathways (C). (D and E) LM2 cells stably expressing Tinagl1-HA were lysed and IP with IgG or anti-HA antibodies. The IP samples were subjected to WB analysis with the indicated antibodies to detect the interaction with EGFR and the integrin b1 subunit (D), and with integrins av, or a5 subunits (E). See also Figure S4.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Tinagl1, in 1:1000 (WB), 2 mg (IP), Rabbit ProteinTech Cat#12077-1-AP; RRID: AB_2058942 Tinagl1, in 1:100 (IHC), Rabbit Sigma-Aldrich Cat#HPA048695; RRID: AB_2680497 b-actin, in 1:10,000 (WB), mouse Abcam Cat#ab6276; RRID: AB_2223210 EGFR, in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#4267; RRID: AB_2246311 p-EGFR (Try1068), in 1:1000 (WB), 1:100 (IHC), Rabbit Cell Signaling Technology Cat#3777; RRID: AB_2096270 FAK, in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#3285; RRID: AB_10694068 p-FAK (Try397), in 1:1000 (WB), 1:100 (IHC), Rabbit Cell Signaling Technology Cat#8556; RRID: AB_10891442 p-FAK (Try925), in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#3284; RRID: AB_2253227 AKT, in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#4691; RRID: AB_915783 p-ATK (S473), in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#4060; RRID: AB_2315049 ERK1/2, in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#4695; RRID: AB_390779 p-ERK1/2 (Thr202/Tyr204), in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#4370; RRID: AB_2315112 Integrin b1 subunit, in 1:1000 (WB), 2 mg (IP), Rabbit Cell Signaling Technology Cat#34971 Integrin a5 subunit, in 1:1000 (WB), 2 mg (IP), Rabbit Cell Signaling Technology Cat#4705; RRID: AB_10827978 Integrin av subunit, in 1:1000 (WB), 2 mg (IP), Rabbit Abcam Cat#ab179475; RRID: AB_2716738 Integrin a3 subunit, in 1:1000 (WB), Rabbit Abcam Cat#ab190731 Integrin a4 subunit, in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#8440P Integrin aM subunit, in 1:1000 (WB), Rabbit Abcam Cat#ab8878; RRID: AB_306831 HA, in 2 mg (IP), Mouse Santa Cruz Biotechnology Cat#sc-7392; RRID: AB_627809 HA, in 1:1000 (WB), Rat Roche, 11867423001 Cat#11867423001; RRID: AB_10094468 MYC, in 1:1000 (WB), 2 mg (IP), Mouse Santa Cruz Biotechnology Cat#sc-40; RRID: AB_627268 FLAG, in 1:1000 (WB), 2 mg (IP), Mouse Sigma-Aldrich Cat#F7425; RRID: AB_439687 His, in 1:1000 (WB), Mouse Sigma-Aldrich Cat#H1029; RRID: AB_260015 GFP, in 1:1000 (WB), Mouse Santa Cruz Biotechnology Cat#sc-9996; RRID: AB_627695 Fibronectin, in 1:1000 (WB), Rabbit ProteinTech Cat#15613-1-AP; RRID: AB_2105691 EGF, in 1:1000 (WB), Mouse Santa Cruz Biotechnology Cat#sc-275; RRID: AB_631417 (Continued on next page) e1 Cancer Cell 35, 1–17.e1–e7, January 14, 2019

Techniques: Expressing, Immunoprecipitation, Control, Silver Staining, Mass Spectrometry, Stable Transfection

Journal: Cancer cell

Article Title: Tinagl1 Suppresses Triple-Negative Breast Cancer Progression and Metastasis by Simultaneously Inhibiting Integrin/FAK and EGFR Signaling.

