Journal: The Journal of Experimental Medicine

Article Title: The human CIB1–EVER1–EVER2 complex governs keratinocyte-intrinsic immunity to β-papillomaviruses

doi: 10.1084/jem.20170308

Figure Lengend Snippet: CIB1 forms a complex with EVER1 and EVER2. (A) CIB1 protein levels in LCLs derived from patients with loss-of-function mutations of EVER1 (D576*) and EVER2 (D362* or T150Mfs*3), and comparison with those in heterozygous carriers, positive controls (ctrl 1 and 2), P4 and P11, and patients with RHOH and MST1 deficiencies. (B) CIB1, EVER1, and EVER2 mRNA levels were assessed by RT-qPCR in controls ( n = 4), CIB1 m/m (P1–P6 and P12), EVER1 m/m, and EVER2 m/m ( n = 3 each) LCLs. Each symbol represents the mean of one cell line measured in three independent experiments. Statistical significance was assessed by one-way ANOVA followed by Dunnett’s multiple comparison tests relative to healthy controls. (C) CIB1 protein levels in LCLs derived from patients with loss-of-function mutations of EVER1 (D576*), EVER2 (T150Mfs*3), and a healthy control after reconstitution with WT EVER1 or EVER2 by retroviral transduction and stable selection. Asterisk indicates a nonspecific band. (D) CIB1, EVER1, and EVER2 mRNA levels were measured by RT-qPCR in LCLs derived from patients with loss-of-function mutations of EVER1 (D576*), EVER2 (T150Mfs*3), or CIB1 and a healthy control after reconstitution with WT EVER1 or EVER2 by retroviral transduction and stable selection. The data were first normalized against RNaseP as a housekeeping gene and then against an appropriate untransduced parental cell line by the ΔΔCt method. Statistical significance was assessed by one-way ANOVA followed by Dunnett’s multiple comparison test relative to the corresponding untransduced control. (B and D) ns, P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001. (E) HEK293T cells were transfected with plasmids encoding CIB1-HA, FLAG-EVER1, and FLAG-EVER2 either separately or together; 24 h after transfection, samples were subjected to immunoprecipitation with FLAG (Fl)- or HA-specific antibodies (+). Samples incubated with nonspecific IgG served as specificity controls. Western blots were performed to detect coimmunoprecipitated CIB1-HA and FLAG-EVER1 or FLAG-EVER2. The immunoprecipitation of EVER1/2 and CIB1 was confirmed by reincubation with antibodies specific for FLAG and HA, respectively. The presence of all proteins was checked by Western blotting of an input sample taken before immunoprecipitation. GAPDH served as a loading control. vec, vector. (F) Healthy control keratinocytes were transfected with plasmids encoding CIB1-HA, FLAG-EVER1, and FLAG-EVER2 either alone or in combination; 24 h after transfection, cells were subjected to immunofluorescence imaging with Alexa Fluor 568–HA and Alexa Fluor 488–FLAG antibody combinations. DAPI was used for counterstaining. Colocalization was assessed by calculating Pearson’s correlation coefficient with Imaris software. Bar, 13 µm. The results shown are representative of three independent experiments.

Article Snippet: The antibodies used were directed against the FLAG (anti-DDK; Origene) and HA epitopes (Sigma-Aldrich) and were used at a dilution of 1:500.

Techniques: Derivative Assay, Comparison, Quantitative RT-PCR, Control, Retroviral, Transduction, Selection, Transfection, Immunoprecipitation, Incubation, Western Blot, Plasmid Preparation, Immunofluorescence, Imaging, Software

Journal: The Journal of Experimental Medicine

Article Title: The human CIB1–EVER1–EVER2 complex governs keratinocyte-intrinsic immunity to β-papillomaviruses

doi: 10.1084/jem.20170308

Figure Lengend Snippet: Analysis of zinc signaling/levels and NF- k B activation in the presence and absence of CIB1. (A) HEK293T cells were transfected with plasmids encoding CIB1, EVER1, and EVER2 either alone or in combination and with a 4×MRE-dependent EGFP reporter construct. After 24 h, cells were stimulated overnight with PMA/ionomycin (10 ng/ml and 50 ng/ml, respectively) or zinc sulfate (ZnSO 4 ; 100 µM). The next day, cells were stained with 1 µg/ml DAPI to exclude dead cells, and GFP fluorescence was determined with an LSRII flow cytometer. The RRR with the value for vector-transfected cells was set at 100%. Statistical significance was assessed by one-way ANOVA followed by Dunnett’s multiple comparison test relative to the appropriate vector-transfected control (ns, P > 0.05; *, P < 0.05; ***, P < 0.001; n = 3). (B) Flow cytometric quantification of absolute amounts of labile zinc in LCLs derived from healthy controls, EVER1-, EVER2-, or CIB1-deficient patients, or in keratinocytes from P14 with 1 µM FluoZin-3 as described by . Statistical significance was assessed by one-way ANOVA followed by Dunnett’s multiple comparison test relative to the healthy controls ( n = 3). (C) Kinetics of zinc flux in LCLs derived from healthy controls and EVER1-, EVER2-, or CIB1-deficient patients. Cells were loaded with 1 µM FluoZin-3 for 30 min. Fluorimetric measurement were performed on a Victor microplate reader. Baseline fluorescence was recorded every minute for 10 min. Cells were then loaded with 100 µM ZnSO 4 and recorded for 15 min. The specificity of the zinc signal was confirmed by adding the calcium-specific chelator BAPTA before the quenching of the signal with the zinc-specific chelator TPEN and recording for 20 min. No significant effect of genotype was detected in two-way repeat-measures ANOVA ( n = 3). (D) HEK293T cells were transfected with plasmids encoding CIB1-FLAG, FLAG-EVER1, and EVER2 either alone or in combination. Cells were stimulated with 50 ng/ml TNFα 6 h after transfection, incubated overnight, and then harvested and processed for Western blotting. Membranes were probed for the canonical NF-κB component (p)IκBa and the noncanonical NF-κB component p100/p52. Expression of the constructs used for transfection was verified by incubation with a FLAG-specific antibody. GAPDH served as a loading control ( n = 3). (E) Primary keratinocytes from unrelated donors (controls 1 and 2), a healthy family member from kindred A1 carrying the mutation in a heterozygous state (A1.viii.2), and one patient each from kindreds A1 and C were stimulated with 10 ng/ml TNFα for 5, 10, or 20 min and then harvested and processed for Western blotting. Membranes were probed for the canonical NF-κB component (p)IκBa. CIB1 levels were assessed with a polyclonal antibody. GAPDH served as a loading control. vec, empty vector ( n = 3).

