Journal: Cell Death & Disease

Article Title: Melanophilin-induced primary cilia promote pancreatic cancer metastasis

doi: 10.1038/s41419-025-07344-2

Figure Lengend Snippet: A , B PLCG1 was activated in -QQ PANC-1 cells. A Phosphorylated PLCG1 increased as shown by human phospho-kinase array analysis in Prt and -QQ PANC-1 cells. The red line shows reference spots, whereas the blue line indicates the phosphorylation of Y783 of PLCG1. B PLCG1 was phosphorylated at tyrosine 783 in -QQ cells. Extracts of parental (Prt) or -QQ PANC-1 cells were analyzed by western blot assay with antibodies against phosphorylated PLCG1 at Y783, PLCG1, and tubulin. C The PLCG1 mRNA level increased in -QQ cells. Quantitative results of relative phospholipase C family mRNA levels were analyzed by qPCR in Prt or -QQ PANC-1 cells. D – F PLCG1 promoted EMT, cell migration, and invasion. D Depletion of PLCG1 alleviated EMT in -QQ cells. Extracts of -QQ PANC-1 cells transfected with siRNA against PLCG1 (siPLCG1) were analyzed by western blot assay with antibodies against PLCG1, E-cadherin (E-cad), and Ku70. E , F Depletion of PLCG1 alleviated cell migration and invasion of -QQ cells. Quantitative results of the relative ( E ) migrated and ( F ) invaded cell numbers in Prt or -QQ PANC-1 cells in the presence or absence of siRNA against PLCG1 (siPLCG1). G Depletion of IFT88 decreased PLCG1 expression. Extracts of -QQ PANC-1 cells in the absence or presence of siRNA against IFT88 (siIFT88) were analyzed by western blot assay with antibodies against phosphorylated PLCG1 (Y783), PLCG1, and Ku70. H MLPH depletion decreased PLCG1 expression and restored EMT. Extracts of -QQ-shScr or -QQ-shMLPH#1 or #2 PANC-1 cells were analyzed by western blot assay with antibodies against MLPH, PLCG1, E-cadherin (E-cad), and actin. I – J PLCG1 and phosphorylated PLCG1 at Y783 (p-PLCG1) localized to the primary cilia. Immunofluorescence staining of Prt or -QQ PANC-1 cells with antibodies against acetylated tubulin (Ac-tub) and ( I ) p-PLCG1 and ( J ) PLCG1. DNA was stained with DAPI (blue). Scale bar, 10 µm. K Depletion of PLCG1 inhibited primary ciliogenesis. Quantitative results of the frequency of ciliated cells of Prt or -QQ PANC-1 cells were shown in the presence or absence of siRNA against PLCG1 (siPLCG1). L Graphic abstract. When PDAC cells suffered a Gln deprivation condition, MLPH was upregulated and recruited to the centrosome to facilitate primary ciliogenesis. The primary cilia induced and activated PLCG1 to promote EMT, invasion, and metastasis. Besides, PLCG1 was recruited to the primary cilia, thereby feedforward maintaining primary ciliogenesis. Data are represented as the mean ± SD of three independent experiments. n.s. no significance, * P < 0.05, *** P < 0.001.

Article Snippet: Anti-IFT88 (13967-1-AP), anti-ARL13B (17711-1-AP), anti-MLPH (10338-1-AP), and anti-MMP9 (10375-2-AP) antibodies were purchased from Proteintech (Chicago, IL, USA). anti-CEP164 (NBP1-81445) and anti-myosin 5a (NBP1-92156) antibodies were purchased from Novus (Littleton, CO, USA). anti-β-actin (AC-15; GTX26276) and anti-PLCγ-1 (phospho-Y783; GTX24828) were purchased from GeneTex (Irvine, CA, USA).

Techniques: Western Blot, Migration, Transfection, Expressing, Immunofluorescence, Staining

Journal: Stem Cells (Dayton, Ohio)

Article Title: Calcium and Calcineurin-NFAT Signaling Regulate Granulocyte-Monocyte Progenitor Cell Cycle via Flt3-L

