Journal: Frontiers in Cell and Developmental Biology

Article Title: Meclozine Attenuates the MARK Pathway in Mammalian Chondrocytes and Ameliorates FGF2-Induced Bone Hyperossification in Larval Zebrafish

doi: 10.3389/fcell.2021.694018

Figure Lengend Snippet: Meclozine attenuates the MAPK pathway of FGF2-treated tibiae in the organ culture system. (A) Upper panels: Representative images of E16.5 tibiae of wild-type mice after 4-day culture. Scale bares indicate 1 mm. Lower panels: Absolute bone length after 4-day treatment. Dots indicate the length. Lines are drawn between dots of the same individual. Statistical significance was analyzed by paired Student’s t -test. (B) Enrichment plots of two MAPK signaling-associated gene sets identified by GSEA between FGF2- and FGF2+, and FGF2+ and FGF2+ meclozine in the articular cartilage of ex vivo cultured tibiae. (C) Heatmap depicting the expression of the genes in REACTOME_MAPK_FAMILY_SIGNALING_CASCADES signature and ST_P38_MAPK_PATHWAY signature between FGF2- and FGF2+, and FGF2+ and FGF2+ meclozine in the articular cartilage of ex vivo cultured tibiae. (D) Enrichment plots of BMP signaling-associated gene set identified by GSEA between FGF2- and FGF2+, and FGF2+ and FGF2+ meclozine in the articular cartilage of ex vivo cultured tibiae. (E) Heatmap depicting the expression of the Ihh , Bmp2 , Bmp4 , and Bmp7 between FGF2- and FGF2+, and FGF2+ and FGF2+ meclozine in the articular cartilage of ex vivo cultured tibiae. MAPK, mitogen-activated protein kinase; GSEA, Gene set enrichment analysis.

Article Snippet: FGF2 (3339-FB-025, R and D Systems) was administered in the presence or absence of 20 μM meclozine (155341, MP Biomedicals) for 4 days.

Techniques: Organ Culture, Ex Vivo, Cell Culture, Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: Meclozine Attenuates the MARK Pathway in Mammalian Chondrocytes and Ameliorates FGF2-Induced Bone Hyperossification in Larval Zebrafish

doi: 10.3389/fcell.2021.694018

Figure Lengend Snippet: Treatment protocol of FGF2 and meclozine are determined for evaluating vertebral ossification in larval zebrafish. (A) Treatment regimen of FGF2 for larval zebrafish. (B) Quantification of ossified vertebrae after FGF2 treatment. Dots indicate the number of ossified vertebrae of each sample, and bars indicate means. Statistical significance was analyzed by one-way ANOVA with post-hoc Tukey HSD. (C) Representative images of larval zebrafish from the lateral view at seven dpf stained with Alizarin red after 30 ng/mL FGF2 treatment from eight hpf to seven dpf. Arrows: ossified vertebrae. Scale bar indicates 500 µm. (D) Survival curve of larval zebrafish treated with each dose of meclozine from eight hpf to seven dpf.

Article Snippet: FGF2 (3339-FB-025, R and D Systems) was administered in the presence or absence of 20 μM meclozine (155341, MP Biomedicals) for 4 days.

Techniques: Staining

Journal: Frontiers in Cell and Developmental Biology

Article Title: Meclozine Attenuates the MARK Pathway in Mammalian Chondrocytes and Ameliorates FGF2-Induced Bone Hyperossification in Larval Zebrafish

doi: 10.3389/fcell.2021.694018

Figure Lengend Snippet: Meclozine attenuates spinal and craniofacial bone ossification in FGF2-treated larval zebrafish. (A) Representative images of larval zebrafish from the lateral view at seven dpf stained with Alizarin red after 30 ng/mL FGF2 treatment, with or without 1 µM meclozine, from eight hpf to seven dpf. Arrows: ossified vertebrae. Scale bar indicates 500 µm. (B) Quantification of ossified vertebrae after FGF2 treatment, with or without meclozine. Dots indicate the number of ossified vertebrae of each sample, and bars indicate means. Statistical significance was analyzed by one-way ANOVA with post-hoc Tukey HSD. hpf, hours post-fertilization; dpf, days post-fertilization. (C) Representative craniofacial bone elements of larval zebrafish from anteroposterior view at seven dpf stained with Alizarin red after FGF2 treatment, with or without meclozine. Scale bar indicates 500 µm. (D) Quantification of the number of each ossified craniofacial bone element, including ceratohyal (ch), hyomandibular (hm), branchiostegal ray (br), dentary (d), entopterygoid (en), maxilla (m), and opercle (o), after FGF2 treatment with or without meclozine. Data values are presented as means and standard deviation (SD). Statistical significance was analyzed by one-way ANOVA with post-hoc Tukey HSD.

Article Snippet: FGF2 (3339-FB-025, R and D Systems) was administered in the presence or absence of 20 μM meclozine (155341, MP Biomedicals) for 4 days.