doi: 10.1016/j.ccell.2018.11.016

Figure Lengend Snippet: Figure 5. Tinagl1 Inhibits integrin/FAK and EGFR Signaling Pathways (A) Gene set enrichment analysis of lung metastatic nodules formed by LM2 cells stably expressing the vector control or Tinagl1 were dissected and digested. n = 3 per group. (B) Heatmap representation of microarray data displaying the expression of EGFR or integrin/FAK regulated genes in the control versus Tinagl1-expressing LM2 cells. (C) Heatmap representation of microarray data displaying the expression of genes compensated by integrin/FAK (left) or EGFR (right) in control versus Tinagl1- expressing LM2 cells.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Tinagl1, in 1:1000 (WB), 2 mg (IP), Rabbit ProteinTech Cat#12077-1-AP; RRID: AB_2058942 Tinagl1, in 1:100 (IHC), Rabbit Sigma-Aldrich Cat#HPA048695; RRID: AB_2680497 b-actin, in 1:10,000 (WB), mouse Abcam Cat#ab6276; RRID: AB_2223210 EGFR, in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#4267; RRID: AB_2246311 p-EGFR (Try1068), in 1:1000 (WB), 1:100 (IHC), Rabbit Cell Signaling Technology Cat#3777; RRID: AB_2096270 FAK, in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#3285; RRID: AB_10694068 p-FAK (Try397), in 1:1000 (WB), 1:100 (IHC), Rabbit Cell Signaling Technology Cat#8556; RRID: AB_10891442 p-FAK (Try925), in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#3284; RRID: AB_2253227 AKT, in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#4691; RRID: AB_915783 p-ATK (S473), in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#4060; RRID: AB_2315049 ERK1/2, in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#4695; RRID: AB_390779 p-ERK1/2 (Thr202/Tyr204), in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#4370; RRID: AB_2315112 Integrin b1 subunit, in 1:1000 (WB), 2 mg (IP), Rabbit Cell Signaling Technology Cat#34971 Integrin a5 subunit, in 1:1000 (WB), 2 mg (IP), Rabbit Cell Signaling Technology Cat#4705; RRID: AB_10827978 Integrin av subunit, in 1:1000 (WB), 2 mg (IP), Rabbit Abcam Cat#ab179475; RRID: AB_2716738 Integrin a3 subunit, in 1:1000 (WB), Rabbit Abcam Cat#ab190731 Integrin a4 subunit, in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#8440P Integrin aM subunit, in 1:1000 (WB), Rabbit Abcam Cat#ab8878; RRID: AB_306831 HA, in 2 mg (IP), Mouse Santa Cruz Biotechnology Cat#sc-7392; RRID: AB_627809 HA, in 1:1000 (WB), Rat Roche, 11867423001 Cat#11867423001; RRID: AB_10094468 MYC, in 1:1000 (WB), 2 mg (IP), Mouse Santa Cruz Biotechnology Cat#sc-40; RRID: AB_627268 FLAG, in 1:1000 (WB), 2 mg (IP), Mouse Sigma-Aldrich Cat#F7425; RRID: AB_439687 His, in 1:1000 (WB), Mouse Sigma-Aldrich Cat#H1029; RRID: AB_260015 GFP, in 1:1000 (WB), Mouse Santa Cruz Biotechnology Cat#sc-9996; RRID: AB_627695 Fibronectin, in 1:1000 (WB), Rabbit ProteinTech Cat#15613-1-AP; RRID: AB_2105691 EGF, in 1:1000 (WB), Mouse Santa Cruz Biotechnology Cat#sc-275; RRID: AB_631417 (Continued on next page) e1 Cancer Cell 35, 1–17.e1–e7, January 14, 2019

Techniques: Protein-Protein interactions, Stable Transfection, Expressing, Plasmid Preparation, Control, Microarray

Journal: Cancer cell

Article Title: Tinagl1 Suppresses Triple-Negative Breast Cancer Progression and Metastasis by Simultaneously Inhibiting Integrin/FAK and EGFR Signaling.