Article Snippet: The antibodies used were directed against the FLAG (anti-DDK; Origene) and HA epitopes (Sigma-Aldrich) and were used at a dilution of 1:500.

Techniques: Activation Assay, Transfection, Construct, Staining, Fluorescence, Flow Cytometry, Plasmid Preparation, Comparison, Control, Derivative Assay, Incubation, Western Blot, Expressing, Mutagenesis

Journal: The Journal of Experimental Medicine

Article Title: The human CIB1–EVER1–EVER2 complex governs keratinocyte-intrinsic immunity to β-papillomaviruses

doi: 10.1084/jem.20170308

Figure Lengend Snippet: PLA and coimmunoprecipitation in HaCaT. (A) HaCaT cells were transfected with plasmids encoding FLAG-HPV5 E1, E2, E6, and E7, FLAG-HPV16 E1, E2, E5, E6, and E7, FLAG-HPV4 E8, FLAG–CRPV E8, and CIB1-HA alone or in combination. The day after transfection, samples were plated on microscopy slides, allowed to adhere, fixed in acetone, permeabilized, and subjected to Duolink PLAs with rabbit-HA– and mouse-FLAG–specific antibodies. Z stacks were acquired with a widefield microscope, and PLA-positive sites (defined as structures >0.35 µM 2 ) were scored with Imaris software for 15–50 cells per condition. These pooled results were obtained in two independent experiments. The orange bars indicate the mean. (B) HaCaT cells were transfected with plasmids encoding CIB1-HA and the FLAG-tagged HPV E ORFs scoring positive in the PLA in A. 1 d after transfection, samples were subjected to immunoprecipitation (IP) with FLAG-specific antibodies. Western blots were performed to detect coimmunoprecipitated HPV5 E1, HPV16 E2, E5, HPV4 E8, and CRPV E8. The immunoprecipitation of CIB1 was confirmed by reincubation with a FLAG-specific antibody. The presence of all proteins was checked by Western blotting analysis on an input sample taken before immunoprecipitation. GAPDH served as a loading control ( n = 3). vec, vector.

Article Snippet: The antibodies used were directed against the FLAG (anti-DDK; Origene) and HA epitopes (Sigma-Aldrich) and were used at a dilution of 1:500.

Techniques: Transfection, Microscopy, Software, Immunoprecipitation, Western Blot, Control, Plasmid Preparation

Journal: Cell Death and Differentiation

Article Title: FGFR4 phosphorylates MST1 to confer breast cancer cells resistance to MST1/2-dependent apoptosis

doi: 10.1038/s41418-019-0321-x

Figure Lengend Snippet: FGFR4 suppresses MST1/2 activation and nuclear localization in cancer cell spheres. a shScr and shFGFR4 MDA-MB-453 cell spheres were cultured under non-adherent conditions (10% or 2% FBS), and subjected to immunoblotting. Arrowhead; cleaved N-terminal MST1/2 (in 2% FBS), brackets highlight the fragments of autoactivated MST1/2. b MDA-MB-453 cell spheres were treated with 100 n m BLU9931 for 15 min, and subjected to immunoblotting. c , d shScr and shFGFR4 MDA-MB-453 and ZR-75.1 spheres were analyzed for MST1 expression by c immunofluorescence, and d MST1 nuclear/cytoplasmic ratio was quantified ( n = 4–6 MDA-MB-453 spheres, ≥ 6 microscopic fields/sphere; n = 2–3 ZR-75.1 spheres, ≥ 8 microscopic fields/ sphere; mean ± SEM of two independent experiments. Scale bar 10 µm. e shScr and shFGFR4 MDA-MB-453 cells were transfected with indicated siRNAs before sphere formation, cultured under non-adherent conditions (1% FBS) for 48 h, and subjected to immunoblotting

Article Snippet: Rabbit polyclonal antibodies against FGFR4 (sc-124; Santa Cruz), phospho-FGFR4 (pY642; CSB-PA008250, Cusabio Technology, Houston, TX, USA) and phospho-FRS2-α (pY196; 3864), MST1 (3682), MST2 (3952), phospho-p44/42 MAPK (phospho-Erk1/2) (pT202/pY204; 9101), phospho-AKT (pS473; 9271), phospho-MST1/2 (pT183/pT180; 3681), phospho-YAP (pS127; 4911) all from Cell Signaling Technology, V5-tag (ab9116; Abcam), and horseradish peroxidase–conjugated secondary antibodies (P044701 and P044801, Dako, Santa Clara, CA, USA) for enhanced chemiluminescence detection of immunoblots.

