Journal: Journal of Molecular Medicine (Berlin, Germany)

Article Title: Targeting EFEMP1 enhances chondrogenesis and inhibits hypertrophic differentiation in a spontaneous osteoarthritis mouse model

doi: 10.1007/s00109-026-02656-y

Figure Lengend Snippet: Effect of EFEMP1 knockdown by small interfering RNA (siRNA) on chondrocyte phenotype. A The human chondrocyte cell line C20A4 was transfected with either EFEMP1 siRNA (siEFEMP1) or scrambled siRNA (siCtrl). EFEMP1 and MMP-13 protein expression was analyzed by Western blotting. B Chondrocyte-related gene expression, including SOX9, aggrecan (ACAN), MMP13, type X collagen (COL10A1), and type II collagen (COL2A1), was assessed by quantitative PCR in siEFEMP1- and siCtrl-treated C20A4 cells. C Phosphokinase protein arrays were used to profile phosphorylation changes in intracellular proteins extracted from C20A4 chondrocytes. D Spot intensities exhibiting changes greater than ± 25% between groups were quantified using ImageJ software. Red lines indicate baseline levels from the siCtrl group. E Phosphorylation of p70S6K at threonine 421/serine 424 (T421/T424) was further examined by Western blot analysis. Data are presented as mean ± standard error of the mean (SEM). Statistical significance was determined using an independent-samples t-test. * P < 0.05, ** P < 0.01, and *** P < 0.001

Article Snippet: Samples were incubated overnight at 4 °C with primary antibodies against EFEMP1 (1:100, NBP1-77040; NOVUS, USA), SOX9 (1:50, sc-166505; Santa Cruz Biotechnology, USA), MMP-13 (1:100, GTX100665; GeneTex, USA), aggrecan fragments (1:100, AF1220; R&D Systems, USA), COL2A1 (1:100, ab34712; Abcam, UK), and COL10A1 (1:100, GTX37732; GeneTex, USA).

Techniques: Knockdown, Small Interfering RNA, Transfection, Expressing, Western Blot, Gene Expression, Real-time Polymerase Chain Reaction, Phospho-proteomics, Software

Journal: Journal of Molecular Medicine (Berlin, Germany)

Article Title: Targeting EFEMP1 enhances chondrogenesis and inhibits hypertrophic differentiation in a spontaneous osteoarthritis mouse model

doi: 10.1007/s00109-026-02656-y

Figure Lengend Snippet: Effect of EFEMP1 knockdown on osteoblast differentiation and mineralization in preosteoblastic cells. A The preosteoblastic MC3T3-E1 cell line was transfected with either EFEMP1 siRNA (siEFEMP1) or scrambled siRNA (siCtrl). EFEMP1 and RUNX2 protein expression was analyzed by Western blotting. B, C Alizarin Red staining was used to assess matrix mineralization in MC3T3-E1 cells cultured in osteogenic induction medium (OIM), with quantification performed on days 21, 28, and 35. D Alkaline phosphatase (ALP) staining was performed to evaluate osteogenic differentiation. E ALP enzymatic activity was measured in cell lysates on days 9 and 14 following siRNA transfection. F Osteopontin (OPN) expression was analyzed on days 3 and 7 during osteogenic induction. Data are presented as mean ± standard error of the mean (SEM). Statistical analyses: Data in A were analyzed using an independent-samples t -test, while data in C , E , and F were analyzed using one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test. * P < 0.05, ** P < 0.01, and *** P < 0.001

Article Snippet: Samples were incubated overnight at 4 °C with primary antibodies against EFEMP1 (1:100, NBP1-77040; NOVUS, USA), SOX9 (1:50, sc-166505; Santa Cruz Biotechnology, USA), MMP-13 (1:100, GTX100665; GeneTex, USA), aggrecan fragments (1:100, AF1220; R&D Systems, USA), COL2A1 (1:100, ab34712; Abcam, UK), and COL10A1 (1:100, GTX37732; GeneTex, USA).