doi: 10.1016/j.ccell.2018.11.016

Figure Lengend Snippet: Figure 6. Tinagl1 Inhibits EGFR Dimerization and Blocks the Interaction between the Integrin b1 Subunit and FN (A) LM2 cells were transfected with plasmids to overexpress GFP-EGFR and EGFR-Myc. 48 hr after transfection, the cells were treated with or without 1 mg/mL of r-Tinagl1 for 1 hr, followed by 10 min of 1 ng/mL EGF treatment. The cells were then collected and immunoprecipitated with either IgG or anti-Myc antibody. IP samples were subjected to WB analysis (top), and the amount of EGFR-GFP that interacts with EGFR-Myc was quantified and normalized to the PBS treatment group (bottom). (B) LM2 cells stably expressing EGFR-Myc were pre-treated with PBS or 1 mg/mL of r-Tinagl1 for 1 hr and then treated with 1 ng/mL of EGF for another 10 min. Next, the cells were collected and the dimers were crosslinked with disuccinimidyl suberate (DSS) treatment, followed by WB analysis (top) and quantification of the ratio of dimerized EGFR in each treatment group (bottom). (C) HEK293T cells overexpressing the integrin b1 subunit were lysed. 20 mg of FN was added into the lysate, and the lysate was divided into eight groups. PBS or the indicated amount of proteins were added into each group followed by IP with IgG or anti-b1 antibody. The IP samples were then subjected to WB analysis. (D) HEK293T cells overexpressing both integrin b1 subunit and Tinagl1-HA were lysed and divided into eight groups. PBS or the indicated amount of proteins were added into the lysate, followed by IP and WB analysis.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Tinagl1, in 1:1000 (WB), 2 mg (IP), Rabbit ProteinTech Cat#12077-1-AP; RRID: AB_2058942 Tinagl1, in 1:100 (IHC), Rabbit Sigma-Aldrich Cat#HPA048695; RRID: AB_2680497 b-actin, in 1:10,000 (WB), mouse Abcam Cat#ab6276; RRID: AB_2223210 EGFR, in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#4267; RRID: AB_2246311 p-EGFR (Try1068), in 1:1000 (WB), 1:100 (IHC), Rabbit Cell Signaling Technology Cat#3777; RRID: AB_2096270 FAK, in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#3285; RRID: AB_10694068 p-FAK (Try397), in 1:1000 (WB), 1:100 (IHC), Rabbit Cell Signaling Technology Cat#8556; RRID: AB_10891442 p-FAK (Try925), in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#3284; RRID: AB_2253227 AKT, in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#4691; RRID: AB_915783 p-ATK (S473), in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#4060; RRID: AB_2315049 ERK1/2, in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#4695; RRID: AB_390779 p-ERK1/2 (Thr202/Tyr204), in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#4370; RRID: AB_2315112 Integrin b1 subunit, in 1:1000 (WB), 2 mg (IP), Rabbit Cell Signaling Technology Cat#34971 Integrin a5 subunit, in 1:1000 (WB), 2 mg (IP), Rabbit Cell Signaling Technology Cat#4705; RRID: AB_10827978 Integrin av subunit, in 1:1000 (WB), 2 mg (IP), Rabbit Abcam Cat#ab179475; RRID: AB_2716738 Integrin a3 subunit, in 1:1000 (WB), Rabbit Abcam Cat#ab190731 Integrin a4 subunit, in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#8440P Integrin aM subunit, in 1:1000 (WB), Rabbit Abcam Cat#ab8878; RRID: AB_306831 HA, in 2 mg (IP), Mouse Santa Cruz Biotechnology Cat#sc-7392; RRID: AB_627809 HA, in 1:1000 (WB), Rat Roche, 11867423001 Cat#11867423001; RRID: AB_10094468 MYC, in 1:1000 (WB), 2 mg (IP), Mouse Santa Cruz Biotechnology Cat#sc-40; RRID: AB_627268 FLAG, in 1:1000 (WB), 2 mg (IP), Mouse Sigma-Aldrich Cat#F7425; RRID: AB_439687 His, in 1:1000 (WB), Mouse Sigma-Aldrich Cat#H1029; RRID: AB_260015 GFP, in 1:1000 (WB), Mouse Santa Cruz Biotechnology Cat#sc-9996; RRID: AB_627695 Fibronectin, in 1:1000 (WB), Rabbit ProteinTech Cat#15613-1-AP; RRID: AB_2105691 EGF, in 1:1000 (WB), Mouse Santa Cruz Biotechnology Cat#sc-275; RRID: AB_631417 (Continued on next page) e1 Cancer Cell 35, 1–17.e1–e7, January 14, 2019

Techniques: Transfection, Immunoprecipitation, Stable Transfection, Expressing

Journal: Cancer cell

Article Title: Tinagl1 Suppresses Triple-Negative Breast Cancer Progression and Metastasis by Simultaneously Inhibiting Integrin/FAK and EGFR Signaling.