Techniques: Activation Assay, Cell Culture, Western Blot, Expressing, Immunofluorescence, Transfection

Journal: Cell Death and Differentiation

Article Title: FGFR4 phosphorylates MST1 to confer breast cancer cells resistance to MST1/2-dependent apoptosis

doi: 10.1038/s41418-019-0321-x

Figure Lengend Snippet: MST1-Y433F phosphosite mutant restores MST1/2 activation in FGFR4 expressing cancer cells. a MDA-MB-231 cells co-transfected with FGFR4 (R) and wild-type or phosphosite mutant MST1-Y433F were subjected to immunoblotting as indicated. Ratio of pMOB1/MOB1 is indicated below the immunoblot panel. b T47D cells (co-)transfected with wild-type or MST1-Y433F alone or with FGFR4 (R) were treated with 1 µ m okadaic acid for 1 h before cell lysis, and subjected to immunoblotting. See corresponding T47D immunoblots without okadaic acid in Fig. S4C. c T47D cells with indicated siRNAs, and (co-)transfected with wild-type or MST1-Y433F alone or with FGFR4 (R) were treated with 1 µ m okadaic acid as above, and subjected to immunoblotting. a–c Brackets and arrowhead indicate the activated pMST1/2 fragments. N = 2 independent repeats

Article Snippet: Rabbit polyclonal antibodies against FGFR4 (sc-124; Santa Cruz), phospho-FGFR4 (pY642; CSB-PA008250, Cusabio Technology, Houston, TX, USA) and phospho-FRS2-α (pY196; 3864), MST1 (3682), MST2 (3952), phospho-p44/42 MAPK (phospho-Erk1/2) (pT202/pY204; 9101), phospho-AKT (pS473; 9271), phospho-MST1/2 (pT183/pT180; 3681), phospho-YAP (pS127; 4911) all from Cell Signaling Technology, V5-tag (ab9116; Abcam), and horseradish peroxidase–conjugated secondary antibodies (P044701 and P044801, Dako, Santa Clara, CA, USA) for enhanced chemiluminescence detection of immunoblots.

Techniques: Phospho-proteomics, Mutagenesis, Activation Assay, Expressing, Transfection, Western Blot, Lysis

Journal: Cell Death and Differentiation

Article Title: FGFR4 phosphorylates MST1 to confer breast cancer cells resistance to MST1/2-dependent apoptosis

doi: 10.1038/s41418-019-0321-x

Figure Lengend Snippet: FGFR4 substrate screen identifies tyrosine-phosphorylated Hippo pathway proteins including MST1/2. a Scheme of the substrate screen with recombinant FGFR4 kinase domain. b Top 10 FGFR4 substrates ranked by the Z-score include Hippo pathway -associated proteins (yellow). See Table S1 for the full substrate list. c , d MST1/2 are tyrosine phosphorylated by FGFR4 in COS-1 cells. Flag-tagged MST1/2 were immunoprecipitated after transfection of MST1 and MST2 alone or in combination with FGFR4 G388 (G), or R388 (R) kinase (wt), or kinase-dead (KD) variants, and detected by immunoblotting. e MST1 immunoprecipitates from COS-1 cells co-transfected with FGFR4 (R)-wt or FGFR4 (R)-KD (See Fig. S1A) were trypsin digested and subjected to phoshopeptide enrichment prior to LC-MS/MS analysis ( N = 3) that identified phosphorylated Y433 (red) on MST1 only with FGFR4 (R)-wt, and phosphorylated S410 (green) only with FGFR4 (R)-KD

Article Snippet: Rabbit polyclonal antibodies against FGFR4 (sc-124; Santa Cruz), phospho-FGFR4 (pY642; CSB-PA008250, Cusabio Technology, Houston, TX, USA) and phospho-FRS2-α (pY196; 3864), MST1 (3682), MST2 (3952), phospho-p44/42 MAPK (phospho-Erk1/2) (pT202/pY204; 9101), phospho-AKT (pS473; 9271), phospho-MST1/2 (pT183/pT180; 3681), phospho-YAP (pS127; 4911) all from Cell Signaling Technology, V5-tag (ab9116; Abcam), and horseradish peroxidase–conjugated secondary antibodies (P044701 and P044801, Dako, Santa Clara, CA, USA) for enhanced chemiluminescence detection of immunoblots.

Techniques: Recombinant, Immunoprecipitation, Transfection, Western Blot, Liquid Chromatography with Mass Spectroscopy

Journal: Cell Death and Differentiation

Article Title: FGFR4 phosphorylates MST1 to confer breast cancer cells resistance to MST1/2-dependent apoptosis

doi: 10.1038/s41418-019-0321-x

Figure Lengend Snippet: List of MST1 phoshopeptides identified by mass spectrometry

Article Snippet: Rabbit polyclonal antibodies against FGFR4 (sc-124; Santa Cruz), phospho-FGFR4 (pY642; CSB-PA008250, Cusabio Technology, Houston, TX, USA) and phospho-FRS2-α (pY196; 3864), MST1 (3682), MST2 (3952), phospho-p44/42 MAPK (phospho-Erk1/2) (pT202/pY204; 9101), phospho-AKT (pS473; 9271), phospho-MST1/2 (pT183/pT180; 3681), phospho-YAP (pS127; 4911) all from Cell Signaling Technology, V5-tag (ab9116; Abcam), and horseradish peroxidase–conjugated secondary antibodies (P044701 and P044801, Dako, Santa Clara, CA, USA) for enhanced chemiluminescence detection of immunoblots.