Techniques: Staining, Standard Deviation

Journal: Frontiers in Cell and Developmental Biology

Article Title: Meclozine Attenuates the MARK Pathway in Mammalian Chondrocytes and Ameliorates FGF2-Induced Bone Hyperossification in Larval Zebrafish

doi: 10.3389/fcell.2021.694018

Figure Lengend Snippet: Meclozine ameliorates FGF2-induced hyper ossification in larval zebrafish. (A) Representative craniofacial cartilage elements of larval zebrafish from ventral view, three-dimensional (3D) view, and single layer at seven dpf in Tg (col2a1a:EGFP) after FGF2 treatment, with or without meclozine. Scale bar indicates 100 µm. (B) Quantification of area of craniofacial cartilage, including ceratohyal (ch), hyosymplectic (h), and palatoquadrate (pq) after FGF2 treatment with or without meclozine. (C) Representative craniofacial cartilage and bone elements of larval zebrafish from ventral view, 3D view, and single layer at seven dpf in Tg (col2a1a:EGFP) stained with Alizarine red after FGF2 treatment, with or without meclozine. Scale bar indicates 100 µm. (D) Quantification of relative ossification area, including ceratohyal (ch), hyomandibular (hm), and quadrate (q) after FGF2 treatment with or without meclozine. Relative ossification area was calculated by dividing each red signal area by each green area. Data values are presented as means and standard deviation (SD). Statistical significance was analyzed by one-way ANOVA with post-hoc Tukey HSD.

Article Snippet: FGF2 (3339-FB-025, R and D Systems) was administered in the presence or absence of 20 μM meclozine (155341, MP Biomedicals) for 4 days.

Techniques: Staining, Standard Deviation

Journal: Nature Communications

Article Title: Paclitaxel drives TREM2 + macrophage expansion underlying its inferior therapeutic efficacy compared to Nab-paclitaxel

doi: 10.1038/s41467-026-69060-5

Figure Lengend Snippet: a Schematic of the experimental setup with THP1 or Raw264.7 cells with indicated treatment. Created in BioRender. Xing, Y. (2026) https://BioRender.com/nsp747p b Quantification of proportion of TREM2 in THP1 and Raw264.7 cells with PTX. n = 3 biological independent samples. c Western blot of THP1 and Raw264.7 cells with indicated treatments. The experiment was independently repeated three times with similar results. d Schematic of the experimental strategy. CM was collected from tumor cells with the indicated treatment. TREM2 expression in macrophages incubated with the CM was assessed. Created in BioRender. Xing, Y. (2026) https://BioRender.com/nsp747p e Quantification of proportion of TREM2 in THP1 incubated with CM of BT549 cells, SUM159 cells and MDA-MB-231 cells treated with PTX, in BMDM incubated with CM of Py8119 treated with PTX, in Raw264.7 incubated with CM from 4T1 and Py8119 cells treated with PTX, respectively. n = 3 biological independent samples. f Western blot analysis of TREM2 and related proteins in THP1, BMDMs, and Raw264.7 cells incubated with CM from BT549, SUM159, Py8119, and 4T1 cells, respectively. The experiment was independently repeated three times with similar results. g Representative immunofluorescence staining of THP1 cells incubated with BT549 CM. n = 3 biological independent samples. h Quantification of proportion of TREM2 in THP1 incubated with CM from BT549, SUM159, and MDA-MB-231 cells treated with Nab-PTX, respectively. n = 3 biological independent samples. i Quantification of the proportion of TREM2 in BMDM incubated with CM of Py8119 treated with Nab-PTX. n = 3 biological independent samples. j Cytokine array analysis of CM from BT549 cells treated indicated treatment. k Western blot analysis of the indicated proteins in Raw264.7 cells and BMDMs treated with recombinant FGF2. The experiment was independently repeated three times with similar results. l Quantification of the proportion of TREM2 in Raw264.7 cells and BMDMs treated with recombinant FGF2. n = 3 biological independent samples. m Schematic of the experimental strategy. CM was collected from tumor cells with the indicated treatment, and then pretreatment using an FGF2 neutralizing antibody. TREM2 expression in macrophages incubated with the CM was assessed. Created in BioRender. Xing, Y. (2026) https://BioRender.com/nsp747p n Western blot analysis of the indicated proteins in Raw264.7 and BMDMs incubated with the indicated CM. The experiment was independently repeated three times with similar results. o Quantification of the proportion of TREM2 in Raw264.7 and BMDMs incubated with the indicated CM. n = 3 biological independent samples. p Representative multiplex immunofluorescence staining of CD68, TREM2 and FGF2 in tumors treated with PTX or Nab-PTX ( n = 3 mice per group). The experiment was independently repeated three times with similar results. Data are shown as means ± S.D. and were analyzed by two-sided unpaired Student’s t test ( b , e , h and i ) and two-sided one-way ANOVA followed by Tukey’s test ( l and o ). Source data are provided as a Source Data file.

Article Snippet: The FGF2 protein (HY-P73052AF) was purchased from MedChemExpress and used with indicated concentration.

Techniques: Western Blot, Expressing, Incubation, Immunofluorescence, Staining, Recombinant, Multiplex Assay

Journal: Nature Communications

Article Title: Paclitaxel drives TREM2 + macrophage expansion underlying its inferior therapeutic efficacy compared to Nab-paclitaxel

doi: 10.1038/s41467-026-69060-5

Figure Lengend Snippet: a Overlap of RNA-seq ( n = 3) and PROMO public database analyses to predict transcription factors regulating TREM2 expression. b Correlations between FGF2 and ATF3 expression in breast cancer using TCGA databases. c qPCR analysis of Atf3 expression in the indicated cells. n = 4 (DMSO and PTX) or 3 (PBS and Nab-PTX) biological independent samples. d Western blot analysis of the indicated proteins in Py8119 cells transfected with ATF3-expressing or control vectors. The experiment was independently repeated three times with similar results. e qPCR analysis of Fgf2 expression in Py8119 cells transfected with Atf3 -expressing or control vectors. n = 3 biological independent samples. f Luciferase activity in HEK293T cells transfected with the indicated reporters and Atf3 -expressing or control vectors. n = 3 biological independent samples. g Abundance of Atf3 bound to the Trem2 promoter in Py8119 cells, as assessed by ChIP-qPCR. n = 3 biological independent samples. h ATAC-seq tracks showing the chromatin accessibility in the ATF3 loci for BT549 cells treated by PTX or Nab-PTX. n = 2 samples per group. Data are shown as means ± S.D. and were analyzed by two-sided unpaired Student’s t test ( c, e and f ) and two-sided two-way ANOVA followed by Šídák’s test ( g ). Source data are provided as a Source Data file.