doi: 10.1016/j.ccell.2018.11.016

Figure Lengend Snippet: Figure 7. Tinagl1 Inhibits TNBC Progression by Simultaneously Targeting the Integrin/FAK and EGFR Signaling Pathways (A) 104 LM2 cells was injected into the MFP of NSG mice. Mice were intravenously treated with the indicated reagents twice per week when tumors reached 2 mm in diameter. n = 6 mice per group. (B) WB analysis for the activation of EGFR and FAK in primary tumor of each group after 5 weeks of the treatments as in (A). (C) Quantification of tumor volumes of each treatment group of (A). n = 12 tumors per group. (D and E) Lungs were collected and spontaneous metastasis was examined by ex vivo BLI at the endpoint. n = 6 lungs per group. Representative lungs (D) and quantitative data (E) is shown. Data represent means ± SEM. n.s., not significant; p > 0.05, **p < 0.001, ***p < 0.0001. Significance determined by one-way ANOVA analysis with Dunnett’s test for multiple comparisons. See also Figure S7.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Tinagl1, in 1:1000 (WB), 2 mg (IP), Rabbit ProteinTech Cat#12077-1-AP; RRID: AB_2058942 Tinagl1, in 1:100 (IHC), Rabbit Sigma-Aldrich Cat#HPA048695; RRID: AB_2680497 b-actin, in 1:10,000 (WB), mouse Abcam Cat#ab6276; RRID: AB_2223210 EGFR, in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#4267; RRID: AB_2246311 p-EGFR (Try1068), in 1:1000 (WB), 1:100 (IHC), Rabbit Cell Signaling Technology Cat#3777; RRID: AB_2096270 FAK, in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#3285; RRID: AB_10694068 p-FAK (Try397), in 1:1000 (WB), 1:100 (IHC), Rabbit Cell Signaling Technology Cat#8556; RRID: AB_10891442 p-FAK (Try925), in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#3284; RRID: AB_2253227 AKT, in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#4691; RRID: AB_915783 p-ATK (S473), in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#4060; RRID: AB_2315049 ERK1/2, in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#4695; RRID: AB_390779 p-ERK1/2 (Thr202/Tyr204), in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#4370; RRID: AB_2315112 Integrin b1 subunit, in 1:1000 (WB), 2 mg (IP), Rabbit Cell Signaling Technology Cat#34971 Integrin a5 subunit, in 1:1000 (WB), 2 mg (IP), Rabbit Cell Signaling Technology Cat#4705; RRID: AB_10827978 Integrin av subunit, in 1:1000 (WB), 2 mg (IP), Rabbit Abcam Cat#ab179475; RRID: AB_2716738 Integrin a3 subunit, in 1:1000 (WB), Rabbit Abcam Cat#ab190731 Integrin a4 subunit, in 1:1000 (WB), Rabbit Cell Signaling Technology Cat#8440P Integrin aM subunit, in 1:1000 (WB), Rabbit Abcam Cat#ab8878; RRID: AB_306831 HA, in 2 mg (IP), Mouse Santa Cruz Biotechnology Cat#sc-7392; RRID: AB_627809 HA, in 1:1000 (WB), Rat Roche, 11867423001 Cat#11867423001; RRID: AB_10094468 MYC, in 1:1000 (WB), 2 mg (IP), Mouse Santa Cruz Biotechnology Cat#sc-40; RRID: AB_627268 FLAG, in 1:1000 (WB), 2 mg (IP), Mouse Sigma-Aldrich Cat#F7425; RRID: AB_439687 His, in 1:1000 (WB), Mouse Sigma-Aldrich Cat#H1029; RRID: AB_260015 GFP, in 1:1000 (WB), Mouse Santa Cruz Biotechnology Cat#sc-9996; RRID: AB_627695 Fibronectin, in 1:1000 (WB), Rabbit ProteinTech Cat#15613-1-AP; RRID: AB_2105691 EGF, in 1:1000 (WB), Mouse Santa Cruz Biotechnology Cat#sc-275; RRID: AB_631417 (Continued on next page) e1 Cancer Cell 35, 1–17.e1–e7, January 14, 2019

Techniques: Protein-Protein interactions, Injection, Activation Assay, Ex Vivo

Journal: Nature Communications

Article Title: IL-21-mediated non-canonical pathway for IL-1β production in conventional dendritic cells