Techniques: Sequencing

Journal: Cell Death and Differentiation

Article Title: FGFR4 phosphorylates MST1 to confer breast cancer cells resistance to MST1/2-dependent apoptosis

doi: 10.1038/s41418-019-0321-x

Figure Lengend Snippet: FGFR4 is overexpressed in HER2 + , MST1/2 low breast cancer cells. a , b FGFR4 and HER2 expression in luminal MDA-MB-453, ZR-75.1, and BT474, MCF7, and T47D, and five triple-negative breast cancer cell lines by a immunoblotting and b immunofluorescence. Scale bar 20 μm. c MST1, MST2, and YAP/TAZ expression in these cell lines, detected by immunoblotting ( N = 3)

Article Snippet: Rabbit polyclonal antibodies against FGFR4 (sc-124; Santa Cruz), phospho-FGFR4 (pY642; CSB-PA008250, Cusabio Technology, Houston, TX, USA) and phospho-FRS2-α (pY196; 3864), MST1 (3682), MST2 (3952), phospho-p44/42 MAPK (phospho-Erk1/2) (pT202/pY204; 9101), phospho-AKT (pS473; 9271), phospho-MST1/2 (pT183/pT180; 3681), phospho-YAP (pS127; 4911) all from Cell Signaling Technology, V5-tag (ab9116; Abcam), and horseradish peroxidase–conjugated secondary antibodies (P044701 and P044801, Dako, Santa Clara, CA, USA) for enhanced chemiluminescence detection of immunoblots.

Techniques: Expressing, Western Blot, Immunofluorescence

Journal: Cell Death and Differentiation

Article Title: FGFR4 phosphorylates MST1 to confer breast cancer cells resistance to MST1/2-dependent apoptosis

doi: 10.1038/s41418-019-0321-x

Figure Lengend Snippet: FGFR4 suppresses MST1/2 activation and cleavage in HER2 + breast cancer cells. a , b MDA-MB-453 cells transfected with indicated siRNAs were subjected to immunoblotting for a T183/180 phosphorylated MST1/2, and b MST1 and MST2. Note cleaved ~ 37 kDa MST1/N in FGFR4 knockdown cells (arrowhead). Thin gray line indicates cropping to leave out irrelevant sample lane; see uncropped immunoblots in Fig. S8. c MDA-MB-453 cells transduced with indicated shRNAs were transfected with siScr or siFGFR4 siRNA to 3’UTR before transfection of mock or FGFR4 (R) or (G) overexpression plasmid for a rescue experiment. Lysates were subjected to immunoblotting as indicated. Brackets indicate the cleaved MST1 and MST2 fragments. See Fig. S2A for phopsho-FRS2α and short exposure of MST1. d MDA-MB-453 and ZR-75.1 cells were transduced with indicated si/shRNAs; upper, indicated immunoblots of lysates; lower, quantification of pMOB1/MOB1 ratio, N = 3, mean ± SEM; * P < 0.05. For MST1/2 knockdown e ZR-75.1 and f MDA-MB-453 were transduced with shRNAs followed by transfection with siRNAs as indicated, and g BT474 cells were transfected with indicated siRNAs, and subjected to immunoblotting for pT183/180 MST1/2, MST1, MST2, and pMOB1 as indicated (in e arrowhead points to a full-length, bracket to the cleaved MST2) a–g . N = 3 independent repeats for all; except N = 2 in f and g

Article Snippet: Rabbit polyclonal antibodies against FGFR4 (sc-124; Santa Cruz), phospho-FGFR4 (pY642; CSB-PA008250, Cusabio Technology, Houston, TX, USA) and phospho-FRS2-α (pY196; 3864), MST1 (3682), MST2 (3952), phospho-p44/42 MAPK (phospho-Erk1/2) (pT202/pY204; 9101), phospho-AKT (pS473; 9271), phospho-MST1/2 (pT183/pT180; 3681), phospho-YAP (pS127; 4911) all from Cell Signaling Technology, V5-tag (ab9116; Abcam), and horseradish peroxidase–conjugated secondary antibodies (P044701 and P044801, Dako, Santa Clara, CA, USA) for enhanced chemiluminescence detection of immunoblots.

Techniques: Activation Assay, Transfection, Western Blot, Knockdown, Transduction, Over Expression, Plasmid Preparation

Journal: Cell Death and Differentiation

Article Title: FGFR4 phosphorylates MST1 to confer breast cancer cells resistance to MST1/2-dependent apoptosis

doi: 10.1038/s41418-019-0321-x

Figure Lengend Snippet: FGFR4 counteracts MST1/2-mediated apoptosis. MDA-MB-453 cells transduced with shScr or shFGFR4 shRNAs were transfected with siRNA pools specific for FGFR4, MST1 or MST2, and analyzed for annexin V and propidium iodide (PI) binding by flow cytometry using two different gating strategies for data visualization. a Gating to populations P1 (smaller) and P2 (larger), and annexin V binding (FL1-A) histograms as a marker for early apoptotic cells. b Quantification (% of total, 100,000 events) of apoptosis based on double-positive (annexin V + PI) cells, including both early and late apoptotic stages. See Fig. S3B for representative contour plots and quadrant gating. Mean ± SD of triplicates shown, ** P < 0.01; (repeated three times; N = 3). FSC-A; forward scatter, and SSC-A; side scatter