Techniques: Knockdown, Transfection, Expressing, Western Blot, Staining, Cell Culture, Activity Assay, Comparison

Journal: Journal of Molecular Medicine (Berlin, Germany)

Article Title: Targeting EFEMP1 enhances chondrogenesis and inhibits hypertrophic differentiation in a spontaneous osteoarthritis mouse model

doi: 10.1007/s00109-026-02656-y

Figure Lengend Snippet: EFEMP1 antibody treatment and cartilage histological assessment in a mouse model of osteoarthritis (OA). A Schematic illustration of the EFEMP1 antibody treatment protocol in STR/ort mice from 16 to 21 weeks of age. B Representative Safranin O–stained knee joint sections, with magnified views of the non-calcified cartilage (NCC) and calcified cartilage (CC) regions. Matrix-producing chondrocytes (MPCs) and matrix-non-producing chondrocytes (MNCs) are indicated by arrows. C Quantification of MPC and MNC numbers in the NCC and CC regions of articular cartilage, with counts combined from medial and lateral compartments. D Osteoarthritis severity was evaluated using Osteoarthritis Research Society International (OARSI) scores across four joint compartments: lateral femoral condyle (LF), medial femoral condyle (MF), lateral tibial plateau (LT), and medial tibial plateau (MT). Data in C are presented as mean ± standard error of the mean (SEM), while data in D are presented as median and quartiles using a violin plot. Statistical analysis was performed using an independent-samples t-test. * P < 0.05 and ** P < 0.01

Article Snippet: Samples were incubated overnight at 4 °C with primary antibodies against EFEMP1 (1:100, NBP1-77040; NOVUS, USA), SOX9 (1:50, sc-166505; Santa Cruz Biotechnology, USA), MMP-13 (1:100, GTX100665; GeneTex, USA), aggrecan fragments (1:100, AF1220; R&D Systems, USA), COL2A1 (1:100, ab34712; Abcam, UK), and COL10A1 (1:100, GTX37732; GeneTex, USA).

Techniques: Staining

Journal: Journal of Molecular Medicine (Berlin, Germany)

Article Title: Targeting EFEMP1 enhances chondrogenesis and inhibits hypertrophic differentiation in a spontaneous osteoarthritis mouse model

doi: 10.1007/s00109-026-02656-y

Figure Lengend Snippet: Immunofluorescence analysis of cartilage markers following EFEMP1 antibody treatment in STR/ort mice. A Representative immunofluorescence (IF) staining for EFEMP1, SOX9, MMP13, aggrecan fragments, COL2A1, and COL10A1 in the medial femoral condyle (MF) and medial tibial plateau (MT). Nuclei were counterstained with DAPI (blue), and articular cartilage regions are outlined by orange dashed lines. B Quantification of fluorescence intensity in articular cartilage regions of the femoral condyle and tibial plateau, with medial and lateral compartments combined. EFEMP1 and SOX9 signals were normalized to DAPI, whereas MMP13, aggrecan fragments, COL10A1, and COL2A1 signals were quantified as absolute FITC intensity. Data are presented as mean ± standard error of the mean (SEM). Statistical analysis was performed using an independent-samples t -test. P < 0.05, P < 0.01, and ** P < 0.001

Article Snippet: Samples were incubated overnight at 4 °C with primary antibodies against EFEMP1 (1:100, NBP1-77040; NOVUS, USA), SOX9 (1:50, sc-166505; Santa Cruz Biotechnology, USA), MMP-13 (1:100, GTX100665; GeneTex, USA), aggrecan fragments (1:100, AF1220; R&D Systems, USA), COL2A1 (1:100, ab34712; Abcam, UK), and COL10A1 (1:100, GTX37732; GeneTex, USA).