doi: 10.1038/ncomms8988

Figure Lengend Snippet: ( a ) Top panel: cDCs were rested 16 h and stimulated as indicated for 15 min. pSTAT3 was evaluated by flow cytometry. cDCs were gated as CD11c hi cells. Bottom panel: cDCs were rested 1 h, treated with 20 ng ml −1 IL-21, IL-6, IL-10 or Flt3L for 4 h, and intracellular pro-IL-1β analysed by flow cytometry. Shown are data representative of three experiments. ( b ) Summary of three experiments from lower panel of a . ** P values of the untreated sample compared with IL-21, IL-6 and IL-10 treated samples are 0.0002, 0.0017 and 0.0049, respectively; NS, P =0.4; error bars are means±s.e.m. ( c – e ) cDCs were stimulated with 100 ng ml −1 IL-21 or IL-10 for 1 h, then stimulated with 100 ng ml −1 LPS for 4 h, and the expression of Il1b ( c ), Il6 ( d ), and Tnf ( e ) mRNA were determined. Shown are combined results of 3 independent experiments; error bars are means±s.e.m. ( f ) Top panel: BMMs were rested without M-CSF for 16 h, treated with IL-21 or LPS for 4 h, and intracellular pro-IL-1β assessed by flow cytometry. Bottom panel: BMMs (gated as CD11c + F4/80 + cells) were rested and treated with IL-21 or LPS for 15 min. pSTAT3 was evaluated by flow cytometry. Data are representative of two experiments (total of 6 individual samples). ( g ) Top panel: CD4 + T cells were pre-activated with 5 μg ml −1 plate-bound anti-CD3+2 μg ml −1 soluble anti-CD28 for 3 days, washed, rested 16 h, treated with IL-21 for 4 h, and intracellular pro-IL-1β assessed by flow cytometry. Bottom panel: Rested CD4 + T cells were treated with IL-21 for 15 min. pSTAT3 was evaluated by flow cytometry. Data are representative of three experiments. Statistical analysis was performed by Student's t -test.

Article Snippet: Total mouse splenic CD4 + T cells were pre-activated with plate-bound anti-CD3+anti-CD28 for 3 days, washed and rested overnight, and not stimulated or stimulated with IL-21 for 4 h. Total RNA was isolated, and 5 μg per sample were used for mRNA purification using Dynal oligo(dT) beads (Invitrogen).

Techniques: Flow Cytometry, Expressing

Journal: Nature Communications

Article Title: IL-21-mediated non-canonical pathway for IL-1β production in conventional dendritic cells

doi: 10.1038/ncomms8988

Figure Lengend Snippet: ( a – g ), cDCs were rested 1 h, treated with IL-21 for 1 h; pre-activated CD4 + T cells were washed, rested 16 h, treated with IL-21 for 1 h, and ChIP-Seq performed for STAT3, H3K4me1, and H3K27ac. ( a ) Venn diagram showing overlapping and distinctive STAT3 binding sites in cDCs and CD4 + T cells. ( b – g ) For IL-21-induced-STAT3 binding sites that differentially exist in cDCs or CD4 + T cells, there were cell type-specific binding profiles of STAT3 ( b versus c ) and H3K4me1 ( d versus e ) and H3K27ac ( f versus g ) enhancer marks. Shown are normalized read densities near peak summits for cDC- or CD4 + T-cell specific STAT3 binding sites. ‘Dips' at the plot centres ( d – g ) represent open chromatin corresponding to nucleosome depletion. Data are representative of two experiments.

Article Snippet: Total mouse splenic CD4 + T cells were pre-activated with plate-bound anti-CD3+anti-CD28 for 3 days, washed and rested overnight, and not stimulated or stimulated with IL-21 for 4 h. Total RNA was isolated, and 5 μg per sample were used for mRNA purification using Dynal oligo(dT) beads (Invitrogen).

Techniques: ChIP-sequencing, Binding Assay

Journal: Nature Communications

Article Title: IL-21-mediated non-canonical pathway for IL-1β production in conventional dendritic cells

doi: 10.1038/ncomms8988

Figure Lengend Snippet: ( a ) Freshly isolated cDCs were rested 1 h, then stimulated with IL-21 for 4 h; CD4 + T cells were pre-activated for 3 days, then washed and rested 16 h, and stimulated with IL-21 for 4 h. Shown are genes differentially regulated by IL-21 in cDCs versus pre-activated CD4 + T cells. For cDCs, gene expression profiling was performed by microarray analysis, where cDCs were pooled from three independent experiments, as described in ref. . For pre-activated CD4 + T cells, gene expression profiling data were generated by RNA-Seq analysis. Shown are data from one of two similar experiments. ( b ) Il1b and Il21 expression in cDCs and CD4 + T cells not treated or stimulated with IL-21 as in a . Data are representative of 3 experiments. Error bars are technical duplicates of the representative experiment. ( c , d ) STAT3 binding, H3K4me1, H3K27ac, H3K4me3, and H3K27me3 marks at the Il1b locus in cDCs ( c ) and CD4 + T cells ( d ). Arrows in c indicate STAT3 binding sites at GAS1, GAS2 and GAS3 regions (GAS1: TTAgggGAA (−155 bp), TACcctGAA (−175 bp), TCCctgGAA (−195 bp); GAS2: TTTgggGAA (−2,452 bp), TTCctcCAA (−2,525 bp), TTCttcAAA (−2,549 bp); GAS3: TTGtgtGAA (−9,761 bp)). Arrows in d indicate the STAT3 binding sites identified in cDCs, but no STAT3 binding was seen at these sites in CD4 + T cells. ( e , f ) STAT3 binding, H3K4me1, H3K27ac, H3K4me3 and H3K27me3 marks at the Il21 gene locus in cDCs ( e ) and CD4 + T cells ( f ). Arrow in f indicates the STAT3 binding site at the GAS motif in the Il21 promoter region. Arrow in e indicates this same site, but no STAT3 binding was seen at this site in cDCs. Data are representative of two experiments. Statistical analysis was performed by Student's t -test.