Article Snippet: Rabbit polyclonal antibodies against FGFR4 (sc-124; Santa Cruz), phospho-FGFR4 (pY642; CSB-PA008250, Cusabio Technology, Houston, TX, USA) and phospho-FRS2-α (pY196; 3864), MST1 (3682), MST2 (3952), phospho-p44/42 MAPK (phospho-Erk1/2) (pT202/pY204; 9101), phospho-AKT (pS473; 9271), phospho-MST1/2 (pT183/pT180; 3681), phospho-YAP (pS127; 4911) all from Cell Signaling Technology, V5-tag (ab9116; Abcam), and horseradish peroxidase–conjugated secondary antibodies (P044701 and P044801, Dako, Santa Clara, CA, USA) for enhanced chemiluminescence detection of immunoblots.

Techniques: Transduction, Transfection, Binding Assay, Flow Cytometry, Marker

Journal: Cell Death and Differentiation

Article Title: FGFR4 phosphorylates MST1 to confer breast cancer cells resistance to MST1/2-dependent apoptosis

doi: 10.1038/s41418-019-0321-x

Figure Lengend Snippet: FGFR4 confers resistance to apoptotic modulators in comprehensive drug screen. a (Phospho)protein changes in TCGA RPPA data associated with FGFR4 upregulation in breast cancer, visualized using cBioPortal (RPPA score change in breast cancer tumors with and without alterations in FGFR4; (mean FGFR4 altered – mean FGFR4 unaltered) [ , ]. The most significantly up- and downregulated proteins are highlighted (pink dots); ERBB2, alternative name of HER2; PR, progesterone receptor. b–g Fibrin embedded single-cell suspensions of b–d MDA-MB-453 and e–g ZR-75.1 cells were treated with 100 n m BLU9931 and/or 30 ng/ml FGF1 over a 13–14-day culture, fixed, embedded into paraffin for sectioning, and subjected to immunohistochemistry for Ki67 and BAX expression. Positively stained vs. total number of cells per colony were counted ( N = 30, mean ± SD, ** P < 0.01). Scale bar 50 µm in b and e . b For comprehensive drug sensitivity testing ( N = 1), MDA-MB-453 cells were treated with 527 compounds in five-point dose either alone or in combination with specific FGFR4 inhibitor BLU9931. Dotplot showing the difference in DSS (drug sensitivity score) for cells in treatment combination with BLU9931 (100 n m ) versus single agent treatments. Negative values are compounds inducing larger decreases in viability as single agents; positive scores indicate compounds yielding larger decreases in viability in the presence of BLU9931. Colors demarcate compounds with similar class

Article Snippet: Rabbit polyclonal antibodies against FGFR4 (sc-124; Santa Cruz), phospho-FGFR4 (pY642; CSB-PA008250, Cusabio Technology, Houston, TX, USA) and phospho-FRS2-α (pY196; 3864), MST1 (3682), MST2 (3952), phospho-p44/42 MAPK (phospho-Erk1/2) (pT202/pY204; 9101), phospho-AKT (pS473; 9271), phospho-MST1/2 (pT183/pT180; 3681), phospho-YAP (pS127; 4911) all from Cell Signaling Technology, V5-tag (ab9116; Abcam), and horseradish peroxidase–conjugated secondary antibodies (P044701 and P044801, Dako, Santa Clara, CA, USA) for enhanced chemiluminescence detection of immunoblots.

Techniques: Immunohistochemistry, Expressing, Staining

Journal: Translational Oncology

Article Title: Downregulation of PIP4K2C inhibits the breast cancer cell proliferation, migration and invasion

doi: 10.1016/j.tranon.2025.102420

Figure Lengend Snippet: The mRNA expression of PIP4K2C in pan-cancer. (A) The mRNA expression of PIP4K2C in 33 tumors in TCGA GTEx samples (ns, p > 0.05; * p < 0.05; ** p < 0.01; *** p < 0.001). (B) PIP4K2C expression in the breast cancer tissues and unpaired normal samples. (C) The expression level of PIP4K2C in the breast cancer tissues and the paired normal samples. ACC, adrenocortical carcinoma; BLCA, bladder urothelial carcinoma; BRCA, breast invasive carcinoma; CESC, cervical and endocervical cancers; CHOL, cholangiocarcinoma; COAD, colon adenocarcinoma; DLBC, lymphoid neoplasm diffuse large B-cell lymphoma; ESCA, esophageal carcinoma; GBM, glioblastoma multiforme; HNSC, head and neck squamous cell carcinoma; KICH, kidney chromophobe; KIRC, kidney renal clear cell carcinoma; KIRP, kidney renal papillary cell carcinoma; LAML, acute myeloid leukemia; LGG, brain lower grade glioma; LIHC, liver hepatocellular carcinoma; LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma; MESO, mesothelioma; OV, ovarian serous cystadenocarcinoma; PAAD, pancreatic adenocarcinoma; PCPG, pheochromocytoma and paraganglioma; PRAD, prostate adenocarcinoma; READ, rectum adenocarcinoma; SARC, sarcoma; SKCM, skin cutaneous melanoma; STAD, stomach adenocarcinoma; STES, stomach and esophageal carcinoma; TGCT, testicular germ cell tumors; THCA, thyroid carcinoma; THYM, thymoma; UCEC, uterine corpus endometrial carcinoma; UCS, uterine carcinosarcoma; UVM, uveal melanoma.

