Journal: Frontiers in Molecular Biosciences

Article Title: Comparison of two immunotoxins against DLL3 receptor; as an inhibitor for small cell lung cancer

doi: 10.3389/fmolb.2025.1506768

Figure Lengend Snippet: (A, B) The cell viability percentage of Rova-GrB and Rova-Typh on A549 and HUVEC cell lines after 24 h of incubation (The control group is without Rova-GrB and Rova-Typh). The significance of the results (p-Value) between the control group and other groups has been indicated as follows: *P < 0.05; **P < 0.01; **P < 0.001.

Article Snippet: The human A549 lung cancer cell lines and the normal HUVEC, Human umbilical vein endothelial cell line, were purchased from the Pasteur Institute and Iranian Biological Resource Center (IBRC), respectively.

Techniques: Incubation, Control

Journal: International Journal of Molecular Sciences

Article Title: Endoglin Promotes Myofibroblast Differentiation and Extracellular Matrix Production in Diabetic Nephropathy

doi: 10.3390/ijms21207713

Figure Lengend Snippet: Endoglin is co-localized with the myofibroblast marker α-SMA. Kidney biopsy samples from non-diabetic controls ( A , E , I , M ) and patients with DN ( B – D , F – H , J – L , N – P ) were co-immunostained for endoglin (red) and the endothelial cell marker CD31 ( A – D ), the macrophage marker CD68 ( E – H ), the fibroblast marker vimentin ( I – L ), or the myofibroblast marker α-SMA ( M – P ). Note the co-localization of endoglin and the endothelial marker CD31 in blood vessels, the co-localization of endoglin and the fibroblast marker vimentin, as well as the co-localization of endoglin and the myofibroblast marker α-SMA, particularly in the patients with DN ( H ). The scale bars represent 50 µm.

Article Snippet: The following primary antibodies were used: Goat anti-human endoglin (1:800, R&D systems), mouse anti-human CD31 (IgG1, clone JC70A, 1:200; Dako, Glostrup, Denmark) to stain endothelial cells, mouse anti-human CD68 (IgG1, clone KP1, 1:2000; Dako) to stain macrophages, mouse anti-human vimentin (IgG2a, clone Vim 3B4, 1:750; Dako) to stain fibroblasts, and mouse anti-human α-SMA (IgG2a, clone 1A4, 1:400; Dako) to stain myofibroblasts.

Techniques: Marker

Journal: International Journal of Cancer

Article Title: FHL2 expression by cancer‐associated fibroblasts promotes metastasis and angiogenesis in lung adenocarcinoma

doi: 10.1002/ijc.35174

Figure Lengend Snippet: FHL2 is highly expressed in lung cancer tissue and is a myofibroblast marker as demonstrated by using a previously published single‐cell RNA‐sequencing dataset. (A) Left, UMAP projection color‐coded by major cell lineages in lung tissue. Right, FHL2 expression in each subset. (B) Violin plot showing the expression level of FHL2 in fibroblasts of normal lung tissue (normal lung) and lung cancer tissue (tumor lung). (C) Upper left, UMAP projection of fibroblasts, color‐coded by subtypes identified after subclustering of the fibroblast subset in (B). Upper right, FHL2 expression of each subset. Lower, Violin plot showing the expression level of FHL2 in subtypes of fibroblasts.

Article Snippet: The sections were then incubated at 4°C overnight with rabbit polyclonal anti‐human FHL2 antibody (1/200) (HPA006028, Atlas antibodies, Stockholm, Sweden) or mouse monoclonal anti‐human CD34 (1/400, clone QBEnd/10, NBP2‐32932, Novus biologicals, Novus Biological, Littleton, CO, USA).

Techniques: Marker, RNA Sequencing, Expressing

Journal: International Journal of Cancer

Article Title: FHL2 expression by cancer‐associated fibroblasts promotes metastasis and angiogenesis in lung adenocarcinoma

doi: 10.1002/ijc.35174

Figure Lengend Snippet: Knock‐down of FHL2 in CAF reduces proliferation of CAFs and establishment of FHL2 KO CAFs and TGF‐β1 upregulate FHL2 expression on CAF and characteristics of FHL2 KO CAFs. (A) Silencing of FHL2 in CAF094 hTERTRFP by siRNA. Western blot analysis showing FHL2 expression in CAF094 hTERTRFP transfected with siRNAs. (B) WST‐8 assay for the assessing the growth of CAF094 hTERTRFP cells transfected with siFHL2 or scrambled siRNA (siScr) after a 96‐h incubation. Data represent the mean ± SEM ( n = 3). * p < .05; ** p < .01. (C) Western blot analysis of FHL2 in CAF094 YFPhTERT and CAF094 YFPhTERT FHL2KO cells. GAPDH was used as an internal control. (D) WST‐8 assay for assessing the growth of CAF094 YFPhTERT cells (WT) and CAF094 YFPhTERT FHL2KO cells (KO) after a 72‐h incubation. (E) Western blot analysis of FHL2 in CAF094 YFPhTERT cells. CAF094 YFPhTERT cells were stimulated with 5 ng/mL of recombinant human TGF‐β1 for 48 h. GAPDH was used as an internal control. (F) SRB assay for the assessing the growth of CAF094 YFPhTERT cells (WT) and CAF094 YFPhTERT FHL2KO cells (KO) cells stimulated with 5 ng/mL of recombinant human TGF‐β1 for 72‐h. Data represent the mean ± SEM (n = 3). * p < .05. (G) Results of a gel contraction assay of CAF094 YFPhTERT cells (WT) and CAF094 YFPhTERT FHL2KO cells (KO). Gel contraction was monitored after 72 h by taking photographs of the gels and the percent contraction was calculated using the formula: 100 × (well area − gel area)/well area. (H) Results of a PDGF‐BB‐induced Transwell migration assay. Human recombinant PDGF‐BB (40 ng/mL) was placed in the lower chamber and cells migrating through the membrane were counted after a 24‐h incubation. Data show the mean ± SEM ( n = 3). * p < .05.

Article Snippet: The sections were then incubated at 4°C overnight with rabbit polyclonal anti‐human FHL2 antibody (1/200) (HPA006028, Atlas antibodies, Stockholm, Sweden) or mouse monoclonal anti‐human CD34 (1/400, clone QBEnd/10, NBP2‐32932, Novus biologicals, Novus Biological, Littleton, CO, USA).

Techniques: Knockdown, Expressing, Western Blot, Transfection, Incubation, Control, Recombinant, Sulforhodamine B Assay, Collagen Gel Contraction Assay, Transwell Migration Assay, Membrane

Journal: International Journal of Cancer

Article Title: FHL2 expression by cancer‐associated fibroblasts promotes metastasis and angiogenesis in lung adenocarcinoma

doi: 10.1002/ijc.35174

Figure Lengend Snippet: FHL2 KO on CAFs decreases CAF‐induced migration and invasion of human lung adenocarcinoma cells in vitro and metastasis in an orthotopic lung cancer model. (A) Migration of lung adenocarcinoma cells induced by CAFs was analyzed. In the lower chamber of the Transwell chamber, CAF094 YFPhTERT cells (WT) or CAF094 YFPhTERT FHL2KO cells (KO) were seeded and incubated for 48 h. Normal medium (DMEM supplemented with 10% FBS) was used as a control (Cont). A110L or H23 adenocarcinoma cells were placed into the upper compartment of a Transwell chamber and the cells migrating through the membrane were counted after a 24‐h incubation. (B) Invasion of lung adenocarcinoma cells induced by CAF were analyzed. Matrigel‐coated cell culture inserts were used. In the lower compartment of the Transwell chamber, CAF094 YFPhTERT cells (WT) or CAF094 YFPhTERT FHL2KO cells (KO) were seeded and incubated for 48 h. Normal medium (DMEM supplemented with 10% FBS) was used as a control (Cont). A110L adenocarcinoma cells were placed into the upper compartment of a Transwell chamber and the cells migrating through the membrane were counted after a 24‐h incubation. Data represent the mean ± SEM ( n = 3). ** p < .01; * p < .05. (C) Assessment of the influence of FHL2 KO in CAFs on pulmonary metastasis using an orthotopic lung cancer model. A110L cells (A110L group) or A110L cells with CAF094 YFPhTERT cells (A110L + CAF group), or A110L cells with CAF094 YFPhTERT FHL2KO cells (A110L + CAFFHL2KO group) were inoculated into the left lungs of mice. All the mice in one experiment were sacrificed once significant morbidity was observed in any mouse in the experimental group. As a result, the experiments were terminated at approximately d37–d46. The formation of a primary lung tumor was confirmed and macroscopic pulmonary metastasis to the right lung was evaluated. (Left) Representative macroscopic view of the primary tumor and pulmonary metastasis in the A110L + CAF group. Arrow: Primary tumor arrowhead: Pulmonary metastasis. (Middle) Representative microscopic view of cytokeratin AE1/AE3 staining of main tumor in the left lung in the A110L group as a positive control of immunohistochemical staining to detect A110L lung cancer cells. Scale bar, 200 μm. (Right) Representative microscopic view of cytokeratin AE1/AE3 staining of micrometastasis in the right lung in the A110L + CAF group. Arrow: Primary pulmonary metastasis. Scale bar, 100 μm. (Table) Incidence of pulmonary metastasis in each group is shown. * p < .05.

Article Snippet: The sections were then incubated at 4°C overnight with rabbit polyclonal anti‐human FHL2 antibody (1/200) (HPA006028, Atlas antibodies, Stockholm, Sweden) or mouse monoclonal anti‐human CD34 (1/400, clone QBEnd/10, NBP2‐32932, Novus biologicals, Novus Biological, Littleton, CO, USA).

Techniques: Migration, In Vitro, Incubation, Control, Membrane, Cell Culture, Staining, Positive Control, Immunohistochemical staining

Journal: International Journal of Cancer

Article Title: FHL2 expression by cancer‐associated fibroblasts promotes metastasis and angiogenesis in lung adenocarcinoma

doi: 10.1002/ijc.35174

Figure Lengend Snippet: FHL2 KO in CAFs reduces CAF‐induced tube formation of MS1 cells and relationship of microvascular density and stromal FHL2 expression in clinical tumor samples. (A, B) Results of an endothelial cell tube formation assay. Murine endothelial MS1 cells were seeded in conditioned medium from CAF094 YFPhTERT cells (WT) or CAF094 YFPhTERT FHL2KO cells (KO). Photographs taken 6 h post‐seeding were analyzed. (A) Representative photographs of WT and KO groups are shown. (B) results of the quantification of tube formation are shown. Data represent the mean ± SEM. (C) Immunohistochemical analysis of CD34 in lung adenocarcinoma tissues from patients who underwent surgery. Scale bar, 50 μm. (Left) Representative image showing a tumor with high microvascular density. (Right) Representative image showing a tumor with low microvascular density. (D) Relationship of microvascular density and FHL2 expression. (Left) Relationship of microvascular density and tumoral FHL2 expression. (Right) Relationship of microvascular density and stromal FHL2 expression. **p < .01; *p < .05.

Article Snippet: The sections were then incubated at 4°C overnight with rabbit polyclonal anti‐human FHL2 antibody (1/200) (HPA006028, Atlas antibodies, Stockholm, Sweden) or mouse monoclonal anti‐human CD34 (1/400, clone QBEnd/10, NBP2‐32932, Novus biologicals, Novus Biological, Littleton, CO, USA).

Techniques: Expressing, Endothelial Cell Tube Formation Assay, Immunohistochemical staining

Journal: International Journal of Cancer

Article Title: FHL2 expression by cancer‐associated fibroblasts promotes metastasis and angiogenesis in lung adenocarcinoma

doi: 10.1002/ijc.35174

Figure Lengend Snippet: Survival analysis according to tumoral or stromal FHL2 expression.

Article Snippet: The sections were then incubated at 4°C overnight with rabbit polyclonal anti‐human FHL2 antibody (1/200) (HPA006028, Atlas antibodies, Stockholm, Sweden) or mouse monoclonal anti‐human CD34 (1/400, clone QBEnd/10, NBP2‐32932, Novus biologicals, Novus Biological, Littleton, CO, USA).

Techniques: Expressing

Journal: International Journal of Cancer

Article Title: FHL2 expression by cancer‐associated fibroblasts promotes metastasis and angiogenesis in lung adenocarcinoma

doi: 10.1002/ijc.35174

Figure Lengend Snippet: FHL2 KO in CAFs reduces OPN secretion from CAF and knock‐down of OPN in CAF or antibody blocking of OPN reduces CAF induced migration of A110L. (A) Relative concentrations of 13 proteins of which concentration was significantly different between CM of CAF094 YFPhTERT cells (WT) and CAF094 YFPhTERT FHL2KO cells (KO). Data represent the mean ± SEM ( n = 3). (B) Western blot analysis of OPN in cell lysate of CAF094 YFPhTERT and CAF094 YFPhTERT FHL2KO cells. GAPDH was used as an internal control. (C) Silencing of OPN in CAF094 hTERTRFP by siRNA. Three different siRNAs and smart pool (SP; mixture of three different siOPN RNAs) were used. Western blot analysis showing OPN expression in CAF094 hTERTRFP transfected with siRNAs. (D) Knock down effect of OPN in CAF on migration of lung adenocarcinoma cell A110L induced by CAF CM was analyzed. In the lower chamber of the Transwell chamber, CM from CAF094 YFPhTERT cells transfected with siScr (control) or siOPN‐SP (mixture of three different siOPN RNAs) were placed. A110L adenocarcinoma cells were placed into the upper compartment of a Transwell chamber and the cells migrating through the membrane were counted after a 24‐h incubation. Data of average of three experiments was shown. ** p < .01. (E) Antibody blocking effect of OPN on migration of lung adenocarcinoma cell A110L induced by CAF CM was analyzed. In the lower chamber of the Transwell chamber, CM from CAF094 YFPhTERT cells incubated with control IgG or anti OPN antibody were placed. A110L adenocarcinoma cells were placed into the upper compartment of a Transwell chamber and the cells migrating through the membrane were counted after a 24‐h incubation. Data of average of three experiments was shown. * p < .05.

Article Snippet: The sections were then incubated at 4°C overnight with rabbit polyclonal anti‐human FHL2 antibody (1/200) (HPA006028, Atlas antibodies, Stockholm, Sweden) or mouse monoclonal anti‐human CD34 (1/400, clone QBEnd/10, NBP2‐32932, Novus biologicals, Novus Biological, Littleton, CO, USA).

Techniques: Knockdown, Blocking Assay, Migration, Concentration Assay, Western Blot, Control, Expressing, Transfection, Membrane, Incubation

Journal: International Journal of Cancer

Article Title: FHL2 expression by cancer‐associated fibroblasts promotes metastasis and angiogenesis in lung adenocarcinoma

doi: 10.1002/ijc.35174

Figure Lengend Snippet: Gene expression signature analysis and comparisons to common lung adenocarcinoma driver alterations. (A) Scatter plots of FHL2 mRNA expression versus six proposed biological metagenes in lung cancer. Correlation was calculated using Pearson correlation. Dotted red line corresponds to a linear regression model of FHL2 versus respective metagene. Both FHL2 mRNA expression and metagene scores were mean‐centered as described in Karlsson et al. ¹⁸ (B) FHL2 mRNA expression stratified by gene mutation status for EGFR , KRAS , STK11 , and TP53 in the Lund lung adenocarcinoma cohort. p ‐Values were calculated using Wilcoxon's test.

Article Snippet: The sections were then incubated at 4°C overnight with rabbit polyclonal anti‐human FHL2 antibody (1/200) (HPA006028, Atlas antibodies, Stockholm, Sweden) or mouse monoclonal anti‐human CD34 (1/400, clone QBEnd/10, NBP2‐32932, Novus biologicals, Novus Biological, Littleton, CO, USA).

Techniques: Gene Expression, Expressing, Mutagenesis

Journal: International Journal of Cancer

Article Title: FHL2 expression by cancer‐associated fibroblasts promotes metastasis and angiogenesis in lung adenocarcinoma

doi: 10.1002/ijc.35174

Figure Lengend Snippet: Expression and significance of FHL2 in clinical tumor samples. (A) Immunohistochemical analysis of FHL2 in lung adenocarcinoma tissues from patients with lung adenocarcinoma who underwent surgery (Osaka cohort). Scale bar, 100 μm. Upper panels show representative images of tumoral and stromal FHL2‐negative, tumoral FHL2‐negative stromal FHL2‐positive, tumoral FHL2‐positive stromal FHL2 negative, and tumoral and stromal FHL2‐positive tumor tissues. Tissues were stained with an anti‐FHL2 antibody (brown) and counterstained with hematoxylin. Lower panels show schematic image of tumoral and stromal components. S, stromal component; T, tumoral component. (B) Relationship between tumoral and stromal FHL2 expression in each tumor. (C) Relationship between stromal FHL2 expression and lymph node metastasis. (D) Relationship between stromal FHL2 expression and histologic subtype. AIS, adenocarcinoma in situ; MIA, minimally invasive adenocarcinoma. (E) (Left) Association of stromal FHL2 expression with recurrence in the Osaka lung adenocarcinoma cohort. Kaplan–Meier plot showing the cumulative recurrence rate in patients with lung adenocarcinoma who underwent surgery relative to stromal FHL2 expression. Patients were divided into four groups according to tumoral and stromal FHL2 expression, that is, both tumoral and stromal FHL2‐negative group (double negative [DN]), both tumoral and stromal FHL2‐positive group (double positive [DP]), tumoral FHL2‐negative and stromal FHL2‐positive group (stromal positive [SP]), and tumoral FHL2‐positive and stromal FHL2‐negative group (tumoral positive [TP]). The 5‐year cumulative recurrence rate of DN, DP, SP, TP groups were 11.6%, 18.2%, 38.1%, and 15.0%, respectively. The 5‐year cumulative recurrence rate of SP group was significantly higher compared with that of DN group ( p < .01), however, the difference between SP and DP, TP groups were not significant. (Right) Association of stromal FHL2 expression with disease‐specific survival in the Osaka cohort. Kaplan–Meier plot of the disease‐specific survival rate in patients with lung adenocarcinoma who underwent surgery according to stromal FHL2 expression. The 5‐year disease‐specific survival rate of DN, DP, SP, TP groups were 96.2%, 88.9%, 88.0%, and 96.3%, respectively. The 5‐year disease‐specific survival rate of SP group was significantly lower compared with that of DN group ( p < .05), however, the difference between SP and DP, TP groups were not significant. DN, double negative; DP, double positive; SP, stromal positive; TP, tumoral positive.

Article Snippet: The sections were then incubated at 4°C overnight with rabbit polyclonal anti‐human FHL2 antibody (1/200) (HPA006028, Atlas antibodies, Stockholm, Sweden) or mouse monoclonal anti‐human CD34 (1/400, clone QBEnd/10, NBP2‐32932, Novus biologicals, Novus Biological, Littleton, CO, USA).

Techniques: Expressing, Immunohistochemical staining, Staining, In Situ

Journal: Cell Death Discovery

Article Title: Venlafaxine antagonizes the noradrenaline-promoted colon cancer progression by inhibiting the norepinephrine transporter

doi: 10.1038/s41420-023-01447-5

Figure Lengend Snippet: After 1-week acclimatization, male BALB/c mice were randomly divided into four groups ( n = 5): control (saline), NE (0.125 mg/kg), VEN (10 mg/kg), and drug combination [NE (0.125 mg/kg) + VEN (10 mg/kg)], and administered intraperitoneally with each drug or combination every day. One week later, 1 × 10 6 CT26 cells were injected subcutaneously into the right oxters of the mice. Furthermore, mice of each group were treated with the above drug or drug combination for additional 17 days. A The volumes of tumor xenografts were measured and revealed (upper left). On day 17, mice were sacrificed, and tumor xenografts were removed. The gross morphology, tumor volume, and tumor weight of xenografts were detected (upper right, lower left, and lower right). B Hematoxylin and eosin (HE) staining and CD34 immunohistochemistry (IHC) analysis of the xenograft sections were performed. Microvessel density (MVD) was counted based on CD34 IHC. C The xenograft lysates were collected, and the expressions of CD34, VEGF, and NET were analyzed by western blotting. The band intensities of each protein were normalized by that of β-actin and the average value was obtained from the repetition in each group. Data are expressed as mean ± SD, * P < 0.05, ** P < 0.01 in A , B , C .

Article Snippet: Antibodies against CD31 (Proteintech Cat# 11265-1-AP, RRID: AB_2299349), CD34 (Proteintech Cat# 14486-1-AP, RRID: AB_2228975), vascular endothelial growth factor (VEGF, Proteintech Cat# 19003-1-AP, RRID: AB_2212657), CDK2 (Proteintech Cat# 10122-1-AP, RRID: AB_2078556), cyclin E (Proteintech Cat# 11554-1-AP, RRID: AB_2071066), Akt (Proteintech Cat# 10176-2-AP, RRID: AB_2224574), and protein phosphatase 2 scaffold subunit alpha (PPP2R1A, Proteintech Cat# 15882-1-AP, RRID: AB_2237574) were purchased from Proteintech Group (Wuhan, Hubei, China).

Techniques: Control, Saline, Injection, Staining, Immunohistochemistry, Western Blot

Journal: Cell Death Discovery

Article Title: Venlafaxine antagonizes the noradrenaline-promoted colon cancer progression by inhibiting the norepinephrine transporter

doi: 10.1038/s41420-023-01447-5

Figure Lengend Snippet: A Following acclimatization, male BALB/c mice were randomly divided into four groups ( n = 5): CT26-Lv-shCtrl.+saline, CT26-Lv-shNET-3+saline, CT26-Lv-shCtrl.+NE, and CT26-Lv-shNET-3+NE. Male BALB/c mice were intraperitoneally administered with saline or NE (0.125 mg/kg) daily. One week later, 1 × 10 6 stable cells (CT26-Lv-shNET-3 or CT26-Lv-shCtrl.) were injected subcutaneously into the mice separately. In addition, mice of each group were treated with saline or NE for 17 days. Volumes of tumor xenografts were measured (upper left). On day 17 following cell inoculation, mice were scarified, and tumor xenografts were removed. The gross morphology of tumors, tumor volume, and tumor weight of each group were detected (upper right, lower left, and lower right). B Hematoxylin and eosin (HE) staining of the xenograft sections and CD34 immunohistochemical analysis was performed. C Xenograft lysates were collected, and the expression levels of PPP2R1A, pAkt, Akt, VEGF, and CD34 were analyzed by western blotting. The band intensities of each protein were normalized by that of β-actin and the average value was obtained from the repetition in each group. Data are expressed as mean ± SD, * P < 0.05, ** P < 0.01, or *** P < 0.001 in A and C .

Article Snippet: Antibodies against CD31 (Proteintech Cat# 11265-1-AP, RRID: AB_2299349), CD34 (Proteintech Cat# 14486-1-AP, RRID: AB_2228975), vascular endothelial growth factor (VEGF, Proteintech Cat# 19003-1-AP, RRID: AB_2212657), CDK2 (Proteintech Cat# 10122-1-AP, RRID: AB_2078556), cyclin E (Proteintech Cat# 11554-1-AP, RRID: AB_2071066), Akt (Proteintech Cat# 10176-2-AP, RRID: AB_2224574), and protein phosphatase 2 scaffold subunit alpha (PPP2R1A, Proteintech Cat# 15882-1-AP, RRID: AB_2237574) were purchased from Proteintech Group (Wuhan, Hubei, China).

Techniques: Saline, Injection, Staining, Immunohistochemical staining, Expressing, Western Blot

Journal: eLife

Article Title: Identification of specific Tie2 cleavage sites and therapeutic modulation in experimental sepsis

doi: 10.7554/eLife.59520

Figure Lengend Snippet:

Article Snippet: Antibody , CD144 (VE-cadherin) , BD Biosciences , Cat#: 555661 , IF:1:100.

Techniques: Enzyme-linked Immunosorbent Assay, Recombinant, Software