Article Snippet: The FGF2 protein (HY-P73052AF) was purchased from MedChemExpress and used with indicated concentration.

Techniques: RNA Sequencing, Expressing, Western Blot, Transfection, Control, Luciferase, Activity Assay, ChIP-qPCR

Journal: Nature Communications

Article Title: Paclitaxel drives TREM2 + macrophage expansion underlying its inferior therapeutic efficacy compared to Nab-paclitaxel

doi: 10.1038/s41467-026-69060-5

Figure Lengend Snippet: a Overlap of GeneCards and EDCODE public databases analyses to predict transcription factors that regulate TREM2. b Western blot analysis of the indicated proteins in BMDMs and Raw264.7 cells incubated with FGF2 (left) and with CM from Py8119 cells treated with PTX (right). The experiment was independently repeated three times with similar results. c Western blot analysis of the indicated proteins in Raw264.7 cells incubated with the indicated treatment. The experiment was independently repeated three times with similar results. d qPCR analysis of Trem2 expression in BMDMs transfected with Egr1 -expressing vectors. n = 3 biological independent samples. e Western blot analysis of TREM2 expression in BMDMs transfected with Egr1 -expressing vectors. The experiment was independently repeated three times with similar results. f Luciferase activity of HEK293T cells transfected with the indicated reporters and EGR1 -expressing or control vectors. n = 3 biological independent samples. g Abundance of EGR1 bound to the TREM2 promoter in BMDMs assessed by ChIP-qPCR. n = 3 biological independent samples. h Schematic of the Transwell assay. The first CM was collected from tumor cells with indicated treatment, and the second CM was collected from macrophages incubated with the first CM. The migration and invasion capabilities of macrophages incubated with the first CM were assessed. Created in BioRender. Xing, Y. (2026) https://BioRender.com/nsp747p i Quantification of migration and invasion of Py8119 cells induced by BMDM CM incubated with CM from Py8119 cells treated with PTX, and BT549, SUM159, and MDA-MB-231 cells induced by THP1 CM incubated with CM from BT549, SUM159, and MDA-MB-231 cells treated with PTX (left) or Nab-PTX (right), respectively. n = 5 biological independent samples. j Cytokine array analysis of CM from BMDMs with or without TREM2. k Quantification of migration and invasion of Py8119 cells incubated with indicated proteins. n = 3 biological independent samples. l Quantification of migration and invasion of Py8119 cells induced by CM from BMDMs ( Trem2 +/+ ) with CDE-096. n = 3 biological independent samples. m Western blot analysis of EMT- stimulating proteins in BMDMs incubated with indicated proteins. The experiment was independently repeated three times with similar results. n Schematic illustration showing that FGF2 promotes ERK1/2 phosphorylation to upregulate EGR1, which increases TREM2 expression in macrophages. Upregulated TREM2 enhances the secretion of Serpin E1, HGF, CCL3, and CXCL2 from macrophages to tumor cells, facilitating tumor metastasis via EMT. Created in BioRender. Xing, Y. (2026) https://BioRender.com/nsp747p . Data are shown as means ± S.D. and were analyzed by two-sided unpaired Student’s t test ( d, f, i and l ), two-sided one-way ANOVA followed by Tukey’s test ( k ) and two-sided two-way ANOVA followed by Šídák’s test ( g ). Source data are provided as a Source Data file.

Article Snippet: The FGF2 protein (HY-P73052AF) was purchased from MedChemExpress and used with indicated concentration.

Techniques: Western Blot, Incubation, Expressing, Transfection, Luciferase, Activity Assay, Control, ChIP-qPCR, Transwell Assay, Migration, Phospho-proteomics

Journal: Nature Communications

Article Title: Paclitaxel drives TREM2 + macrophage expansion underlying its inferior therapeutic efficacy compared to Nab-paclitaxel

doi: 10.1038/s41467-026-69060-5

Figure Lengend Snippet: PTX, but not Nab-PTX, promotes lung metastasis by inducing TREM2 + macrophage recruitment. Mechanistically, PTX enhances the ATF3-FGF2 axis in breast cancer cells; secreted FGF2 activates the EGR1–TREM2–EMT cytokine axis in macrophages. Created in BioRender. Xing, Y. (2026) https://BioRender.com/6hxlbow .

Article Snippet: The FGF2 protein (HY-P73052AF) was purchased from MedChemExpress and used with indicated concentration.