Article Snippet: In this assay, primary antibody PIP4K2C (1:100, CUSABIO, CSB-PA819455LA01HU) and secondary antibodies Goat anti-rabbit IgG H&L (Alexa Fluor® 488) (1:400, abcam, ab150077) were used.

Techniques: Expressing

Journal: Translational Oncology

Article Title: Downregulation of PIP4K2C inhibits the breast cancer cell proliferation, migration and invasion

doi: 10.1016/j.tranon.2025.102420

Figure Lengend Snippet: mRNA expression and protein levels of PIP4K2C in breast cancer cell lines and tissues. (A) The expression level of PIP4K2C in the normal mammary gland cell line MCF-10A and breast cancer cell lines (MDA-MB-231, MDA-MB-468, MCF7, ZR751 and BT20) was determined using qPCR. * P < 0.05, *** P < 0.01 vs MCF-10A. (B) The protein levels of PIP4K2C in cell lines were measured by western blot. (C) The expression level of PIP4K2C in the breast cancer tissues and the paired normal samples. (D) The protein levels of PIP4K2C in the breast cancer tissues and the paired normal samples. (E) The immunofluorescence staining of the breast cancer tissues and the paired normal samples. (F) The IHC images of PIP4K2C in normal and tumor tissues.

Article Snippet: In this assay, primary antibody PIP4K2C (1:100, CUSABIO, CSB-PA819455LA01HU) and secondary antibodies Goat anti-rabbit IgG H&L (Alexa Fluor® 488) (1:400, abcam, ab150077) were used.

Techniques: Expressing, Western Blot, Immunofluorescence, Staining

Journal: Translational Oncology

Article Title: Downregulation of PIP4K2C inhibits the breast cancer cell proliferation, migration and invasion

doi: 10.1016/j.tranon.2025.102420

Figure Lengend Snippet: PIP4K2C was knocked down by siRNA. (A-B) The transfection efficiency of MDA-MB-468 was detected at mRNA expression and protein levels, respectively. (C-D) The transfection efficiency of MCF7 was detected at mRNA expression and protein levels (48 h), respectively.

Article Snippet: In this assay, primary antibody PIP4K2C (1:100, CUSABIO, CSB-PA819455LA01HU) and secondary antibodies Goat anti-rabbit IgG H&L (Alexa Fluor® 488) (1:400, abcam, ab150077) were used.

Techniques: Transfection, Expressing

Journal: Translational Oncology

Article Title: Downregulation of PIP4K2C inhibits the breast cancer cell proliferation, migration and invasion

doi: 10.1016/j.tranon.2025.102420

Figure Lengend Snippet: PIP4K2C was overexpressed in MCF 10A by transfection. (A) The transfection efficiency was detected at mRNA expression. (B) overexpression of PIP4K2C resulted in increased proliferation.

Article Snippet: In this assay, primary antibody PIP4K2C (1:100, CUSABIO, CSB-PA819455LA01HU) and secondary antibodies Goat anti-rabbit IgG H&L (Alexa Fluor® 488) (1:400, abcam, ab150077) were used.

Techniques: Transfection, Expressing, Over Expression

Journal: Translational Oncology

Article Title: Downregulation of PIP4K2C inhibits the breast cancer cell proliferation, migration and invasion

doi: 10.1016/j.tranon.2025.102420

Figure Lengend Snippet: Inhibition of PIP4K2C suppressed the proliferation, migration and invasion of MDA-MB-468 and MCF7 cells. (A) Knockdown of PIP4K2C by siRNA resulted in reduced proliferation. (B) The reduced cell migration rate was evaluated by wound healing assay. The percentage of wound closure at 24 and 48 h was calculated using ImageJ based on the change in scratch area from time 0 h. (C-D) The cell migration and invasion ability were detected by transwell assay.

Article Snippet: In this assay, primary antibody PIP4K2C (1:100, CUSABIO, CSB-PA819455LA01HU) and secondary antibodies Goat anti-rabbit IgG H&L (Alexa Fluor® 488) (1:400, abcam, ab150077) were used.

Techniques: Inhibition, Migration, Knockdown, Wound Healing Assay, Transwell Assay

Journal: Translational Oncology

Article Title: Downregulation of PIP4K2C inhibits the breast cancer cell proliferation, migration and invasion

doi: 10.1016/j.tranon.2025.102420

Figure Lengend Snippet: Down-regulation of PIP4K2C enhanced the protein levels of LC3II/LC3I.

Article Snippet: In this assay, primary antibody PIP4K2C (1:100, CUSABIO, CSB-PA819455LA01HU) and secondary antibodies Goat anti-rabbit IgG H&L (Alexa Fluor® 488) (1:400, abcam, ab150077) were used.