Article Snippet: Total mouse splenic CD4 + T cells were pre-activated with plate-bound anti-CD3+anti-CD28 for 3 days, washed and rested overnight, and not stimulated or stimulated with IL-21 for 4 h. Total RNA was isolated, and 5 μg per sample were used for mRNA purification using Dynal oligo(dT) beads (Invitrogen).

Techniques: Isolation, Expressing, Microarray, Generated, RNA Sequencing Assay, Binding Assay

Journal: Nature Communications

Article Title: IL-21-mediated non-canonical pathway for IL-1β production in conventional dendritic cells

doi: 10.1038/ncomms8988

Figure Lengend Snippet: ( a ) cDCs were rested 1 h, treated with 100 ng ml −1 IL-21 or LPS at the indicated time points, and intracellular pro-IL-1β expression was determined. β-actin was used as control. Shown is one of two similar experiments. ( b ) cDCs were treated as in a , with 5 mM ATP added 1 h prior indicated time points in LPS-stimulated samples, and the secretion of IL-1β was determined by enzyme-linked immunosorbent assay (ELISA). Shown are combined results of two independent experiments; error bars are means±s.e.m. ( c ) CD4 + T cells from WT or Il1r −/− mice were cultured in Th17 cell differentiation conditions for 2 days, then supernatant from a 24 h, IL-21-treated cDC culture was added to the Th17 cells and incubated for 2 days, with or without addition of 10 μg ml −1 of anti-IL-1β. Expression of IL-2Rα (MFI) was determined by flow cytometry. The amount of biologically active IL-1β was determined using a standard curve constructed by assaying recombinant IL-1β. Shown are the combined results of two independent experiments with total of six samples. ( d , e ) WT, Casp1 −/− , Nlrp3 −/− and Pycard −/− cDCs were rested 1 h. In d , cDCs were then treated with 100 ng ml −1 LPS for 20–24 h with 5 mM ATP added in the final 1 h, and IL-1β assessed. Data are from two experiments; error bars are means±s.e.m. In e , cDCs were then treated with IL-21 for 20–24 h and IL-1β protein determined. Data are from five experiments. P values of IL-21-treated WT samples as compared with Casp1 −/− , Nlrp3 −/− and Pycard −/− samples are 0.99, 0.22 and 0.96, respectively; error bars are means±s.e.m. ( f ) cDCs from Ripk3 −/− , Ripk3 +/− Casp8 +/− and Ripk3 −/− Casp8 −/− mice were treated as in e , and IL-1β assessed. Data shown are from three experiments. P values of IL-21-treated Ripk3 −/− sample compared with Ripk3 +/− Casp8 +/− and Ripk3 −/− Casp8 −/− samples are 0.57 and 0.93, respectively. In b and d – f , IL-1β production in the culture supernatant was determined by ELISA. Pro-IL-1β induced by IL-21 in the culture supernatant was minimal, based on a pro-IL-1β-specific ELISA. Statistical analysis was performed by Student's t -test.

Article Snippet: Total mouse splenic CD4 + T cells were pre-activated with plate-bound anti-CD3+anti-CD28 for 3 days, washed and rested overnight, and not stimulated or stimulated with IL-21 for 4 h. Total RNA was isolated, and 5 μg per sample were used for mRNA purification using Dynal oligo(dT) beads (Invitrogen).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Cell Culture, Cell Differentiation, Incubation, Flow Cytometry, Construct, Recombinant