Techniques:

Journal: Journal of Biomedical Science

Article Title: The signals of FGFs on the neurogenesis of embryonic stem cells

doi: 10.1186/1423-0127-17-33

Figure Lengend Snippet: The FGF effects on the neurogenesis of ES cells and the FGFR expressions in ES cells . (A) After treatment with FGF1, FGF2, FGF4, and FGF8b from day 1 to day 3 using the SFEB method, the numbers of 46C ES-derived Sox1-GFP + cells were estimated by flow cytometry on day 6 (n = 3 for each panel). (B) On indicated days, FGFRs in 46C ES cells were analyzed by RT-PCR. (C) Expression of FGFRs and the GFP + ES cells was analyzed by immunostaining on day 6 or day 2. Single GFP positive cells were indicated by arrow. Nuclei of all cells are revealed by DAPI staining in blue. Scale bar, 10 μm in C. *, p < 0.01, Anova test.

Article Snippet: Human recombinant FGF2, FGF4 and FGF8b were all from R&D Systems.

Techniques: Derivative Assay, Flow Cytometry, Reverse Transcription Polymerase Chain Reaction, Expressing, Immunostaining, Staining

Journal: The Journal of Biological Chemistry

Article Title: Reactivation of Mitogen-activated Protein Kinase (MAPK) Pathway by FGF Receptor 3 (FGFR3)/Ras Mediates Resistance to Vemurafenib in Human B-RAF V600E Mutant Melanoma *

doi: 10.1074/jbc.M112.377218

Figure Lengend Snippet: Enhanced FGFR3 activation in vemurafenib-resistant B-RAF V600E melanoma cells. A, phospho-RTK antibody array analysis. Cell lysates from A375, M14, A375-R1, and M14-R cell lines were incubated on RTK antibody array for 16 h and phosphorylation status was determined as described under “Experimental Procedures.” Each RTK antibody is spotted in duplicate. Supplemental Table S2 describes the list of RTKs and the layout of the antibody array. B, confirmation of phospho-FGFR3 levels by Western blot analysis. Protein levels of total and phosho-FGFR3 were assessed using immunoblotting. C, ELISA analysis of secreted FGF2 in the conditioned media obtained from A375, A375-R1, M14, and M14-R cells. ELISA was performed as described in “Experimental Procedures.”

Article Snippet: Cells were cultured for 48 h in growth medium described above, and the conditioned medium samples (cell free culture supernatant) were analyzed for concentrations of human FGF2 using human FGF2 Quantikine ELISA Kit (R&D Systems).

Techniques: Activation Assay, Ab Array, Incubation, Phospho-proteomics, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Oncogene

Article Title: Transduction of the SkBr3 breast carcinoma cell line with the HOXB7 gene induces bFGF expression, increases cell proliferation and reduces growth factor dependence.

doi: 10.1038/sj.onc.1201875

Figure Lengend Snippet: Figure 1 Expression of HOXB7 and bFGF genes in A375 melanoma, SkBr3 mammary carcinoma and HOXB7-transduced SkBr3. (a) RNase protection analysis. Riboprobes (R) and protected fragments (P) are shown on the right, and molecular size (base pairs) markers (MWM) (pGEM4Z HpaII) on the left. b-actin expression is shown as internal control. (b) Northern blot analysis. b2 microglobulin expression is shown as internal control. (c) Western blot analysis of bFGF produced by A375 melanoma, SkBr3 mammary carcinoma and HOXB7-transduced SkBr3 cells

Article Snippet: Intracellular and secreted bFGF were quanti®ed by a human bFGF ELISA from R&D systems (Minneapolis, MN).

Techniques: Expressing, Control, Northern Blot, Western Blot, Produced

Journal: Oncogene

Article Title: Transduction of the SkBr3 breast carcinoma cell line with the HOXB7 gene induces bFGF expression, increases cell proliferation and reduces growth factor dependence.

doi: 10.1038/sj.onc.1201875

Figure Lengend Snippet: Figure 3 (a) Role of bFGF for SkBr3/HOXB7 cell growth in low serum. Growth kinetics of parental and HOXB7-transduced SkBr3 cells maintained in 10% or 1% FCS were compared. Cell growth was monitored over the indicated time intervals by methylene blue inclusion, as indicated in Materials and methods. (b) Eect of exogenous rbFGF on SkBr3 cell growth. Growth curves of cells maintained in 1% FCS plus dierent concentra- tions of rbFGF evaluated by methylene blue inclusion

Article Snippet: Intracellular and secreted bFGF were quanti®ed by a human bFGF ELISA from R&D systems (Minneapolis, MN).

Techniques:

Journal: Oncogene

Article Title: Transduction of the SkBr3 breast carcinoma cell line with the HOXB7 gene induces bFGF expression, increases cell proliferation and reduces growth factor dependence.

doi: 10.1038/sj.onc.1201875

Figure Lengend Snippet: Figure 4 Inhibition of SkBr3/HOXB7 cell proliferation upon treatment with 30 mM of bFGF antisense (a) or of sense (s) oligomers. Evidence of bFGF intracrine loop operating in cells kept in low serum. Mean+s.d. of three separate experiments is shown

Article Snippet: Intracellular and secreted bFGF were quanti®ed by a human bFGF ELISA from R&D systems (Minneapolis, MN).

Techniques: Inhibition

Journal: Journal of Cancer Prevention

Article Title: Nuclear Localization of Fibroblast Growth Factor Receptor 1 in Breast Cancer Cells Interacting with Cancer Associated Fibroblasts

doi: 10.15430/jcp.2022.27.1.68

Figure Lengend Snippet: Figure 1. Involvement of FGF2-FGFR1 axisin Akt activation. (A) The effect of CAF-CM on proliferation of breast cancer (MCF-7, MDA-MB-231, and MDA-MB-468) cells was determined by the MTT assay. Cells were incubated with or without CAF-CM for 72 hours. ***Significantly different be- tween the groups compared (P < 0.001). (B) MDA-MB-231 cells were incubated with CAF-CM for the indicated time periods. Phosphorylation of Akt and STAT3 were detected by Western blot analysis. (C) MDA-MB-231 cells were exposed to CAF-CM with or without FGF-2-neutralizing antibody for 3 hours. Phosphorylation of Akt was detected by Western blot analysis. *,***Significantly different between the groups compared (*P < 0.05; ***P < 0.001). (D) MDA-MB-231 cells were treated with 20 ng/mL of FGF2 for the indicated time periods. The phosphorylation of FRS2α as well as Akt was analyzed by Western blot. (E) RNA-seq data set of TCGA breast invasive carcinoma was downloaded from XenaBrower (https://xenabrowser.net). mRNA expression levels of total 1,097 samples (Illumina HiSeq log [normalized counts + 1]) were prepared by quantile normalization. Pearson cor- relation coefficient was calculated to assess the relationship between FGF2 and FGFR1. (F, G) Correlation of FGFR1 protein expression with FGF2 (F) and Akt (G), based on 105 breast invasive carcinoma protein specimens (TCGA, Pan-Cancer Atlas) from the cBioportal database (www.cbiopor- tal.org). FGF2, fibroblast growth factor 2; FGFR1, FGF receptor 1; CAFs, cancer-associated fibroblasts; NFs, normal fibroblasts; CM, conditioned medium; ns, not significantly different; FRS2, FGFR substrate 2; TCGA, The Cancer Genome Atlas; CPTAC, the Clinical Proteomic Tumor Analysis Consortium.

Article Snippet: For neutralization of FGF2 in the CM of CAFs, CM was pre-incubated with 25 μg/mL of human FGF2 antibody or its IgG control (R&D Systems, Inc., Minneapolis, MN, USA) for 1 hour at room temperature prior to use.

Techniques: Activation Assay, MTT Assay, Incubation, Phospho-proteomics, Western Blot, RNA Sequencing, Expressing

Journal: Journal of Cancer Prevention

Article Title: Nuclear Localization of Fibroblast Growth Factor Receptor 1 in Breast Cancer Cells Interacting with Cancer Associated Fibroblasts

doi: 10.15430/jcp.2022.27.1.68

Figure Lengend Snippet: Figure 2. Role of FGFR1 in Akt phosphorylation and breast cancer cell growth and progression. (A) MDA-MB-231 cells were transfected with scrambled or FGFR1 si-RNA for 24 hours. Cells were then incubated with 20 ng/mL of FGF2 for 15 minutes to measure phosphorylated FRS2α. (B) Mice were subjected to xenograft co-injecting with fibroblasts and MDA-MB-231 breast cancer cells. A complex collagen network was detected in H&E-stained tumors by an intense pink and in Masson’s trichrome stain by a blue stain (arrows). Stromal compartment was also detected by α-SMA immunostaining. Magnification, x100. Bars, 100 μm. (C) Phosphorylated Akt in the xenograft tumors was determined by Western blot analysis. *Sig- nificantly different between the groups compared (P < 0.05). (D) Enrichment plots of hallmark gene sets in the high FGFR1-expressing group. FGF2, fibroblast growth factor 2; FGFR1, FGF receptor 1; FRS2, FGFR substrate 2; α-SMA, alpha-smooth muscle actin; CONT, control; EMT, epithelial- mesenchymal transition.

Article Snippet: For neutralization of FGF2 in the CM of CAFs, CM was pre-incubated with 25 μg/mL of human FGF2 antibody or its IgG control (R&D Systems, Inc., Minneapolis, MN, USA) for 1 hour at room temperature prior to use.

Techniques: Phospho-proteomics, Transfection, Incubation, Staining, Immunostaining, Western Blot, Expressing, Control

Journal: Journal of Cancer Prevention

Article Title: Nuclear Localization of Fibroblast Growth Factor Receptor 1 in Breast Cancer Cells Interacting with Cancer Associated Fibroblasts

doi: 10.15430/jcp.2022.27.1.68

Figure Lengend Snippet: Figure 3. The involvement of FGF2-induced ROS generation in nuclear localization of FGFR1. (A) MDA-MB-231 cells were co-cultured with NFs or CAFs for 24 hours. MDA-MB-231 (5 x 10 3 cells) and NFs or CAFs (5 x 10 3 cells) were mixed prior to seeding and incubated for 24 hours. Immunocytochemical analysis was performed using anti-FGFR1 antibody. Cells were then stained with DAPI for detection of nuclei. Magnification, x100. Bars, 200 μm. (B) MDA-MB-231 cells were incubated with FGF2 for 1 hour. Immunocytochemical analysis was performed using anti-FGFR1 antibody. Cells were then stained with PI for detection of nuclei. Magnification, x100. Bars, 200 μm. (C) MDA-MB-231 cells were treated with 20 ng/ mL of FGF2 for 1 hour, followed by Western blot analysis of FGFR1 in cytosolic and nuclear extracts. Lamin B was used as a nuclear marker. *Sig- nificantly different between the groups compared (P < 0.05). (D, E) MDA-MD-231 cells were incubated with CAF-CM or FGF2 for 3 hours and 1 hour, respectively. After staining with DCF-DA for 30 minutes, fluorescent microscopic (D) or flow cytometric (E) analysis was performed to detect intracellu- lar ROS accumulation. Magnification, x40. (F) After pretreatment with NAC for 3 hours, cells were exposed to FGF2 for additional 1 hour. Nuclear ex- tracts were subjected to Western blot analysis to detect the presence of FGFR1 and Nrf2 in the nucleus. **Significantly different between the groups compared (P < 0.01). (G) MDA-MB-231 cells were exposed to FGF2 (20 ng/mL) for 1 hour. Cell lysates were subjected to immunoprecipitation using CBP antibody for 16 hours followed by immunoblotting with. FGFR1 or Nrf2 antibody. FGF2, fibroblast growth factor 2; FGFR1, FGF receptor 1; ROS, reactive oxygen species; CAFs, cancer-associated fibroblasts; CM, conditioned medium; NFs, normal fibroblasts; DAPI, 4′,6-diamidino-2-phenylindole; PI, propidium iodide; CONT, cotrol; DCF-DA, 2’,7’-dichlorodihydrofluorescein diacetate; NAC, N-acetylcysteine; CBP, CREB-binding protein.

Article Snippet: For neutralization of FGF2 in the CM of CAFs, CM was pre-incubated with 25 μg/mL of human FGF2 antibody or its IgG control (R&D Systems, Inc., Minneapolis, MN, USA) for 1 hour at room temperature prior to use.

Techniques: Cell Culture, Incubation, Staining, Western Blot, Marker, Immunoprecipitation, Binding Assay

Journal: Journal of Cancer Prevention

Article Title: Nuclear Localization of Fibroblast Growth Factor Receptor 1 in Breast Cancer Cells Interacting with Cancer Associated Fibroblasts

doi: 10.15430/jcp.2022.27.1.68

Figure Lengend Snippet: Figure 4. Possible association between nuclear FGFR1 and Nrf2. (A) TNBC patient cohorts were validated based on the mean expression value of the indicated single genes (FGFR1 or NFE2L2) or as a signature of two genes together and patient survival was analyzed (n = 255). (B, C) MDA- MB-231 cells were transfected with scrambled or Nrf2 si-RNA for 24 hours. Cells were then incubated with 20 ng/mL of FGF2 for 3 hours. The mRNA (B) and protein (C) expression of cyclin D1 was assessed by RT-PCR and Western blot analyses, respectively. The expression of cyclin D1 was mea- sured by RT-PCR (B) and Western blot (C) analyses. (D) In tumor microenvironment, fibroblasts are activated to form CAFs, which secrete FGF2. CAF-derived FGF2 could induces nuclear translocation as well as de novo synthesis of FGFR1, ultimately contributing to cancer cell proliferation, mi- gration and tumor growth. While membrane bound FGFR1 may translocate to nucleus as a complex with FGF2 which has nuclear localization signal (NLS), the complex is likely rather to stimulate the intracellular signaling via FRS2α, which induces transcription of FGFR-1 gene. On the other hand, newly synthesized FGFR-1 is speculated to enter the nucleus as a complex with a cargo protein harboring NLS. FGFR-1 is translocated to the inner nuclear membrane through the nuclear pore complexes (NPCs), which is regulated by importin β. FGF2, fibroblast growth factor 2; FGFR1, FGF receptor 1; TNBC, triple negative breast cancer; HR, hazard ratio; CAFs, cancer-associated fibroblasts; ER, endoplasmic reticulum; FRS2, FGFR substrate 2; CBP, CREB-binding protein.

Article Snippet: For neutralization of FGF2 in the CM of CAFs, CM was pre-incubated with 25 μg/mL of human FGF2 antibody or its IgG control (R&D Systems, Inc., Minneapolis, MN, USA) for 1 hour at room temperature prior to use.

Techniques: Expressing, Transfection, Incubation, Reverse Transcription Polymerase Chain Reaction, Western Blot, Derivative Assay, Translocation Assay, Membrane, Synthesized, Binding Assay

Journal: JCI Insight

Article Title: Fibroblast deletion of ROCK2 attenuates cardiac hypertrophy, fibrosis, and diastolic dysfunction

doi: 10.1172/jci.insight.93187

Figure Lengend Snippet: Fibroblast-specific ROCK2-deficient (ROCK2Postn–/–) and littermate control (ROCK2flox/flox) mice (A–H), and fibroblast-specific constitutively active ROCK knock-in (caROCKPostn–/–) and littermate control (caROCKflox/flox) mice (I–N) were treated with saline or Ang II for 4 wk. (A and I) Representative immunoblots of ROCK1, ROCK2, and ROCK activity, as assessed by the ratio of phosphorylated form of the myosin-binding subunit (MBS) to total MBS (p-MBS/t-MBS), in heart tissues from each experimental genotype. (B and C) Quantification of ROCK1 and ROCK2 protein expression, and (D and J) ROCK activity levels by densitometry (n = 4–6 each). (E and K) Representative immunoblots of FGF2, CTGF, and α-SMA in heart tissues from each experimental genotype. (F–H and L–N) Quantification of FGF2, CTGF, and α-SMA protein expression by densitometry (n = 4–6 each). *P < 0.05 vs. saline-treated each genotype. #P < 0.05 vs. Ang II–treated respective controls. Data are expressed as mean ± SEM. P values were calculated using one-way ANOVA with Tukey’s HSD test.

Article Snippet: FGF2 protein in this NaCl wash buffer was quantitated using rat FGF2 ELISA kit according to manufacturer’s instructions (MFB00; R&D Systems).

Techniques: Control, Knock-In, Saline, Western Blot, Activity Assay, Binding Assay, Expressing

Journal: JCI Insight

Article Title: Fibroblast deletion of ROCK2 attenuates cardiac hypertrophy, fibrosis, and diastolic dysfunction

doi: 10.1172/jci.insight.93187

Figure Lengend Snippet: (A and B) Quantitative PCR analysis mRNA levels of Rock1 and Rock2; (C) a prohypertrophic mediator of Fgf2 (encoding fibroblast growth factor 2); (D) a profibrotic mediator of Ctgf (connective tissue growth factor); (E) a fibroblast-myoblast differentiation marker, Acta2 (α-smooth muscle actin); (F and G) hypertrophic markers of Nppa (atrial natriuretic factor) and Acta1 (skeletal muscle α-actin); and (H and I) fibrotic markers of Col1a (collagen type I) and Fn1 (fibronectin 1) in heart tissues from ROCK2Postn–/– and littermate control (ROCK2flox/flox) mice at 4 wk after saline or Ang II infusion (n = 4–7 each). *P < 0.05, **P < 0.01 vs. saline-treated ROCK2flox/flox mice. #P < 0.05 vs. Ang II-treated ROCK2flox/flox mice. Data are expressed as mean ± SEM. P values were calculated using one-way ANOVA with Tukey’s HSD test.

Article Snippet: FGF2 protein in this NaCl wash buffer was quantitated using rat FGF2 ELISA kit according to manufacturer’s instructions (MFB00; R&D Systems).

Techniques: Real-time Polymerase Chain Reaction, Marker, Control, Saline

Journal: JCI Insight

Article Title: Fibroblast deletion of ROCK2 attenuates cardiac hypertrophy, fibrosis, and diastolic dysfunction

doi: 10.1172/jci.insight.93187

Figure Lengend Snippet: (A and B) Representative immunoblots and densitometric quantification of ROCK activity, as assessed by the ratio of phosphorylated form of the myosin-binding subunit (MBS) to total MBS (p-MBS/t-MBS), stimulated by 1 μM Ang II or 10 ng/ml TGF-β1 for 24 hours, with or without 10 μM Y27632, a specific ROCK inhibitor, in MEFs isolated from WT mice (n = 3–4 each). (C–F) Representative immunoblots and densitometric quantification of FGF2, CTGF, and α-SMA protein expression, stimulated by Ang II or TGF-β1, with or without Y27632, in WT MEFs (n = 3–4 each). *P < 0.05, **P < 0.01 vs. vehicle-stimulated WT MEFs. ##P < 0.01 vs. the same stimulated WT MEFs without Y27632. ††P < 0.01 vs. Ang II–stimulated WT MEFs without Y27632. (G–J) Representative immunoblots and densitometric quantification of ROCK1 and ROCK2 protein expression and ROCK activity, as assessed by the ratio of phosphorylated form of the myosin-binding subunit (MBS) to total MBS (p-MBS/t-MBS), stimulated by Ang II or TGF-β1, in MEFs isolated from WT, global Rock1-KO (ROCK1–/–), and global Rock2-KO (ROCK2–/–) mice (n = 3–4 each). (K–N) Representative immunoblots and densitometric quantification of FGF2, CTGF, and α-SMA protein expression, stimulated by Ang II or TGF-β1, in WT, global ROCK1–/–, and global ROCK2–/– MEFs (n = 3–4 each). **P < 0.01 vs. vehicle-stimulated WT MEFs. #P < 0.05, ##P < 0.01 vs. the same stimulated WT MEFs. ††P < 0.01 vs. Ang II–stimulated WT MEFs. ‡P < 0.05, ‡‡P < 0.01 vs. TGF-β1-stimulated global ROCK1–/– MEFs. Data are expressed as mean ± SEM. P values were calculated using one-way ANOVA with Tukey’s HSD test.

Article Snippet: FGF2 protein in this NaCl wash buffer was quantitated using rat FGF2 ELISA kit according to manufacturer’s instructions (MFB00; R&D Systems).

Techniques: Western Blot, Activity Assay, Binding Assay, Isolation, Expressing

Journal: JCI Insight

Article Title: Fibroblast deletion of ROCK2 attenuates cardiac hypertrophy, fibrosis, and diastolic dysfunction

doi: 10.1172/jci.insight.93187

Figure Lengend Snippet: (A–C) Quantitative PCR analysis of Fgf2 (encoding fibroblast growth factor 2), Ctgf (connective tissue growth factor), and Acta2 (α-smooth muscle actin) mRNA expression stimulated by 1 μM Ang II or 10 ng/ml TGF-β1 for 24 hours, with or without 10 μM Y27632, a specific ROCK inhibitor, in MEFs isolated from WT (n = 3–4 each). **P < 0.01 vs. vehicle-stimulated WT MEFs. #P < 0.05, ##P < 0.01 vs. the same stimulated WT MEFs without Y27632. †P < 0.05, ††P < 0.01 vs. Ang II–stimulated WT MEFs without Y27632. (D–F) Quantitative PCR analysis of Fgf2, Ctgf, and Acta2 mRNA expression stimulated by 1 μM Ang II or 10 ng/ml TGF-β1 for 24 hours in MEFs isolated from WT, global Rock1-KO (ROCK1–/–), and global Rock2-KO (ROCK2–/–) mice (n = 3–4 each). *P < 0.01, **P < 0.01 vs. vehicle-stimulated WT MEFs. #P < 0.05, ##P < 0.01 vs. the same stimulated WT MEFs. †P < 0.05, ††P < 0.01 vs. Ang II–stimulated WT MEFs. ‡P < 0.05 vs. TGF-β1–stimulated global ROCK1–/– MEFs. Data are expressed as mean ± SEM. P values were calculated using one-way ANOVA with Tukey’s HSD test.

Article Snippet: FGF2 protein in this NaCl wash buffer was quantitated using rat FGF2 ELISA kit according to manufacturer’s instructions (MFB00; R&D Systems).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Isolation

Journal: JCI Insight

Article Title: Fibroblast deletion of ROCK2 attenuates cardiac hypertrophy, fibrosis, and diastolic dysfunction

doi: 10.1172/jci.insight.93187

Figure Lengend Snippet: (A and B) Representative immunoblots and densitometric quantification of ROCK activityas assessed by the ratio of phosphorylated form of the myosin-binding subunit (MBS) to total MBS (p-MBS/t-MBS), stimulated by 10 ng/ml TGF-β1 for 24 hours, with or without 10 μM Y27632, a specific ROCK inhibitor, in RNCFs (n = 3–4 each). (C–F) Representative immunoblots and densitometric quantification of FGF2, CTGF, and α-SMA protein expression, stimulated by TGF-β1, with or without Y27632, in RNCFs (n = 3–4 each). **P < 0.01 vs. vehicle-stimulated RNCFs. ##P < 0.01 vs. TGF-β1–stimulated RNCFs without Y27632. (G–J) Representative immunoblots and densitometric quantification of ROCK1 and ROCK2 protein expression and ROCK activity, stimulated by TGF-β1, in RNCFs transfected with control, ROCK1, or ROCK2 siRNA (n = 3–4 each). (K–N) Representative immunoblots and densitometric quantification of FGF2, CTGF, and α-SMA protein expression, stimulated by TGF-β1, in RNCFs transfected with control, ROCK1, or ROCK2 siRNA (n = 3–4 each). **P < 0.01 vs. vehicle-stimulated RNCFs transfected with control siRNA. ##P < 0.01 vs. TGF-β1–stimulated RNCFs transfected with control siRNA. †P < 0.05, ††P < 0.01 vs. TGF-β1–stimulated RNCFs transfected with ROCK1 siRNA. Data are expressed as mean ± SEM. P values were calculated using one-way ANOVA with Tukey’s HSD test.

Article Snippet: FGF2 protein in this NaCl wash buffer was quantitated using rat FGF2 ELISA kit according to manufacturer’s instructions (MFB00; R&D Systems).

Techniques: Western Blot, Binding Assay, Expressing, Activity Assay, Transfection, Control

Journal: JCI Insight

Article Title: Fibroblast deletion of ROCK2 attenuates cardiac hypertrophy, fibrosis, and diastolic dysfunction

doi: 10.1172/jci.insight.93187

Figure Lengend Snippet: (A) Detection of CTGF and FGF2 protein expression in the mixture of supernatant and eluted extracellular matrix of RNCFs, stimulated by 10 ng/ml TGF-β1 for 24 hours, with or without 2.5 μM Y27632. Ponceau S staining of the membrane shows equal loading. (B) ELISA quantification of FGF2 concentration in eluted extracellular matrix of RNCFs, stimulated by TGF-β1 with or without Y27632 (n = 6–9, each in triplicate). **P < 0.01 vs. vehicle-stimulated RNCFs. ##P < 0.01 vs. TGF-β1-stimulated RNCFs without Y27632. (C) Detection of CTGF and FGF2 protein expression in the mixture of supernatant and eluted extracellular matrix of TGF-β1–stimulated RNCFs, transfected with control, ROCK1, or ROCK2 siRNA. (D) ELISA quantification FGF2 concentration in eluted extracellular matrix of TGF-β1–stimulated RNCFs, transfected with control, ROCK1, or ROCK2 siRNA (n = 5–8, each in triplicate). **P < 0.01 vs. vehicle-stimulated RNCFs transfected with each siRNA. ##P < 0.01 vs. TGF-β1–stimulated RNCFs transfected with control siRNA. †P < 0.05 vs. TGF-β1–stimulated RNCFs transfected with ROCK1 siRNA. Data are expressed as mean ± SEM. (E and F) Representative fluorescent images and quantification of cellular hypertrophy of rat H9C2 cardiomyocytes stained with sarcomeric α-actinin (green), in response to 0–100 ng/ml FGF2 for 24 hours. Nuclei are stained with DAPI (blue). (n = 30–50 each). Scale bars: 25 μm. (G–I) Quantification of RT-PCR analysis of hypertrophic markers of Acta1 (encoding skeletal muscle α-actin), Myh7 (β-myosin heavy chain), and Nppa (atrial natriuretic factor) in H9C2 cells stimulated by FGF2 (n = 3–4 each). *P < 0.05, **P < 0.01 vs. vehicle-stimulated H9C2 cells. Data are expressed as mean ± SEM. P values were calculated using one-way ANOVA with Tukey’s HSD test.

Article Snippet: FGF2 protein in this NaCl wash buffer was quantitated using rat FGF2 ELISA kit according to manufacturer’s instructions (MFB00; R&D Systems).

Techniques: Expressing, Staining, Membrane, Enzyme-linked Immunosorbent Assay, Concentration Assay, Transfection, Control, Reverse Transcription Polymerase Chain Reaction