Techniques:

Journal: Advanced Science

Article Title: Serine/Threonine Kinase 33 as a Novel Target of Bufalin in Treatment of Triple‐Negative Breast Cancer

doi: 10.1002/advs.202506253

Figure Lengend Snippet: STK33 promotes TNBC cell proliferation by increasing the protein stability of CCAR1. A) The proteins immunoprecipitated by anti‐Flag antibody were analyzed by mass spectrometry, and the number of peptides for each protein identified was listed. B) 293T cells were transfected with Flag‐STK33 and HA‐CCAR1 plasmids, and then subjected to immunoprecipitation with anti‐Flag or anti‐HA antibodies. The lysates and immunoprecipitates were then blotted. C) MDA‐MB‐231 cells were transfected with an HA‐CCAR1 plasmid, and immunofluorescence staining was performed to detect the localization of HA (red) and STK33 (green) within the cells. D) The expression of STK33 and CCAR1 in MDA‐MB‐231 xenograft were measured by western blot. E) MDA‐MB‐231 and HCC1806 cells were transfected with STK33 siRNA or non‐targeting siRNA, and the expression of STK33 and CCAR1 was measured by western blot. F) 293T cells were transfected with Flag‐STK33 and HA‐CCAR1, and then subjected to immunoprecipitation with anti‐HA antibody, and the phosphorylation of CCAR1 was measured by western blot using p‐Ser/Thr antibody. G) MDA‐MB‐231 and HCC1806 cells were transfected with STK33 siRNA, followed by treatment with 10 µ m MG132 for 4h before harvest. The expression of STK33 and CCAR1 was measured by western blot. H) MDA‐MB‐231 cells were transfected with STK33 siRNA or non‐targeting siRNA, and followed treated with 10µg mL −1 of cycloheximide (CHX) and harvested at the indicated time points. The protein levels of CCAR1 and STK33 were detected by western blot. I) 293T cells were transfected with HA‐CCAR1, MYC‐Ub, or Flag‐STK33 plasmids, followed by treatment with MG132 (10 µ m ) for 10 h before harvest. Then the cell lysates were subjected to immunoprecipitation with anti‐HA antibody and blotted with anti‐MYC antibody. J) MDA‐MB‐231 cells were transfected with STK33 siRNA, and then transfected with HA‐CCAR1, the expression of STK33 and HA was measured by western blot. K) MDA‐MB‐231 cells were transfected with STK33 siRNA, and then transfected with HA‐CCAR1, and cell proliferation was measured by the colony formation assay. The data are presented as mean ± SD of three independent experiments. One‐way ANOVA was used to determine statistical significance, *** P < 0.001, **** P < 0.0001.

Article Snippet: The CCAR1 antibody (CSB‐PA816898ESR1HU) was purchased from CUSABIO (https://www.cusabio.com/).

Techniques: Immunoprecipitation, Mass Spectrometry, Transfection, Plasmid Preparation, Immunofluorescence, Staining, Expressing, Western Blot, Phospho-proteomics, Colony Assay

Journal: Advanced Science

Article Title: Serine/Threonine Kinase 33 as a Novel Target of Bufalin in Treatment of Triple‐Negative Breast Cancer

doi: 10.1002/advs.202506253

Figure Lengend Snippet: High expression of the STK33‐CCAR1 axis is correlated with poor survival in patients with TNBC. A) Representative IHC staining for STK33 and CCAR1 in TNBC. Cases 1 and 2 are representative of a patient with STK33‐high TNBC. Cases 3 and 4 are representative of a patient with STK33‐low TNBC. B, C) Pearson's correlation analyses of STK33 and CCAR1. D) IHC analyses of STK33 and CCAR1 levels in breast cancer tissues from the Human Protein Atlas database (https://www.proteinatlas.org/). E) Pearson's correlation analyses of STK33 and CCAR1 mRNA levels in liver hepatocellular carcinoma from the TIMER web server (https://cistrome.shinyapps.io/timer/). F) Pearson's correlation analyses of STK33 and CCAR1 mRNA levels in diffuse large B‐cell lymphoma from the TIMER web server (https://cistrome.shinyapps.io/timer/). G) The correlation between STK33 expression and the histology grade in TNBC patients, the data are presented as mean ± SD, one‐way ANOVA was used to determine statistical significance, P < 0.05 was considered to be statistically significant. H) The correlation between CCAR1 expression and the histology grade in TNBC patients, the data are presented as mean ± SD, one‐way ANOVA was used to determine statistical significance, P < 0.05 was considered to be statistically significant. I) Kaplan–Meier plots of the overall survival based on CCAR1 expression in TNBC patients. J) Kaplan–Meier curves of overall survival based on STK33 and CCAR1 expression in TNBC patients.

Article Snippet: The CCAR1 antibody (CSB‐PA816898ESR1HU) was purchased from CUSABIO (https://www.cusabio.com/).

Techniques: Expressing, Immunohistochemistry

Journal: Advanced Science

Article Title: Serine/Threonine Kinase 33 as a Novel Target of Bufalin in Treatment of Triple‐Negative Breast Cancer