Techniques: Immunofluorescence, Staining, Fluorescence

Journal: Journal of Molecular Medicine (Berlin, Germany)

Article Title: Targeting EFEMP1 enhances chondrogenesis and inhibits hypertrophic differentiation in a spontaneous osteoarthritis mouse model

doi: 10.1007/s00109-026-02656-y

Figure Lengend Snippet: Cytokine profiling following EFEMP1 antibody treatment in STR/ort mice with osteoarthritis. A Heat-map representation of serum cytokine expression profiles measured using a multiplex assay. B Quantification of serum cytokine concentrations determined by Luminex xMAP analysis. Data are presented as mean ± standard error of the mean (SEM). Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test. P < 0.05, P < 0.01, and P < 0.001

Article Snippet: Samples were incubated overnight at 4 °C with primary antibodies against EFEMP1 (1:100, NBP1-77040; NOVUS, USA), SOX9 (1:50, sc-166505; Santa Cruz Biotechnology, USA), MMP-13 (1:100, GTX100665; GeneTex, USA), aggrecan fragments (1:100, AF1220; R&D Systems, USA), COL2A1 (1:100, ab34712; Abcam, UK), and COL10A1 (1:100, GTX37732; GeneTex, USA).

Techniques: Expressing, Multiplex Assay, Luminex

Journal: JHEP Reports

Article Title: Biliary atresia susceptibility gene EFEMP1 regulates extrahepatic bile duct elastic fiber formation and mechanics

doi: 10.1016/j.jhepr.2024.101215

Figure Lengend Snippet: Elastic fibers are reduced in adult Efemp1 +/- EHBDs but elastin expression is unchanged. (A) Representative staining of elastin in WT and Efemp1 +/- EHBDs. (B) Quantification of percent area of staining. (C) Representative elastic fiber staining of WT and Efemp1 +/- EHBDs. Arrowheads show examples of elastic fibers. Note organized fibers under the basement membrane in WT. Additional examples shown in Fig. S2 . (D) Quantification of the number of elastic fibers per 50 μm 2 . (E) Representative images of collagen by SHG imaging in WT and Efemp1 +/- EHBDs. (F) Quantification of percent collagen area of SHG signal. Forward signal in red and backward signal in green. Dotted lines and L denote lumens. 6-8 ducts were analyzed per genotype for all stains, with 3-5 images taken per duct. All scale bars 50 μm. Data shown are mean ± SEM. Significance in B and D determined by unpaired t-test. Significance in F determined using the Mann-Whitney test since the data were not normally distributed. EHBD, extrahepatic bile duct; SHG, second harmonic generation; WT, wild-type.

Article Snippet: Heat-mediated antigen retrieval with 10 mM citric acid pH 6.0 in a pressure cooker for 2 h was used for staining with antibodies against EFEMP1 (fibulin-3) (1:100, Thermo Fisher, Waltham, MA, #PA5-29347), fibulin-4 (1:100, Abcam, Waltham, MA, #Ab1250730), or fibulin-5 (1:200, Proteintech, Rosement, IL, #12188-1-AP).

Techniques: Expressing, Staining, Membrane, Imaging, MANN-WHITNEY

Journal: JHEP Reports

Article Title: Biliary atresia susceptibility gene EFEMP1 regulates extrahepatic bile duct elastic fiber formation and mechanics

doi: 10.1016/j.jhepr.2024.101215

Figure Lengend Snippet: Mechanical properties measured by pressure myography are altered in Efemp1 +/- EHBDs. (A) Measured outer diameter of WT and Efemp1 +/- adult mice EHBDs at increasing pressures. (B) Calculated circumferential stress of WT and Efemp1 +/- adult mice EHBDs at increasing pressures. (C) Calculated circumferential stretch of WT and Efemp1 +/- adult mice EHBDs at increasing pressures. (D) Relative change in inner diameter of WT vs. Efemp1 +/- treated EHBD as pressure is increased from unloaded (0 mm Hg) condition to physiological pressure (2 mmHg). Data shown are mean ± SEM. Six mice used per condition. ## represent uneven x axes. p values in graphs A-C represent two-way ANOVA between profile of WT vs. Efemp1 +/- . p value in D represents Fisher's least significant difference after two-way ANOVA. EHBD, extrahepatic bile duct; WT, wild-type.

Article Snippet: Heat-mediated antigen retrieval with 10 mM citric acid pH 6.0 in a pressure cooker for 2 h was used for staining with antibodies against EFEMP1 (fibulin-3) (1:100, Thermo Fisher, Waltham, MA, #PA5-29347), fibulin-4 (1:100, Abcam, Waltham, MA, #Ab1250730), or fibulin-5 (1:200, Proteintech, Rosement, IL, #12188-1-AP).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Clinicopathological Appearance of Epidermal Growth-Factor-Containing Fibulin-like Extracellular Matrix Protein 1 Deposition in the Lower Gastrointestinal Tract: An Autopsy-Based Study

doi: 10.3390/ijms25147581

Figure Lengend Snippet: Representative microphotographs showing elastofibrosis of the muscularis propria and subserosa associated with EFEMP1 deposition. ( a – h ) Colon: ( a , b , e , f ) elastica–Masson staining; ( c , g ) Congo red staining; ( d , h ) IHC for EFEMP1. ( a – d ) 31 colon cases; ( e – h ) 37 colon cases. Panels ( b – d ) and panels ( f – h ) show the same sites in serial sections, respectively. ( a – d ) Elastofibrosis is observed in the outer part of the longitudinal muscular layer to the superficial part of the subserosa ( a , b ). In this lesion, congophilia was nearly negative, whereas obvious immunoreactivity for EFEMP1 was identified. ( e – h ) Proliferation of elastic fibers is obvious compared with the first case ( e , f ). However, congophilia and immunoreactivity for EFEMP1 are similar to the first case ( g , h ). Scale bar = 200 μm ( a , e ); 100 μm ( b – d , f – h ).

Article Snippet: Single IHC was performed using primary antibodies against fibulin-3 (EFEMP1) (mouse, clone mab3-5, 1:2000, Santa Cruz, TX, USA), and phosphorylated α-synuclein (clone LB508, 1:500; Zymed, San Francisco, CA, USA) was performed in all cases.

Techniques: Staining

Journal: International Journal of Molecular Sciences

Article Title: Clinicopathological Appearance of Epidermal Growth-Factor-Containing Fibulin-like Extracellular Matrix Protein 1 Deposition in the Lower Gastrointestinal Tract: An Autopsy-Based Study

doi: 10.3390/ijms25147581

Figure Lengend Snippet: Representative microphotographs of ATTR and EFEMP1 deposition. ( a – f ) Small intestine; ( g , h ) colon. ( a , c , d ) Congo red staining under bright-field illumination ( a , c ) and under polarized light ( d ); (panel a , inset) IHC for transthyretin; ( e – h ) double IHC for transthyretin and EFEMP1. ( a , b ) ATTR deposition pattern in the submucosa. ATTR deposition is identified mainly in the vessels and involves relatively small arteries (arrowheads in panel b ). Involvement of the veins (arrows in panel b ) is not obvious. ( c , d ) ATTR deposition pattern in the subserosa. ATTR deposition (arrowhead) shows stronger congophilia than EFEMP1 deposition (arrow) and exhibits clear apple-green birefringence under polarized light (arrowhead in panel d ). ( e , f ) Most ATTR and EFEMP1/AEFEMP1 deposits are observed independently (arrowheads and arrows indicate depositions of ATTR and EFEMP1/AEFEMP1, respectively). ( g , h ) However, colocalization of the two deposition types is identified in some vessels (arrowhead indicates ATTR deposition; arrow indicates ATTR and EFEMP1/AEFEMP1 deposition). Scale bar = 200 μm ( a , e ); 100 μm ( b – d , f – h ).

Article Snippet: Single IHC was performed using primary antibodies against fibulin-3 (EFEMP1) (mouse, clone mab3-5, 1:2000, Santa Cruz, TX, USA), and phosphorylated α-synuclein (clone LB508, 1:500; Zymed, San Francisco, CA, USA) was performed in all cases.

Techniques: Staining

Journal: International Journal of Molecular Sciences

Article Title: Clinicopathological Appearance of Epidermal Growth-Factor-Containing Fibulin-like Extracellular Matrix Protein 1 Deposition in the Lower Gastrointestinal Tract: An Autopsy-Based Study

doi: 10.3390/ijms25147581

Figure Lengend Snippet: Summary of clinical and pathological data in patients with definite or possible constipation.

Article Snippet: Single IHC was performed using primary antibodies against fibulin-3 (EFEMP1) (mouse, clone mab3-5, 1:2000, Santa Cruz, TX, USA), and phosphorylated α-synuclein (clone LB508, 1:500; Zymed, San Francisco, CA, USA) was performed in all cases.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Clinicopathological Appearance of Epidermal Growth-Factor-Containing Fibulin-like Extracellular Matrix Protein 1 Deposition in the Lower Gastrointestinal Tract: An Autopsy-Based Study

doi: 10.3390/ijms25147581

Figure Lengend Snippet: Schematic presentation of the hypothesized pathological effects of EFEMP1/AEFEMP1 deposition in the lower gastrointestinal tract. It is hypothesized that EFEMP1/AEFEMP1 deposition in blood vessels causes impaired blood flow, whereas deposition around the Auerbach plexus, muscularis propria, subserosa, and serosa reduces peristalsis and causes wall vulnerability. Complications such as hypothyroidism, LBD, and diabetes are presumed to be exacerbating factors in these conditions. A combination of these pathologies may result in ischemia of the intestinal tract and associated perforation, ileus, intestinal volvulus, constipation, and diverticulum formation.

Article Snippet: Single IHC was performed using primary antibodies against fibulin-3 (EFEMP1) (mouse, clone mab3-5, 1:2000, Santa Cruz, TX, USA), and phosphorylated α-synuclein (clone LB508, 1:500; Zymed, San Francisco, CA, USA) was performed in all cases.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Clinicopathological Appearance of Epidermal Growth-Factor-Containing Fibulin-like Extracellular Matrix Protein 1 Deposition in the Lower Gastrointestinal Tract: An Autopsy-Based Study

doi: 10.3390/ijms25147581

Figure Lengend Snippet: Representative microphotographs of EFEMP1 on elastica–Masson and Congo red staining and IHC for EFEMP1 in the lower gastrointestinal tract. Deposits in the vessel ( a – c ); mucosa and muscularis mucosae ( d – f ); muscularis propria ( g – i ); Auerbach plexus ( j – l ); serosa ( m – o ); and interstitium of the subserosa (including the mesentery) ( p – r ). ( a , d , g , j , m , p ) Elastica–Masson staining; ( b , e , h , k , n , q ) Congo red staining under bright-field illumination; (insets in ( b , n )) Congo red staining under polarized light; ( c , f , i , l , o , r ) IHC for EFEMP1. Notably, areas positive for EFEMP1 deposition show proliferation (panel d ), contortion, thickening, and fragmentation of elastic fibers (panels a , g , j , m , p ). Deposits in the muscularis mucosae show no congophilia ( e ), and deposits in the muscularis propria ( h ), peri-Auerbach plexus ( k ), and interstitium of the subserosa ( q ) exhibit weak congophilia compared with that in the vessel ( b ) and serosa ( n ). Arrowheads indicate EFEMP1 deposits, and the area indicated by the black square is the region shown in the inset. Scale bar = 200 μm ( d – f , g – i ); 100 μm ( a – c , g – i , m – r ).

Article Snippet: Single IHC was performed using primary antibodies against fibulin-3 (EFEMP1) (mouse, clone mab3-5, 1:2000, Santa Cruz, TX, USA), and phosphorylated α-synuclein (clone LB508, 1:500; Zymed, San Francisco, CA, USA) was performed in all cases.

Techniques: Staining

Journal: International Journal of Molecular Sciences

Article Title: Clinicopathological Appearance of Epidermal Growth-Factor-Containing Fibulin-like Extracellular Matrix Protein 1 Deposition in the Lower Gastrointestinal Tract: An Autopsy-Based Study

doi: 10.3390/ijms25147581

Figure Lengend Snippet: Representative microphotographs of EFEMP1 deposition grading used in this study: ( a – n ) IHC for EFEMP1; ( a , f , k ) vessel; ( b , g ) muscularis propria; ( c , h , l ) Auerbach plexus; ( d , i , m ) serosa; and ( e , j , n ) mesentery (interstitium). Arrowheads indicate EFEMP1 deposits. Scale bar = 200 μm ( a – n ).

Article Snippet: Single IHC was performed using primary antibodies against fibulin-3 (EFEMP1) (mouse, clone mab3-5, 1:2000, Santa Cruz, TX, USA), and phosphorylated α-synuclein (clone LB508, 1:500; Zymed, San Francisco, CA, USA) was performed in all cases.

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Novel markers of MCL1 inhibitor sensitivity in triple-negative breast cancer cells

doi: 10.1016/j.jbc.2024.107375

Figure Lengend Snippet: A 4 gene signature (GS) correlates with AZD5991 sensitivity and MCL-1 dependency in TNBC cell lines. A , analysis of gene expression correlation with TIMER software in 139 TNBC tumors (TCGA) showed that the 4 genes positively correlate with each other. B , 18 TNBC cell lines from GDSC are divided into AZD5991 resistant ( green ) and sensitive ( yellow ) groups. mRNA levels (RPKM) of AXL, ETS1, IL6, and EFEMP1 in all cell lines were extracted from CCLE RNAseq database. The median level of each gene is calculated and listed on top. Gene levels above the median is marked in red and scored 1 and below median is scored 0. The sum of the 4 gene scores is listed on right . C , correlation of GS scores with AZD5991 sensitivity was analyzed with ROC curve with sensitive cells defined as event 1. The results showed a significant correlation ( p = 0.002) with an ROC area 0.95 (ROC area 1.0 indicates perfect correlation). D, 13 TNBC cell lines with MCL-1 CRISPR (DepMap public 23Q2) are divided into MCL-1 dependent ( green ) and independent ( yellow ) groups based on their Chromos scores. Gene scores are calculated as described in B. Correlation of GS scores with MCL-1 dependency was analyzed with ROC curve ( E ). The results showed a significant correlation ( p = 0.04) with a ROC area 0.79).

Article Snippet: Antibodies to BCL2 (124), MCL1 (D35A5), pAKT (S473) (D9E), AKT (11E1), pJAK3 (Y980/981) (D44E3), JAK3 (D7B12), pSTAT3 (Y705) (D3A7), STAT3, pERK1/2 (T202/Y204) (197G2), ERK1/2 (137F5), AXL (C89E7) and ETS1 (D808A) were from Cell Signaling; EFEMP1 (fibulin-3, mAb35) and β-actin (C4) antibodies were from Santa Cruz.

Techniques: Expressing, Software, CRISPR

Journal: The Journal of Biological Chemistry

Article Title: Novel markers of MCL1 inhibitor sensitivity in triple-negative breast cancer cells

doi: 10.1016/j.jbc.2024.107375

Figure Lengend Snippet: Inhibition of ETS1 reduces AXL, IL6, and EFEMP1 expression and increases MCL1 inhibitor sensitivity via expression of BCL-2. A , HS578T and MDA231 cells were transfected with control siRNA or ETS1 siRNA for 24 h. B, HS578T and MDA231 cells were treated with vehicle or TK216 (1 μM) for 24 h. mRNA were qPCR analyzed for the indicated genes. Average (triplicate) relative mRNA is presented with SD indicated. Asterisks indicate statistical significance. Representative results of two independent experiments. C , HS578T and MDA231 cells were treated with vehicle, TK216 (1μM), S63845 (1μM), or TK216 + S63845 for 3 days and then analyzed for sub-G1 cells. % sub-G1 cells are presented. Asterisks indicate statistical significance. Representative results of three independent experiments. D , MDA231 cells were treated with vehicle, S63845 (1μM), or S63845 plus BGB324 (1 μM) or TK216 (1μM), for 24 h. Lysates were immunoblotted for the indicated proteins. Representative results of two independent experiments. Original images are presented in Fig. S5 . E , The cells were treated with vehicle or the ERK inhibitor ulixertinib (2 μM) for 24 h. Lysates were immunoblotted for the indicated proteins. Representative results of two independent experiments. Original images are presented in Fig. S6 . F , MDA231 cells were treated with vehicle, S63845 (1 μM), or S63845 plus ABT-199 (2 μM and 5 μM) for 72 h. G , The cells were transfected with control siRNA or the indicated siRNAs against AXL, ETS1, IL6, or EFEMP1 and then treated with vehicle or S63845 for 72 h. Cells were analyzed with FACS for the cell cycle. The average % Sub-G1 cells were presented with SD indicated. Asterisks indicate statistical significance. Representative results of two independent experiments.

Article Snippet: Antibodies to BCL2 (124), MCL1 (D35A5), pAKT (S473) (D9E), AKT (11E1), pJAK3 (Y980/981) (D44E3), JAK3 (D7B12), pSTAT3 (Y705) (D3A7), STAT3, pERK1/2 (T202/Y204) (197G2), ERK1/2 (137F5), AXL (C89E7) and ETS1 (D808A) were from Cell Signaling; EFEMP1 (fibulin-3, mAb35) and β-actin (C4) antibodies were from Santa Cruz.

Techniques: Inhibition, Expressing, Transfection, Control

Journal: The Journal of Biological Chemistry

Article Title: Novel markers of MCL1 inhibitor sensitivity in triple-negative breast cancer cells

doi: 10.1016/j.jbc.2024.107375

Figure Lengend Snippet: Overexpression of ETS1 and exogenous EFEMP1 and IL6 increases BCL2 expression and promotes MCL1 inhibitor resistance. A , control BT20 cells and BT20-pLIX-ETS1 cells were treated with vehicle or doxycycline (DOX, 0.5 μg/ml) for 24 h. Lysates were immunoblotted for the indicated proteins. Representative images of two independent experiments. B , control BT20 cells and BT20-pLIX-ETS1 cells were treated with vehicle or doxycycline (DOX, 0.5 μg/ml) in combination with vehicle or S63845 for 72 h. Cells were analyzed with FACS for cell cycle. Average % Sub-G1 cells (apoptosis) were presented with SD indicated. Asterisks indicate statistical significance. Representative results of two independent experiments. BT20 cells were FBS-starved for 24 h and then treated with vehicle or recombinant EFEMP1 (50 ng/ml) for 30 min ( C ) or recombinant IL6 (50 ng/ml) for 10 min ( D ). E , BT20 cells were treated with vehicle or S63845 (1 μM) or S63845 in combination with EFEMP1 plus IL6 for 24 h. Lysates were immunoblotted for the indicated proteins. Representative results of two independent experiments. F , BT20 cells were treated with vehicle or S63845 (1 μM) or S63845 in combination with EFEMP1 plus IL6 for 72 h. Cells were analyzed with FACS for the cell cycle. Average % Sub-G1 cells (apoptosis) were presented with SD indicated. Asterisks indicate statistical significance. Representative results of two independent experiments.

Article Snippet: Antibodies to BCL2 (124), MCL1 (D35A5), pAKT (S473) (D9E), AKT (11E1), pJAK3 (Y980/981) (D44E3), JAK3 (D7B12), pSTAT3 (Y705) (D3A7), STAT3, pERK1/2 (T202/Y204) (197G2), ERK1/2 (137F5), AXL (C89E7) and ETS1 (D808A) were from Cell Signaling; EFEMP1 (fibulin-3, mAb35) and β-actin (C4) antibodies were from Santa Cruz.

Techniques: Over Expression, Expressing, Control, Recombinant

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Unveiling the role of osteosarcoma-derived secretome in premetastatic lung remodelling

doi: 10.1186/s13046-023-02886-9

Figure Lengend Snippet: Proteomic analysis identified EFEMP1 as a potential metastatic-related biomarker in osteosarcoma. A, B Gene ontology analysis (GO) output. Biological process (BP) and molecular function (MF) of differently expressed proteins (DEPs) in the two pairwise-comparison groups (SCR 143B vs. MG-63; Plasma PT vs. CTR). Circle sizes in BP denote the number of genes involved in the process. C Reactome pathway enrichment analysis of identified proteins in the two pairwise- comparison groups. Circle sizes denote the number of genes included in a group and colour indicates the p-value. The common pathways are highlighted. D Venn diagram showing specific and common proteins among the two pairwise groups: SCR 143B vs. MG-63 and Plasma PT vs. CTR. E EFEMP1 levels in the SCR of the metastatic 143B and non-metastatic MG-63 OS cells. F Representative western blot of EFEMP1 in the metastatic 143B and non-metastatic MG-63 OS cells. G Plasma levels of EFEMP1 in control mice (CTR), mice treated with the 143B SCR, bearing a primary tumour (PT) or with lung metastasis (Lung Met). H Representative western blot of EFEMP1 expression in 143B cells (CTR), non-targeting (NT) and siRNA knockdown of EFEMP1 cells. I EFEMP1 levels in the SCR of 143B cells, NT and siRNA EFEMP1-knockdown cells. J Representative bioluminescence images of mice treated with the SCR of control 143B (SCR + i.v. group) or EFEMP1-knockdown cells (SCR siRNA EFEMP1 + i.v. group) followed i.v. injection of 143B-Luc + cells. Data are presented as mean ± SEM, from 7 to 8 independent experiments. **** p < 0.0001 were significantly different when compared with the SCR from MG-63 (unpaired t-test (E)); *p < 0.05, **p < 0.01 and ***p < 0.001 were significantly different when compared with the values present in the plasma from healthy mice (Kruskal-Wallis test (G); *p < 0.05, **p < 0.01 were significantly different when compared with 143B SCR (Kruskal-Wallis test (I))

Article Snippet: The samples were paraffin-embedded and sectioned into 4 μm tissue slices for immunostaining with anti-EFEMP1/fibulin 3 monoclonal antibody (1:500, ab256457, Abcam).

Techniques: Biomarker Assay, Comparison, Western Blot, Expressing, Injection

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Unveiling the role of osteosarcoma-derived secretome in premetastatic lung remodelling

doi: 10.1186/s13046-023-02886-9

Figure Lengend Snippet: EFEMP1 expression in primary tumours correlates with poor prognosis in OS patients. A Representative images of EFEMP1 staining and H&E in biopsy samples of high-grade OS patients at x100 magnification (Scale bar: 50 μm). B, C Kaplan-Meier analysis of overall survival in chondroblastic, fibroblastic and osteoblastic OS patient samples (n = 73 patients) and with metastatic disease (n = 37 patients). Scan cut-off was used to group samples into high (blue) and low (red) EFEMP1 expressions. p-values were determined by a log-rank test

Article Snippet: The samples were paraffin-embedded and sectioned into 4 μm tissue slices for immunostaining with anti-EFEMP1/fibulin 3 monoclonal antibody (1:500, ab256457, Abcam).

Techniques: Expressing, Staining