doi: 10.1002/stem.1813

Figure Lengend Snippet: Flt3-L triggers signaling leading to nuclear factor of activated T cells (NFAT) translocation in granulocyte–monocyte progenitors (GMPs). (A): Levels of intracellular Ca 2+ measured as Fluo4 fluorescence in sorted lin − Sca1 + cKIT + bone marrow (BM) cells (LSKs), common myeloid progenitors (CMPs), and GMPs. After 20 seconds of measurements cells were triggered with FLT3-L, ionomycin, thapsigargin, or Hanks' balanced salt solution (HBSS), and Ca 2+ levels were assessed for another 5 minutes. (B): Intracellular Ca 2+ chelator BAPTA was used to block Ca 2+ release induced by FLT3-L in sorted GMPs. (C): Flt3-L induces Ca 2+ release in GMPs sorted as lin − , cKit + , Sca-1 − , CD34 + , CD16/32 high . Graph of Ca 2+ release analysis in GMPs from lineage-depleted BM cells. Flt3-L (1μg/ml) was added after 1 minute of measurement followed by 4 minutes Ca 2+ release measurement. Representative of three independent experiments, where BM cells from five mice were pooled. (D): Different levels of phospholipase Cγ (PLCγ 2 ) phosphorylation (pPLCγ 2 ) in LSKs (pool of hematopoietic stem cell [HSCs] and multipotent progenitors [MPPs]; lin − , cKit + , Sca-1 + ), CMPs, and GMPs in freshly isolated BM. (E–G): Different levels of pPLCγ 1 in progenitor populations before and after Flt3-L administration. Lineage-depleted BM cells were cultured for 4 hours in HSC medium, Flt3-L (1 μg/ml) was added 15 minutes before fixing and labeling with antibodies for progenitor markers, PLCγ 1 and pPLCγ 2 . Cells were gated as LSKs (pool of HSCs and MPPs; lin − , cKit + , Sca-1 + ), CMPs (lin − , cKit + , Sca-1 − , CD34 + , CD16/32 int ), and GMPs (lin − , cKit + , Sca-1 − , CD34 + , CD16/32 high ). (E, F): Percentage of pPLCγ 1 cells and Gm of fluorescence of pPLCγ 1 and double labeling of total PLCγ 1 with pPLCγ 1 upon trigger with Flt3-L are shown. Data are presented as mean ± SE, *, p < .05 and **, p < .01 in an unpaired Student's t -test. (G): Histogram overlay of pPLCγ 1 intensity in GMPs after the Flt3-L trigger. Representative experiment from three independent experiments is shown. (H): Flt3-L induces NFAT translocation in cKIT + -enriched BM cells. BM cells were depleted of lineaged cells and enriched with cKIT + beads, maintained in HSC medium and transduced with NFAT reporter constructs. Transfected cells were kept in HSC medium for 48 hours, and 1 μg/ml of Flt3-L was added for last 4 hours of culture before nuclear translocation of NFAT reflecting activity of luciferase reporter gene was measured. Data are presented as mean ± SE from one representative of three independent experiments, *, p < .05 in an unpaired Student's t -test. BM pooled from 10 mice was used for each experiment. (I): Proposed graphical scheme of cell cycle regulation in GMPs. Flt3-L activates PLCγ 1 and induce increase in intracellular Ca 2+ levels, this further activates calcineurin and NFAT translocation. When calcineurin-NFAT interaction is blocked, expression of cell cycle regulation genes in GMPs is changed to promote further proliferation. Abbreviations: CMP, common myeloid progenitors; CsA, Cyclosporine A; FITC, fluorescein isothiocyanate; GMP, granulocyte–monocyte progenitor; HBSS, Hanks' balanced salt solution; LSK, lin − Sca1 + cKIT + bone marrow cells; NFAT, nuclear factor of activated T cells; NT, non-treated; PLC, phospholipase Cγ.

Article Snippet: Progenitor subsets were labeled with antibodies, cells were fixed and incubated with anti-pPLCγ 1 -FITC and total PLCγ 1 -PE and analyzed using the FlowCellect PLCγ 1 Activation Dual Detection Kit (Merck Millipore, Billerica, MA, www.emdmillipore.com ) according to manufacturer's instructions.

Techniques: Translocation Assay, Fluorescence, Blocking Assay, Isolation, Cell Culture, Labeling, Transduction, Construct, Transfection, Activity Assay, Luciferase, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Systematic pharmacological analysis of agonistic and antagonistic fibroblast growth factor receptor 1 MAbs reveals a similar unique mode of action

doi: 10.1016/j.jbc.2022.102729

Figure Lengend Snippet: Impact of R1 and IMC-H7 MAbs and FAbs on FGFR1-dependent signaling in the presence of FGF1 or FGF2. A , schematic representation of FGFR1 signaling pathways activated in response to FGF1/2 binding. Measurement of ( B ) FRS2 phosphorylation in COS7 cells, ( C ) Elk1 activity in COS7 cells, and ( D ) PLCγ1 recruitment in U2OS cells expressing FGFR1c and incubated in the presence of a constant amount of FGF2 and increasing amounts of R1 and IMC-H7 MAbs or FAbs. For ( B – D ), data are normalized to the maximum signal obtained in the absence of Abs. Data are means ± S.D. of at least three independent experiments performed in triplicate. E , immunoblots performed on lysates prepared from CAL-120 cells incubated in the presence of 0.3 nM FGF1 and 0.08 to 80 nM of FP-1039, 1 to 1000 nM of AZD4547, or 0.1 to 100 nM of R1MAb2 or IMC-H7 MAb. Immunoblots are representative of three independent experiments. The quantitation data from these three independent experiments are shown in Fig. S2 . F , CAL-120 cells 7-days proliferation assay in the absence or presence of FGF1 (0.3 nM) and R1MAb2, IMC-H7 MAb, FGF ligand trap (FP-1039), or AZD4547. Data are normalized as follows: 100% = 0.3 nM FGF1 and 0% = no FGF1. Data are means ± S.D and are representative of three independent experiments. FGF, fibroblast growth factor; FGFR1, FGF receptor 1.

Article Snippet: Engineered U2OS cells expressing ProLink (PK)-tagged FGFR1 and an enzyme acceptor–tagged PLCγ1 (DiscoverX) were plated (40,000 cells/well) in a 384-well plate and incubated overnight at 37 °C, 5% CO 2 .

Techniques: Binding Assay, Activity Assay, Expressing, Incubation, Western Blot, Quantitation Assay, Proliferation Assay

Journal: The Journal of Biological Chemistry

Article Title: Systematic pharmacological analysis of agonistic and antagonistic fibroblast growth factor receptor 1 MAbs reveals a similar unique mode of action

doi: 10.1016/j.jbc.2022.102729

Figure Lengend Snippet: Assess ment of differences in FGFR1 signaling pathways triggered by R1MAb2 or IMC-H7 MAb versus FGF ligands. A , cell-surface SNAP-tagged FGFR1c interaction determined using TR-FRET in the presence of buffer or isotype controls ( blue histobars ), FGF2 ( purple histobar ), or R1MAb2, FAb2, IMC-H7 MAb, and FAb ( black histobars ). Results are normalized to the TR-FRET signal obtained in the absence of stimulation. B , Western blot analysis performed using lysates prepared from FGFR1-amplified CAL-120 cells incubated with a concentration range of FGF1 starting at 1 nM or a concentration range of R1MAb2 or IMC-H7 MAb starting at 10 nM. Immunoblots are representative of three independent experiments. The quantitation data from these three independent experiments are shown in Fig. S5 . C , PLCγ1 recruitment measured in U2OS cells expressing ProLink tagged FGFR1c and an Enzyme Acceptor tagged SH2 domain in the presence of increasing amounts of R1MAb2, IMC-H7 MAb, or FGF2. D , phosphorylation status of cancer-related kinases assessed using phospho-kinase arrays in CAL-120 cells treated with FGF1/heparin or R1MAb2. Array Part A contains 29 Abs printed in duplicate, and array Part B contains 16 Abs printed in duplicate. All arrays were processed at the same time. E , immunoblots and quantitation data of FGFR1 downstream signaling molecules performed with lysates from CAL-120 cells transfected with either a nontargeting control (NTC) or PLCγ1 siRNA and treated with FGF1 or R1MAb2 48 h posttransfection. The quantitation data are means ± S.D. of three independent experiments. Student’s t -tests for 2 comparisons: ∗ p < 0.05. F , calcium release signal measured upon stimulation of FGFR1c-expressing HEK293 cells with increasing amounts of R1MAb2, IMC-H7 MAb, or FGF2. For (C and F), data are normalized to the maximum signal obtained with FGF2. For ( A , C , and F ) data are means ± S.D. of at least three independent experiments performed in triplicate. One-way ANOVA with Tukey’s multiple comparison test: ∗∗∗∗ p < 0.0001. FGF, fibroblast growth factor; FGFR1, FGF receptor 1; TR-FRET, time-resolved FRET.

Article Snippet: Engineered U2OS cells expressing ProLink (PK)-tagged FGFR1 and an enzyme acceptor–tagged PLCγ1 (DiscoverX) were plated (40,000 cells/well) in a 384-well plate and incubated overnight at 37 °C, 5% CO 2 .

Techniques: Western Blot, Amplification, Incubation, Concentration Assay, Quantitation Assay, Expressing, Transfection

Journal: The Journal of Biological Chemistry

Article Title: Systematic pharmacological analysis of agonistic and antagonistic fibroblast growth factor receptor 1 MAbs reveals a similar unique mode of action

doi: 10.1016/j.jbc.2022.102729

Figure Lengend Snippet: Proposed model of FGFR1 activation in the presence of FGF ligand, FGFR1 Ab alone (R1MAb or IMC-H7 MAb), or FGF ligand and FGFR1 MAb. In the presence of FGF, FGFR forms stable dimers with FGF contacting both receptors and both FGFR1 extracellular domains interacting through a small patch in D2. A conformational change occurs at the TM level that translates into full activation of the FRS2- and PLCγ1-dependent signaling pathways. In the presence of the FGFR1 MAb, the ECDs are further apart and cannot interact through D2, which may result in a KD configuration favorable to phosphorylation events supporting FRS2-dependent signaling only and cell proliferation. When both MAb and FGF ligand are present, the MAb indirectly competes with FGF binding, and at high MAb concentration, a similar phenotype as previously described with MAb alone is observed. FGF, fibroblast growth factor; FGFR1, FGF receptor 1; KD, kinase domain.

Article Snippet: Engineered U2OS cells expressing ProLink (PK)-tagged FGFR1 and an enzyme acceptor–tagged PLCγ1 (DiscoverX) were plated (40,000 cells/well) in a 384-well plate and incubated overnight at 37 °C, 5% CO 2 .

Techniques: Activation Assay, Binding Assay, Concentration Assay