doi: 10.1002/advs.202506253

Figure Lengend Snippet: Bufalin exerts anti‐cancer activity in animal TNBC model and in patient‐derived TNBC organoids. 4‐week‐old female nude mice were inoculated with MDA‐MB‐231 cells. The tumor‐bearing mice were subsequently given the indicated treatment. A) Subcutaneous tumors were excised and photographed at the end of the experiment. B) Tumor sizes were measured on the specified days, and the data are presented as mean ± SD of 7 mice. One‐way ANOVA was used to determine statistical significance; P < 0.05 was considered to be statistically significant. C) Tumor weights were measured at the end of the experiments, and the data are presented as mean ± SD of 7 mice. One‐way ANOVA was used to determine statistical significance; P < 0.05 was considered to be statistically significant. D) Representative immunohistostaining images for detecting Ki67 expression in the tumor specimens. E) Representative immunohistostaining images for detecting STK33 expression in the tumor specimens. F) Western blot analysis of the STK33 and CCAR1 protein expression in xenograft tumors following the indicated treatment. G) Mice liver functions were measured at the end of the experiments, and the data are presented as mean ± SD of 7 mice. One‐way ANOVA was used to determine statistical significance, ns, P > 0.05. ALT, alanine aminotransferase; AST, aspartate aminotransferase. H) Mice kidney functions were measured at the end of the experiments, and the data are presented as mean ± SD of 7 mice. One‐way ANOVA was used to determine statistical significance, ns, P > 0.05. BUN, blood urea nitrogen. I) Representative images of TNBC patient‐derived organoids (Scale bar 50µm). J–L) The proliferation curve of TNBC PDOs treated with Bufalin, the data are presented as mean ± SD of three independent experiments. One‐way ANOVA was used to determine statistical significance; P < 0.05 was considered to be statistically significant. M) Spearman correlation analysis between STK33 expression and the IC 50 of TNBC PDOs.

Article Snippet: The CCAR1 antibody (CSB‐PA816898ESR1HU) was purchased from CUSABIO (https://www.cusabio.com/).

Techniques: Activity Assay, Derivative Assay, Expressing, Western Blot

Journal: Advanced Science

Article Title: Serine/Threonine Kinase 33 as a Novel Target of Bufalin in Treatment of Triple‐Negative Breast Cancer

doi: 10.1002/advs.202506253

Figure Lengend Snippet: Regulatory signaling pathway of Bufalin in TNBC. In this study, we identified STK33 as a putative target of Bufalin. Bufalin disrupts the interaction between STK33 and HSP90, thereby promoting the ubiquitination and proteasomal degradation of STK33. Furthermore, STK33 is highly expressed in TNBC and enhances TNBC cell proliferation by phosphorylating and stabilizing CCAR1. Targeted degradation of STK33 by Bufalin significantly inhibits TNBC growth, highlighting its potential as a promising therapeutic candidate for TNBC treatment. Created in BioRender. Jiang, S. (2025) https://BioRender.com/00nde1g .

Article Snippet: The CCAR1 antibody (CSB‐PA816898ESR1HU) was purchased from CUSABIO (https://www.cusabio.com/).

Techniques: Ubiquitin Proteomics

Journal: International Journal of Molecular Sciences

Article Title: Canonical and Non-Canonical Localization of Tight Junction Proteins during Early Murine Cranial Development

doi: 10.3390/ijms25031426

Figure Lengend Snippet: ZO-1 shows the strongest expression on the apical surface of the mouse neuroepithelium. ( A ): Confocal images show a representative coronal section of mouse cranial neural folds with immunofluorescence co-staining detecting ZO-1 (yellow) and integrin alpha 4 (ITGA4, magenta) at embryonic stage E 8.5 (9 somites, 9s). The overlay image of all channels (merge) is shown at the top of panel ( A ), followed below by the individual channel images, showing the signals for ITGA4 (magenta) and ZO-1 (yellow). Nuclei are stained with DAPI (cyan). Scale bar: 50 µm. Immunofluorescence staining for ITGA4 was performed to label the basolateral cellular domains. ZO-1 showed the strongest signals in the neuroectoderm (NE) and less strong signals in the non-neuronal ectoderm (NNE) and the mesenchymal cells, underlying the neuronal ectoderm. ( A′ ): Higher magnification of boxed area in ( A ). The overlay image of all channels is shown at the top of panel ( A′ ), followed below by the individual channel image, showing the signals for ZO-1 (yellow). Scale bar: 5 µm. ZO-1 was localized to the apical cell–cell junctions. ( B ): Confocal images show representative coronal section of mouse cranial neural folds with immunofluorescence co-staining detecting ZO-1 (yellow) and ITGA4 (magenta) at embryonic stage E 9.0 (13 somites, 13s). The overlay image of all channels is shown at the left of panel ( B ), followed to the right by the individual channel images, showing the signals for ITGA4 (magenta) and ZO-1 (yellow). Nuclei are stained with DAPI (cyan). Scale bar: 50 µm. ZO-1 showed the strongest signals in the neuroectoderm (NE) and less strong signals in the non-neuronal ectoderm (NNE) and mesenchymal cells. ( C ): E 8.0 whole-mount mouse embryos (6 somites, 6s) were immunofluorescence co-labelled for ZO-1 (yellow) and ITGA4 (magenta). The overlay image of both channels is shown at the top of panel ( C ), followed below by the individual channel image showing the signals for ZO-1 (yellow). The frontal view on the whole-mount forebrains, imaged using confocal microscopy, is shown. Scale bar: 50 µm. ( C′ ): Magnification of boxed area indicated in ( C ) with merged-channel image at the top, showing ITGA4 (magenta) and ZO-1 (yellow) signals and ZO-1 single-channel image below. Scale bar: 10 µm.

Article Snippet: Rabbit ITGA4 , Cusabio, Houston TX, USA, CSB-PA011867LA01HU , 1:200.

Techniques: Expressing, Immunofluorescence, Staining, Confocal Microscopy

Journal: International Journal of Molecular Sciences

Article Title: Canonical and Non-Canonical Localization of Tight Junction Proteins during Early Murine Cranial Development

doi: 10.3390/ijms25031426

Figure Lengend Snippet: List of antibodies.

Article Snippet: Rabbit ITGA4 , Cusabio, Houston TX, USA, CSB-PA011867LA01HU , 1:200.

Techniques: