Journal: Aging

Article Title: Age-related defects in autophagy alter the secretion of paracrine factors from bone marrow mononuclear cells.

doi: 10.18632/aging.203127

Figure Lengend Snippet: Figure 3. Atg7 knockdown in Y-Sca-1+ BMCs blocks paracrine stimulation of old cardiac fibroblasts. (A) Atg7 expression was measured in Y-Sca-1+ bone marrow cells (BMCs), treated with Atg7-siRNA or sc-siRNA, by RT-qPCR. n=3-4. (B–E) Quantification and representative images from (B) scratch wound assay, (C) proliferation assay, (D) stress fiber formation or (E) β-galactosidase staining of old cardiac fibroblasts, treated with Y-Sca-1+ Atg7-siRNA CM or Y-Sca-1+ sc-siRNA CM for 24-48 hours. Dashed yellow line in (B) indicates the wound edge at 0 hours. Scale bar represents 100 μm. Data analysis was carried out by t-test. Data presented as mean ± SEM; n=4-6; **p≤0.01.

Article Snippet: The nonspecific siRNA oligonucleotides (scrambled siRNA) and siRNA oligonucleotides targeting mouse Atg7 were purchased from OriGene (#SR427399). siRNA was transfected into Y-Sca-1+ BMCs using Lipofectamine 2000 (#11668-027, Invitrogen) according to the manufacturer’s protocol.

Techniques: Knockdown, Expressing, Quantitative RT-PCR, Scratch Wound Assay Assay, Proliferation Assay, Staining

Journal: Development (Cambridge, England)

Article Title: Syndecan 4 controls lymphatic vasculature remodeling during mouse embryonic development

doi: 10.1242/dev.140129

Figure Lengend Snippet: Lymphatic vessels show remodeling defects in Sdc4−/− mice during late embryonic development. (A-D) Whole-mount immunofluorescence staining for Vegfr3, showing a hierarchal lymphatic vascular network in the mesentery of control mice at E18.5 (A,B); yellow arrow, capillaries; white arrows, lymphatic collecting vessels. In Sdc4 nulls, by contrast, lymphatic vessels are abnormally branched and tend to form a vascular plexus (C, arrows). Enlarged lymphatic vessels were also seen in Sdc4−/− mice (D, arrows). (E) Prox1high lymphatic valve-forming ECs reorient perpendicular to flow direction at E17.5 in control animals. The nuclei of valve-forming cells are highly elongated and are tightly packed (a, arrowheads). At E18.5, valve-forming cells reorganize again and form mature lymphatic valves containing two leaflets (b, arrowheads). Prox1high valve-forming cells are concentrated in valve areas in the control (c, arrows). (F) The nuclei of Prox1high valve-forming cells are rounded (a, arrowheads), randomly oriented and loosely arranged in Sdc4−/− mice at E17.5. Nuclei of similar morphology and organization were also seen in the mutants at E18.5 (b). Some of the Prox1high valve-forming cells in Sdc4−/− embryos do not reorient, and remain parallel to the longitudinal axis of lymphatic vessels (c, arrow). (G) Nuclear orientation of Prox1high cells in E17.5 mesenteric lymphatic vessels of control and Sdc4−/− mice (n=5). The percentage of nuclei reoriented >45° relative to flow direction is indicated. Mann–Whitney test. (H) Quantification of nuclear shape (roundness) of Prox1high cells in E17.5 mesenteric lymphatic vessels (n=5). Mann–Whitney test. Scale bars: 200 μm in A-D; 30 μm in Ea,b,Fa,b; 100 μm in Ec,Fc.

Article Snippet: Transfected cells were harvested for experiments 72 h after transfection. siRNAs used for gene KD were from OriGene Technologies: scrambled siRNA (NC1 Control SR30004) and siRNA against human SDC4 (SR304301), human VANGL2 (SR311428) or human PECAM1 (SR303439), each at 15 nM.

Techniques: Immunofluorescence, Staining, MANN-WHITNEY

Journal: Development (Cambridge, England)

Article Title: Syndecan 4 controls lymphatic vasculature remodeling during mouse embryonic development

doi: 10.1242/dev.140129

Figure Lengend Snippet: Abnormal alignment of actin filaments and compromised formation of lymphatic valves in Sdc4−/− mice. (A) Phalloidin labeling showing that long, fine actin fibers of LECs align with the flow direction in control animals. (B) In Sdc4 nulls, actin filaments are thick, short and randomly organized. Yellow arrows (A,B) indicate flow direction in lymphatic vessel. (C) Representative V-shaped mature lymphatic valves in control mice (arrows). (D,E) Ring-shaped immature lymphatic valves (D) and abnormal valves consisting of randomly oriented Prox1high lymphatic valve-forming ECs (E) in Sdc4−/− animals. (F) Quantification of lymphatic valves formed in mesentery at E18.5 based on Prox1 and VE-cadherin staining. The proportion (%) of different types of valves relative to the total number of valves formed per mesentery is shown. V-shaped valves are defined as mature valves. Immature valves are ring-shaped. Valves consisting of randomly organized Prox1high valve-forming ECs or Prox1high cells remaining parallel to the flow direction are considered as abnormal valves. Student's t-test (two-tailed). Data are mean±s.e.m. (n=3). Scale bars: 10 μm in A,B; 15 μm in C-E.

Article Snippet: Transfected cells were harvested for experiments 72 h after transfection. siRNAs used for gene KD were from OriGene Technologies: scrambled siRNA (NC1 Control SR30004) and siRNA against human SDC4 (SR304301), human VANGL2 (SR311428) or human PECAM1 (SR303439), each at 15 nM.

Techniques: Labeling, Staining, Two Tailed Test

Journal: Development (Cambridge, England)

Article Title: Syndecan 4 controls lymphatic vasculature remodeling during mouse embryonic development

doi: 10.1242/dev.140129

Figure Lengend Snippet: Sdc4−/−; Pecam1−/− double nulls show a severe lymphatic phenotype. (A) Mesenteric lymphatic vessels in Sdc4−/−; Pecam1−/− embryos show irregular morphology (middle, white arrow), increased diameter (middle, yellow arrow) and abnormal branching (bottom, arrows) at E18.5, which were not seen in controls (top, arrows). (B) Sparse mural cell (α-SMA+) coverage in mesenteric lymphatic vessels in control mice at E18.5. (C) Extensive mural cell recruitment in lymphatic vessels of Sdc4−/−; Pecam1−/− double nulls. A, artery; V, vein; L, lymphatic vessel. (D) Mature V-shaped valves in mesenteric lymphatic vessels of control mice at E18.5 (arrows). (E) Abnormal lymphatic valves containing randomly oriented Prox1high valve-forming ECs formed in Sdc4−/−; Pecam1−/− mice at E18.5 (arrows). (F) Quantification of lymphatic valves formed in the mesentery of control and Sdc4−/−; Pecam1−/− mice at E18.5 according to antibody staining for Prox1 and VE-cadherin. The proportion (%) of different types of valves relative to the total number of valves formed per mesentery is shown. Mature valves are V-shaped; immature valves are ring shaped; abnormal valves are those consisting of randomly organized Prox1high valve-forming cells or Prox1high cells aligned parallel to the longitudinal axis of lymphatic vessels. Student's t-test (two-tailed). Data represent mean±s.e.m. (n=3). Scale bars: 200 μm in A; 100 μm in B,C; 15 μm in D,E.

Article Snippet: Transfected cells were harvested for experiments 72 h after transfection. siRNAs used for gene KD were from OriGene Technologies: scrambled siRNA (NC1 Control SR30004) and siRNA against human SDC4 (SR304301), human VANGL2 (SR311428) or human PECAM1 (SR303439), each at 15 nM.

Techniques: Staining, Two Tailed Test

Journal: Development (Cambridge, England)

Article Title: Syndecan 4 controls lymphatic vasculature remodeling during mouse embryonic development

doi: 10.1242/dev.140129

Figure Lengend Snippet: The PCP protein Vangl2 is upregulated in LECs in Sdc4−/− mice. (A) qRT-PCR analysis of relative VANGL2 expression in human LECs after being subjected to laminar flow (8 dynes/cm2) for 20 h versus static cells. mRNA expression was normalized to endogenous β-actin (ACTB). Mann–Whitney test (n=6). *P<0.05. Values represent mean±s.e.m. (B) qRT-PCR for Vangl2 and Celsr1 in thoracic ducts of WT and Sdc4−/− mice. mRNA expression was normalized to endogenous 18S rRNA and Cdh5. Mann–Whitney test (n=4). *P<0.05. Values are mean±s.e.m. (C,D) Visualization of the orientation of control LECs (pLenti-control) (left) and cells overexpressing VANGL2 (pLenti-VANGL2) (right) by phalloidin labeling (green) and DAPI staining (blue). VANGL2-overexpressing LECs do not align to flow direction (D, right). Scale bars: 20 μm. (E) Quantification of cell orientation of control and VANGL2-overexpressing LECs. Cells were either maintained in no flow conditions (static) or subjected to laminar flow (8 dynes/cm2) for 16 h. One-way ANOVA. ns, not significant; **P<0.01. n>2700 cells/condition/experiment. Four independent experiments were performed. Values are mean±s.e.m.

Article Snippet: Transfected cells were harvested for experiments 72 h after transfection. siRNAs used for gene KD were from OriGene Technologies: scrambled siRNA (NC1 Control SR30004) and siRNA against human SDC4 (SR304301), human VANGL2 (SR311428) or human PECAM1 (SR303439), each at 15 nM.

Techniques: Quantitative RT-PCR, Expressing, MANN-WHITNEY, Labeling, Staining

Journal: Development (Cambridge, England)

Article Title: Syndecan 4 controls lymphatic vasculature remodeling during mouse embryonic development

doi: 10.1242/dev.140129

Figure Lengend Snippet: SDC4 controls flow sensing in LECs through VANGL2. (A,B) Phalloidin labeling (green) and DAPI staining (blue) show the arrangement of human LECs under static conditions (A) and sheared with laminar flow (8 dynes/cm2) for 16 h (B). Scale bars: 20 μm. (C) Quantification of cell orientation of LECs transfected with a scrambled, SDC4, or SDC4 and VANGL2 siRNA. Cells were either cultured at a no flow condition (static) or subjected to laminar flow (8 dynes/cm2) for 16 h. Two-way ANOVA. ns, not significant; **P<0.01, ***P<0.001. n>1800 cells/condition/experiment. Four independent experiments were performed. Values are mean±s.e.m.

Article Snippet: Transfected cells were harvested for experiments 72 h after transfection. siRNAs used for gene KD were from OriGene Technologies: scrambled siRNA (NC1 Control SR30004) and siRNA against human SDC4 (SR304301), human VANGL2 (SR311428) or human PECAM1 (SR303439), each at 15 nM.

Techniques: Labeling, Staining, Transfection, Cell Culture

Journal: Pathogens

Article Title: Cross-Sectional Study on the Prevalence of PCV Types 2 and 3 DNA in Suckling Piglets Compared to Grow–Finish Pigs in Downstream Production

doi: 10.3390/pathogens11060671

Figure Lengend Snippet: Cq values of tissue pools (PCV2 and PCV3 PCR) in regard to the age (1–7 days of age) of the sampled piglets (circle and asterisk indicate outliers).

Article Snippet: For virus detection, we used a quantitative duplex PCR (Virotype ® PCV2/PCV3, INDICAL BIOSCIENCE GmbH, Leipzig, Germany).

Techniques:

Journal: Scientific Reports

Article Title: Vitamin D receptor and its antiproliferative effect in human pulmonary arterial hypertension

doi: 10.1038/s41598-024-78380-9

Figure Lengend Snippet: Calcitriol inhibits PASMC proliferation via BMPR2. Human control PASMC were transfected with siRNA for BMPR2 (siBMPR2) or control siRNA (scramble). ( A ) BMPR2 mRNA expression assessed by qRT-PCR after 48 h post-transfection. Data is expressed as scatter plots and bars with medians, * p < 0.05, Mann–Whitney test. ( B ) Effects of calcitriol on proliferation in silenced BMPR2 PASMC measured by MTT assay. Results are expressed as mean ± SEM. *p < 0.05, t-test vs scramble. ( C ) and ( D ) Effects of calcitriol (1–100 nmol/l) on proliferation in PASMC transfected with siBMPR2 or scramble, measured by MTT and BrdU assays, respectively. * p < 0.05, two-way ANOVA, Bonferroni post hoc test, vs scramble. ( E ) Effects of calcitriol on proliferation in PASMC in the presence or absence of DMH1 (5 µmol/l), by MTT and BrdU assay; *** p < 0.001 calcitriol vs vehicle (black column), two way-ANOVA. n = 4–5 different cultures in duplicate or triplicate.

Article Snippet: Human control PASMC were transfected with specific siRNA against BMPR2 (SR300456B, OriGene, Maryland, USA), KCNK3 (gene encoding TASK-1, SASI_Hs01_00108786, Sigma-Aldrich, St. Louis, USA), BIRC5 ( gene encoding survivin, SASI_Hs01_00052229, Sigma-Aldrich, St. Louis, USA), and control non-targeting siRNA (SIC001 scramble siRNA, Sigma-Aldrich, St. Louis, USA), using LipofectamineTM RNAiMAX (Thermo Fisher Scientific, Massachusetts, USA) according to the manufacturer’s instructions as previously reported .

Techniques: Control, Transfection, Expressing, Quantitative RT-PCR, MANN-WHITNEY, MTT Assay, BrdU Staining

Journal: Carcinogenesis

Article Title: The p53 target gene desmocollin 3 acts as a novel tumor suppressor through inhibiting EGFR/ERK pathway in human lung cancer.

doi: 10.1093/carcin/bgs273

Figure Lengend Snippet: Fig. 1. Downregulation of DSC3 expression and aberrant methylation of DSC3 in lung cancer cell lines. (A) DSC3 mRNA expression in 11 lung cancer cell lines was detected by real-time RT–PCR. Normalized mRNA levels are presented relative to normal HBECs arbitrarily set to 1.0. Glyceraldehyde 3-phosphate dehydrogenase was used as control. The data are presented as the means of three independent experiments ± standard deviation. (B) DSC3 protein expression was detected by western blotting in lung cancer cell lines and HBEC. Two bands represent ‘a’ and ‘b’ isoforms of DSC3 protein. β-actin was used as loading control. (C) Real-time RT–PCR showed that after treatment with 10 µM DAC for 96 h, DSC3 mRNA expression was upregulated in seven cell lines. (−) = untreated; (+) = treated with DAC. The data are expressed as the mean values of three independent experiments ± standard deviation. (D) Methylation pattern of DSC3 in the promoter region. Black square: totally methylated CpG site; gray square: partially methylated CpG site; white square: unmethylated CpG site.

Article Snippet: The human DSC3 small interfering RNA (siRNA) duplexes (3 unique 27mer) were synthesized by OriGene Technologies Inc (Rockville, MD USA).

Techniques: Expressing, Methylation, Quantitative RT-PCR, Control, Standard Deviation, Western Blot

Journal: Carcinogenesis

Article Title: The p53 target gene desmocollin 3 acts as a novel tumor suppressor through inhibiting EGFR/ERK pathway in human lung cancer.

doi: 10.1093/carcin/bgs273

Figure Lengend Snippet: Fig. 2. Expression of DSC3 in primary lung tumors. (A) Representative IHC results of DSC3 protein expression from primary lung tumors. IHC was scored semiquantitatively as negative (score 0), weak (score 1), moderate (score 2) and strong expression (score 3). Image with ×40 magnification of the corresponding case is shown in the inset. (B) Kaplan–Meier curves showed that patients whose tumors harbored methylated DSC3 DNA had a worse overall survival than patients whose tumors harbored unmethylated DSC3 DNA, indicating DSC3 methylation status is a prognostic marker for primary lung tumors.

Article Snippet: The human DSC3 small interfering RNA (siRNA) duplexes (3 unique 27mer) were synthesized by OriGene Technologies Inc (Rockville, MD USA).

Techniques: Expressing, Methylation, Marker

Journal: Carcinogenesis

Article Title: The p53 target gene desmocollin 3 acts as a novel tumor suppressor through inhibiting EGFR/ERK pathway in human lung cancer.

doi: 10.1093/carcin/bgs273

Figure Lengend Snippet: Fig. 3. The effect of p53 on DSC3 expression. (A) Induction of p53 protein expression was measured by western blotting after transfection of the two lung cancer cell lines H2170 and H1299 with an expression vector encoding wild-type p53. Empty vector was used as control. (B) Induction of DSC3 mRNA expression was measured by quantitative RT–PCR after transfection with the p53 expression vector. Empty vector was used as control. Expression of DSC3 was upregulated in H2170, but not in H1299. (C) Methylation status of DSC3 in the p53 binding site was analyzed by bisulfite sequencing. H2170 was unmethylated, whereas H1299 was methylated. Black square: totally methylated CpG site; gray square: partially methylated CpG site; white square: unmethylated CpG site. (D) After DAC treatment, p53-induced DSC3 expression was enhanced in H1299.

Article Snippet: The human DSC3 small interfering RNA (siRNA) duplexes (3 unique 27mer) were synthesized by OriGene Technologies Inc (Rockville, MD USA).

Techniques: Expressing, Western Blot, Transfection, Plasmid Preparation, Control, Quantitative RT-PCR, Methylation, Binding Assay, Methylation Sequencing

Journal: Carcinogenesis

Article Title: The p53 target gene desmocollin 3 acts as a novel tumor suppressor through inhibiting EGFR/ERK pathway in human lung cancer.

doi: 10.1093/carcin/bgs273

Figure Lengend Snippet: Fig. 4. Effects of ectopic DSC3 expression on the proliferation, clonogenicity, migration and invasion of lung cancer cells. DSC3 re-expression in stably transfected cell lines H2170 and H1299 was confirmed by western blotting (A) and immunofluorescence (B). (C) Growth curves of mock transfectants, and DSC3 transfectants showed that DSC3 transfectants grew slower than mock transfectants. The data are shown as the mean values of three independent experiments ± standard deviation. (D) Quantitative analyses of colony numbers were performed. A significant reduction in colony numbers was observed in DSC3 transfectants compared with mock transfectants. (E) Cell motility was determined by wound-healing assay in the presence of mitomycin C. The ability of DSC3 transfectants spreading along the wound edges was significantly reduced compared with control cells. (F) Quantification of the migrated and invaded cells. All the data present are the mean values of three independent experiments ± standard deviation. *,P < 0.05; **, P < 0.01.

Article Snippet: The human DSC3 small interfering RNA (siRNA) duplexes (3 unique 27mer) were synthesized by OriGene Technologies Inc (Rockville, MD USA).

Techniques: Expressing, Migration, Stable Transfection, Transfection, Western Blot, Immunofluorescence, Standard Deviation, Wound Healing Assay, Control

Journal: Carcinogenesis

Article Title: The p53 target gene desmocollin 3 acts as a novel tumor suppressor through inhibiting EGFR/ERK pathway in human lung cancer.

doi: 10.1093/carcin/bgs273

Figure Lengend Snippet: Fig. 5. DSC3 attenuated the activation of EGFR/ERK pathway. (A) DSC3 transfectant cells and mock transfectant cells were treated with 50 ng/ml of EGF for 30 min, and the expression levels of EGFR, phospho-EGFR and DSC3 were analyzed by western blotting. EGF treatment led to EGFR phosphorylation and decreased DSC3. (B) The cells were treated with 10 µM gefitinib for 24 h alone or together with 50 ng/ml of EGF for 30 min. The expression levels of phospho- EGFR and phospho- ERK1/2 were inhibited by gefitinib in both transfectant cells and mock transfectants. The expression of DSC3 was upregulated in the combination of gefitinib and EGF treatment. (C) In H226 cells with endogenous expression of DSC3, treatment with 50ng/ml of EGF resulted in decreased DSC3 expression. (D) The expression levels of phospho-ERK1/2 were reduced in DSC3 transfectants compared with mock transfectants. (E) Cells were treated with 50 µM of PD98059, an ERK inhibitor, alone for 60 min or together with 50 ng/ml of EGF for 30 min. In PD98059 pre-treated cells, the upregulation of phospho- ERK1/2 by EGF stimulation decreased. β-Actin was used as loading control for western blotting. (F) H226 cells with endogenous expression of DSC3 were transfected with DSC3 siRNA in parallel with a non-targeting siRNA control. Knockdown of DSC3 expression was confirmed by western blotting, and DSC3- knockdown cells displayed higher level of phospho-ERK1/2 compared with control cells.

Article Snippet: The human DSC3 small interfering RNA (siRNA) duplexes (3 unique 27mer) were synthesized by OriGene Technologies Inc (Rockville, MD USA).

Techniques: Activation Assay, Transfection, Expressing, Western Blot, Phospho-proteomics, Control, Knockdown

Journal: The Journal of biological chemistry

Article Title: Mitochondrial cytochrome P450 1B1 is involved in pregnenolone synthesis in human brain cells.

doi: 10.1016/j.jbc.2023.105035

Figure Lengend Snippet: Figure 1. Effect of CYP27A1 inhibitors and siRNA knockdown on pregnenolone synthesis in MGM-1 cells. A–D, ELISA measurements of pregnenolone in culture media of MGM-1 cells treated with four different doses of anastrozole (A and B) or dexmedetomidine (C and D) for 2 h, under basal conditions (A and C) or with 50 μM 22(R)-hydroxycholesterol treatment (B and D). E–G, siRNA knockdown of CYP27A1 in MGM-1 cells. Knockdown efficiency was determined by qRT-PCR (E) and immunoblot (F). G, ELISA measurements of secreted pregnenolone in transfected MGM-1 cells. Each data point represents the average from one experiment, where each treatment was performed in triplicate within each experiment. Data are presented as mean ± SD, N = 3. Statistics performed compared to no-inhibitor treatment group (A–D) or scrambled negative transfection control (E and G). (* p < 0.05, ** p < 0.01, *** p < 0.001). qRT-PCR, quantitative reverse transcription PCR.

Article Snippet: The cells were transfected with 5 nM scrambled negative control siRNA duplexes or genespecific siRNA duplexes (Origene, #SR301124 for CYP27A1, #SR301093 for CYP1A1, and #SR301095 for CYP1B1) using siTran 2.0 transfection reagents (Origene, #TT320001), according to the manufacturer’s instructions.

Techniques: Knockdown, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Western Blot, Transfection, Control, Reverse Transcription

Journal: The Journal of biological chemistry

Article Title: Mitochondrial cytochrome P450 1B1 is involved in pregnenolone synthesis in human brain cells.

doi: 10.1016/j.jbc.2023.105035

Figure Lengend Snippet: Figure 3. Effect of CYP1A1 or CYP1B1 siRNA knockdown on pregnenolone synthesis in MGM-1 cells. Knockdown of CYP1A1 or CYP1B1 expression determined by qRT-PCR (A and B). Pregnenolone synthesis of transfected MGM-1 cells measured by ELISA (C and D). Each data point represents the average from one experiment, where each treatment was performed in triplicate within each experiment. Data are presented as mean ± SD, N = 3. Statistics performed compared to scrambled negative control. (*p < 0.05, ***p < 0.001). qRT-PCR, quantitative reverse transcription PCR.

Article Snippet: The cells were transfected with 5 nM scrambled negative control siRNA duplexes or genespecific siRNA duplexes (Origene, #SR301124 for CYP27A1, #SR301093 for CYP1A1, and #SR301095 for CYP1B1) using siTran 2.0 transfection reagents (Origene, #TT320001), according to the manufacturer’s instructions.

Techniques: Knockdown, Expressing, Quantitative RT-PCR, Transfection, Enzyme-linked Immunosorbent Assay, Negative Control, Reverse Transcription

Journal: The Journal of biological chemistry

Article Title: Mitochondrial cytochrome P450 1B1 is involved in pregnenolone synthesis in human brain cells.

doi: 10.1016/j.jbc.2023.105035

Figure Lengend Snippet: Figure 4. Effect of CYP1A1/CYP1B1 inhibitors or siRNA knockdown on pregnenolone synthesis in H295R-S1 cells. A–D, ELISA measurements of pregnenolone in culture media of H295R-S1 cells treated with different doses of ANF (A and C) or TMS (B and D) for 2 h, at basal (A and B) levels or with 50 μM 22(R)-HC treatment (C and D). E–G, knockdown of CYP1A1 or CYP1B1 via siRNA and its effect on pregnenolone synthesis (G) in H295R-S1 cells. Knockdown efficiency was determined by qRT-PCR (E and F). (**p < 0.01, ***p < 0.001). ANF, α-napthoflavone; qRT-PCR, quantitative reverse transcription PCR; TMS, 2,4,30,50-tetramethoxystilbene.

Article Snippet: The cells were transfected with 5 nM scrambled negative control siRNA duplexes or genespecific siRNA duplexes (Origene, #SR301124 for CYP27A1, #SR301093 for CYP1A1, and #SR301095 for CYP1B1) using siTran 2.0 transfection reagents (Origene, #TT320001), according to the manufacturer’s instructions.

Techniques: Knockdown, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Reverse Transcription

Journal: Journal of Clinical Investigation

Article Title: Transcription factor TBX4 regulates myofibroblast accumulation and lung fibrosis

doi: 10.1172/jci85328

Figure Lengend Snippet: Figure 2. Tbx4-lineage cells in the lung are the major source of myofibroblasts. (A) Sche- matic depicting lineage analysis methodology. Tbx4-CreLME Rosa26-tdTomato, αSMA-GFP Tbx4LME-Cre Rosa26-tdTomato, and αSMA-GFP Tbx4-CreKi Rosa26-tdTomato mice were used for all experiments in this figure. (B) Representative histology images of uninjured or bleomycin-treated (d21) adult αSMA-GFP Tbx4LME-Cre Rosa26-tdTomato mouse lung. (C) Representative histology images of uninjured or bleomycin-treated (d21) adult αSMA-GFP Tbx4-CreKi Rosa26-tdTomato mouse lung. (D) Representative FACS plots of mouse lung single cells isolated from uninjured or ble- omycin-treated (d21) αSMA-GFP Tbx4-CreKi Rosa26-tdTomato mice. (E) Quantification of tdT+ cells among αSMA-GFP+ cells in D (n = 3 mice analyzed, mean ± SEM). Arrows show cells with overlaps. Scale bars: 10 μm (B) and 100 μm (C). aw, airway; bv, blood vessel; f, fibrotic foci; m, mesenchyme.

Article Snippet: Tbx4 siRNA effects on 3T3 cell line and human IPF lung fibroblast HA production were assessed with siRNA spe cific to Tbx4 (mTbx4 siRNA: 5′rGrCrArCrUrGrCrCrArArGrArAr ArCrArUrGrGrArArArGGT-3′; hTbx4 siRNA: 5′rUrGrCrArArUrU rArUrCrUrArArGrArArGrUrGrArCrUrUTG-3′; Universal scrambled negative control siRNA duplex, SR30004, OriGene Technologies).

Techniques: Isolation

Journal: Journal of Clinical Investigation

Article Title: Transcription factor TBX4 regulates myofibroblast accumulation and lung fibrosis

doi: 10.1172/jci85328

Figure Lengend Snippet: Figure 3. Tbx4-lineage cells in the lung are the major source of COL1α1+ fibroblasts. (A) Schematic depicting lineage analysis methodology. COL1α1-GFP Tbx4-CreKi Rosa26-tdTomato mice were used for all experiments in this figure. (B–D) Representative histology images of uninjured or bleomycin-treated (d21) adult COL1α1-GFP Tbx4-CreKi Rosa26-tdTomato mouse lung. (E) Representative FACS plots of mouse lung single cells isolated from uninjured or ble- omycin-treated (d21) COL1α1-GFP Tbx4-CreKi Rosa26-tdTomato mice. (F) Quantification of tdT+ cells among COL1α1-GFP+ cells, and COL1α1-GFP+ cells within tdT+ cells in E (n = 3 mice analyzed, mean ± SEM). Arrows show cells with overlaps. Scale bars: 1 mm (B) and 10 μm (C and D). a, alveoli; aw, airway.

Article Snippet: Tbx4 siRNA effects on 3T3 cell line and human IPF lung fibroblast HA production were assessed with siRNA spe cific to Tbx4 (mTbx4 siRNA: 5′rGrCrArCrUrGrCrCrArArGrArAr ArCrArUrGrGrArArArGGT-3′; hTbx4 siRNA: 5′rUrGrCrArArUrU rArUrCrUrArArGrArArGrUrGrArCrUrUTG-3′; Universal scrambled negative control siRNA duplex, SR30004, OriGene Technologies).

Techniques: Isolation

Journal: Journal of Clinical Investigation

Article Title: Transcription factor TBX4 regulates myofibroblast accumulation and lung fibrosis

doi: 10.1172/jci85328

Figure Lengend Snippet: Figure 4. Tbx4-lineage cells are highly proliferative upon bleomycin injury in the lung. (A) Experimental design for inducible TBX4 cell labeling using 1 or 4 doses of tamoxifen 1 week before bleomycin injury for Tbx4LME-CreER Rosa26-tdTomato mice. Lungs were harvested on d21. Tbx4-CreKi Rosa26-tdTomato (Tam, 0.2 mg/g/dose) and Tbx4LME-CreER Confetti (Tam, 0.1 mg/g/dose) were used for all experiments in this figure. (B and D) Representative lung sections from Tbx4LME-CreER Rosa26-tdTomato mice with 1 (B) or 4 (D) doses of tamoxifen injection showing Tbx4-lineage cells in red and nuclei in blue. (C and E) Quantification of Tbx4 cells counted in B and D (from 2 lobes per mouse, n = 9 mice; ****P ≤ 0.0001 by 2-tailed Student’s t test; mean ± SEM). (F) Repre- sentative immunofluorescent images of mice from B staining with αSMA, COL1α1, desmin, vimentin, PDGFRβ, and NG2. Arrows show cells with overlap in staining (n = 6 lungs examined). (G) TBX4 cells were marked in Tbx4LME-CreER Confetti mice using 4 doses of tamoxifen. Representative confocal images of a typical colony of TBX4 cells. Cells in clone are enumerated. (H) TBX4 colony size at baseline and d21 after bleomycin injury. n = 9 lungs examined. ****P ≤ 0.0001 by Student’s t test; mean ± SEM. Scale bars: 100 μm (B and D) and 10 μm (F and G). a, alveoli; aw, airway; f, fibrotic foci; m, mesenchyme.

Article Snippet: Tbx4 siRNA effects on 3T3 cell line and human IPF lung fibroblast HA production were assessed with siRNA spe cific to Tbx4 (mTbx4 siRNA: 5′rGrCrArCrUrGrCrCrArArGrArAr ArCrArUrGrGrArArArGGT-3′; hTbx4 siRNA: 5′rUrGrCrArArUrU rArUrCrUrArArGrArArGrUrGrArCrUrUTG-3′; Universal scrambled negative control siRNA duplex, SR30004, OriGene Technologies).

Techniques: Labeling, Injection, Staining

Journal: Journal of Clinical Investigation

Article Title: Transcription factor TBX4 regulates myofibroblast accumulation and lung fibrosis

doi: 10.1172/jci85328

Figure Lengend Snippet: Figure 6. Ablation of TBX4 cells decreases HAS2 and HA expression. (A) Verification of ablation of Tbx4-lineage cells using Tbx4LME-CreER Rosa26- tdTomato and Tbx4LME-CreER Rosa26-tdTomato Rosa26-DTA mice. Five doses of tamoxifen were injected, and lungs were harvested 1 week later. (B) Representative images were taken from Tbx4LME-CreER Rosa26-tdTomato and Tbx4LME-CreER Rosa26-tdTomato Rosa26-DTA mouse lung (n = 4 lungs examined). (C) Quantification of Tbx4 lineage tracing and ablation, expressed as cells counted in B (n = 4 in each group of mice). (D and E) Tbx4 mRNA (D) and Has2 mRNA (E) expression of lung fibroblasts from Tbx4LME-CreER Rosa26-tdTomato and Tbx4LME-CreER Rosa26-tdTomato Rosa26-DTA were analyzed by quantitative RT-PCR, and normalized by GAPDH (n = 4 mice per group examined). (F) HA contents in conditioned media of lung fibroblasts from Tbx4LME-CreER Rosa26-tdTomato and littermate controls were analyzed by HA ELISA. n = 6 mice per group examined. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 by 2-tailed Student’s t test, mean ± SEM.

Article Snippet: Tbx4 siRNA effects on 3T3 cell line and human IPF lung fibroblast HA production were assessed with siRNA spe cific to Tbx4 (mTbx4 siRNA: 5′rGrCrArCrUrGrCrCrArArGrArAr ArCrArUrGrGrArArArGGT-3′; hTbx4 siRNA: 5′rUrGrCrArArUrU rArUrCrUrArArGrArArGrUrGrArCrUrUTG-3′; Universal scrambled negative control siRNA duplex, SR30004, OriGene Technologies).

Techniques: Expressing, Injection, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Journal of Clinical Investigation

Article Title: Transcription factor TBX4 regulates myofibroblast accumulation and lung fibrosis

doi: 10.1172/jci85328

Figure Lengend Snippet: Figure 7. Ablation of TBX4 cells attenuates pulmonary fibrosis. (A) Strategy for inducible ablation of TBX4 cells. Tbx4LME-CreER Rosa26-DTA mice were used in the subsequent experiment. The above-mentioned transgenic mice and their WT littermates (8–16 weeks old) were treated with bleomycin (2.5 U/kg), followed by 5 doses of tamoxifen (0.2 mg/g/injection) every other day starting on d7. The lungs were collected for hydroxyproline content deter- mination and Masson’s trichrome staining on d21 after bleomycin. DTA, diphtheria toxin fragment A. (B–G) Representative Masson’s trichrome staining images of bleomycin-treated (d21) adult littermate control (B–D) or Tbx4LME-CreER Rosa26-DTA (E–G) mouse lung. (H) Targeting TBX4 cells reduced lung fibrosis (means ± SEM, ***P ≤ 0.001, 1-way ANOVA with Bonferroni test; uninjured littermate control, n = 6; bleomycin [Bleo] littermate control, n = 20; Tbx4LME-CreER Rosa26-DTA, n = 16). (I–K) Representative images of COL1α1 (I), αSMA (J), and HA (K) immunofluorescence staining of d21 Tbx4LME-CreER Rosa26-DTA mouse lung. n = 6 mice per group examined. Scale bars: 1 mm (B and E) and 100 μm (C, D, F, G, and I–K).

Article Snippet: Tbx4 siRNA effects on 3T3 cell line and human IPF lung fibroblast HA production were assessed with siRNA spe cific to Tbx4 (mTbx4 siRNA: 5′rGrCrArCrUrGrCrCrArArGrArAr ArCrArUrGrGrArArArGGT-3′; hTbx4 siRNA: 5′rUrGrCrArArUrU rArUrCrUrArArGrArArGrUrGrArCrUrUTG-3′; Universal scrambled negative control siRNA duplex, SR30004, OriGene Technologies).

Techniques: Transgenic Assay, Injection, Staining, Control, Immunofluorescence

Journal: Journal of Clinical Investigation

Article Title: Transcription factor TBX4 regulates myofibroblast accumulation and lung fibrosis

doi: 10.1172/jci85328

Figure Lengend Snippet: Figure 8. Loss of TBX4 expression in COL1α2-expressing cells attenuates pulmonary fibrosis. (A) Strategy for inducible knockout of TBX4 expression in COL1α2+ cells. Col1a2-CreER Tbx4fl/fl mice were used in these experiments. The above-mentioned mice and their WT littermates (8–16 weeks old) were treated with bleomycin (2.5 U/kg), followed by 5 doses of tamoxifen (0.2 mg/g/injection) every other day starting on d7. The lungs were collected for hydroxyproline content determination on d21 after bleomycin. (B) Knockdown of Tbx4 in COL1α2-expressing cells decreased hydroxyproline content (means ± SEM, ***P ≤ 0.001, ****P ≤ 0.0001, n = 6 in uninjured Tbx4fl/fl group, n = 4 in uninjured Col1a2-CreER Tbx4fl/fl group, n = 23 in bleo Tbx4fl/fl group, n = 27 in bleo Col1a2-CreER Tbx4fl/fl group). (C) Representative Masson’s trichrome staining from lungs at d21 after bleomycin injection, showing decreased collagen deposition (blue) in Col1a2-CreER Tbx4fl/fl. (D and E) Representative images of COL1α1 and αSMA antibody staining for bleo d21 Col1a2-CreER Tbx4fl/fl mouse lung. n = 6 mice per group examined. Scale bars: 100 μm (C–E).

Article Snippet: Tbx4 siRNA effects on 3T3 cell line and human IPF lung fibroblast HA production were assessed with siRNA spe cific to Tbx4 (mTbx4 siRNA: 5′rGrCrArCrUrGrCrCrArArGrArAr ArCrArUrGrGrArArArGGT-3′; hTbx4 siRNA: 5′rUrGrCrArArUrU rArUrCrUrArArGrArArGrUrGrArCrUrUTG-3′; Universal scrambled negative control siRNA duplex, SR30004, OriGene Technologies).

Techniques: Expressing, Knock-Out, Injection, Knockdown, Staining

Journal: Journal of Clinical Investigation

Article Title: Transcription factor TBX4 regulates myofibroblast accumulation and lung fibrosis

doi: 10.1172/jci85328

Figure Lengend Snippet: Figure 9. Inhibition of Tbx4 in αSMA+ cells attenuates lung fibrosis. (A) Strategy for inducible knockout of Tbx4 expression in αSMA+ cells. Acta2-CreER Tbx4fl/fl mice were used in these experiments. The above-mentioned mice and their WT littermates (8–16 weeks old) were treated with bleomycin (2.5 U/ kg), followed by 5 doses of tamoxifen (0.2 mg/g/injection) every other day starting on d7. The lungs were collected for hydroxyproline content determina- tion on d21 after bleomycin. (B) Knockdown of Tbx4 in αSMA-expressing cells decreased hydroxyproline content (means ± SEM, *P ≤ 0.05, ****P ≤ 0.0001, n = 6 in uninjured Tbx4fl/fl group, n = 4 in uninjured Acta2-CreER Tbx4fl/fl group, n = 17 in bleo Tbx4fl/fl group, n = 16 in bleo Acta2-CreER Tbx4fl/fl group). Hydroxyproline content values were expressed as percentage of control Tbx4fl/fl group, which is set to 100%. One-way ANOVA with Bonferroni test was used. (C) Representative Masson’s trichrome staining from lungs at d21 after bleomycin injection, showing decreased collagen deposition (blue) in Acta2- CreER Tbx4fl/fl mice. (D and E) Representative images of COL1α1 and αSMA antibody staining for bleo d21 Acta2-CreER Tbx4fl/fl mouse lung. n = 6 mice per group examined. Scale bars: 100 μm (C–E).

Article Snippet: Tbx4 siRNA effects on 3T3 cell line and human IPF lung fibroblast HA production were assessed with siRNA spe cific to Tbx4 (mTbx4 siRNA: 5′rGrCrArCrUrGrCrCrArArGrArAr ArCrArUrGrGrArArArGGT-3′; hTbx4 siRNA: 5′rUrGrCrArArUrU rArUrCrUrArArGrArArGrUrGrArCrUrUTG-3′; Universal scrambled negative control siRNA duplex, SR30004, OriGene Technologies).

Techniques: Inhibition, Knock-Out, Expressing, Injection, Knockdown, Control, Staining

Journal: Journal of Clinical Investigation

Article Title: Transcription factor TBX4 regulates myofibroblast accumulation and lung fibrosis

doi: 10.1172/jci85328

Figure Lengend Snippet: Figure 10. TBX4 regulates fibroblast invasion through regulation of Has2. (A) Tbx4 mRNA expression was increased in sorted tdT+ cells from Tbx4-CreTg Rosa26-tdTomato mice. (B) tdT+ fibroblasts were more invasive. Invasion capacity values were expressed as relative fold change of control, which is set to 1. (C) Has2 mRNA level was elevated in tdT+ fibroblasts. (D) Tbx4 siRNA reduced TGF-β1–induced HA release by mouse fibroblast 3T3 cells. Cells were transfected with either Tbx4 siRNA or control siRNA (Con). Forty-eight hours later cells were treated with TGF-β1 at 1 ng/ml or 10 ng/ml concentration for 24 hours. HA was measured in conditioned media. CT, control. (E) TBX4 enhanced Has2 promoter activity. All Has2 promoter constructs showed increased activity over the promoterless vector pGL3 when cotransfected with a TBX4-expressing vector in HEK 293 cells. (F–H) MEF cells were isolated from E14 embryo of CMV-Cre Tbx4f/w and WT mice. MEF cells at passage 3 were used for experiments. (F) Tbx4 mRNA levels were compared between Tbx4-defi- cient and WT MEFs. (G) MEF cells were treated with or without TGF-β1 (5 ng/ml) overnight. Has2 mRNA expression was analyzed using RT-PCR. (H) HA in supernatants was measured with HA ELISA. (I) TBX4 gene expression was elevated in invasive human fibroblasts from IPF patients. (J) Invasion assay for IPF fibroblasts was performed 48 hours after transfection of control and TBX4 siRNA. Knocking down TBX4 with siRNA reduced the invasiveness of IPF fibroblasts. (K) Knocking down TBX4 with siRNA reduced HA production of primary fibroblasts from IPF patients with or without 1 ng/ml TGF-β1 treatment after 48 hours. n = 4 in each group were analyzed. All results are the mean of triplicate experiments ± SEM. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, as analyzed by 2-tailed Student’s t test (B, F, I, and J) or 1-way ANOVA with Bonferroni test (D, E, G, H, and K).

Article Snippet: Tbx4 siRNA effects on 3T3 cell line and human IPF lung fibroblast HA production were assessed with siRNA spe cific to Tbx4 (mTbx4 siRNA: 5′rGrCrArCrUrGrCrCrArArGrArAr ArCrArUrGrGrArArArGGT-3′; hTbx4 siRNA: 5′rUrGrCrArArUrU rArUrCrUrArArGrArArGrUrGrArCrUrUTG-3′; Universal scrambled negative control siRNA duplex, SR30004, OriGene Technologies).

Techniques: Expressing, Control, Transfection, Concentration Assay, Activity Assay, Construct, Plasmid Preparation, Isolation, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Gene Expression, Invasion Assay

Journal: Oncotarget

Article Title: Multiplicity of acquired cross-resistance in paclitaxel-resistant cancer cells is associated with feedback control of TUBB3 via FOXO3a-mediated ABCB1 regulation

doi: 10.18632/oncotarget.9118

Figure Lengend Snippet: ( A ) Characterization of indicated parental or drug-resistant phenotype cell lines for TUBB3 expression at both mRNA (upper panel) and protein (lower panel) levels. ( B ) Characterization of indicated parental or drug-resistant phenotype cell lines for FOXO3a expression at both mRNA (upper panel) and protein (lower panel) levels. ( C ) Identification of ABCB1 -association expression in a panel of indicated parental or drug resistant cancer cell lines at the mRNA level. ( D ) Confirmation of P-gp protein overexpression in drug resistant cancer cell lines. ( E ) Intracellular distribution and localization of P-gp expression in both wild-type and drug-resistant phenotype cell lines. Cells were stained with human P-gp antibody and DAPI and analyzed through confocal microscopy. Images shown were magnified at ×200.

Article Snippet: For ABCB1 silencing, cells were transfected with scramble siRNA (FITC-conjugate) or human-specific ABCB1 (MDR1) siRNA duplexes (Santa Cruz Biotechnology) using Lipofectamine 3000 [ ].

Techniques: Expressing, Over Expression, Staining, Confocal Microscopy

Journal: Oncotarget

Article Title: Multiplicity of acquired cross-resistance in paclitaxel-resistant cancer cells is associated with feedback control of TUBB3 via FOXO3a-mediated ABCB1 regulation

doi: 10.18632/oncotarget.9118

Figure Lengend Snippet: ( A and B ) Fraction of drug-intolerant A549, A549-PacR (A) and HEK293, HEK293/ ABCB1 (B) cells. Cells were treated with PTX, 5-FU, DCT, or CIS for 24 hr, and the cell viability was determined by MTT assay. Data are represented as means ± SEM. ( C ) Representative drug intolerant cell colonies (right) and quantified colony numbers of A549-PacR cells (left). Cells were exposed to 5-FU-conditioned media (CM) and continuously grown for > 7 days and formed colonies of established PTX-resistant cells were stained with sapphire 700. Data are represented as means ± SEM. ( D and E ) ATP-dependent P-gp efflux activity. Cells were treated with 60 μM 5-FU for 24 hr and assayed for Rho-123 incorporation. Flow cytometry was used to quantify Rho-123 fluorescence (D). Drug-resistant cells were treated with DMSO, 60 μM 5-FU, or 50 μM DCT for 24 hr and was assayed for ABCB1 ATPase activity (E). Data are represented as means ± SEM. ( F and G ) Association of P-gp expression with occurrence of cross-resistance. Drug-resistant cells were treated with 5-FU, DCT, or CIS for 24 hr (F) and time-dependently treated with 5-FU for (G). Cells were treated with 20 μM 5-FU then assayed for qRT-PCR using ABCB1 -specific primer. ( H ) P-gp-specific ATPase activity. Cells were treated with increasing 5-FU concentration for 24 hr and cells were subjected to P-gp luminescent ATPase assay. Data are represented as means ± SEM.

Article Snippet: For ABCB1 silencing, cells were transfected with scramble siRNA (FITC-conjugate) or human-specific ABCB1 (MDR1) siRNA duplexes (Santa Cruz Biotechnology) using Lipofectamine 3000 [ ].

Techniques: MTT Assay, Staining, Activity Assay, Flow Cytometry, Fluorescence, Expressing, Quantitative RT-PCR, Concentration Assay, ATPase Assay

Journal: Oncotarget

Article Title: Multiplicity of acquired cross-resistance in paclitaxel-resistant cancer cells is associated with feedback control of TUBB3 via FOXO3a-mediated ABCB1 regulation

doi: 10.18632/oncotarget.9118

Figure Lengend Snippet: Cross-drug resistance profile of cells with resistance to paclitaxel or gefitinib, and of ABCB1 transiently transfected cells and association with P-gp and MRP protein expressions

Article Snippet: For ABCB1 silencing, cells were transfected with scramble siRNA (FITC-conjugate) or human-specific ABCB1 (MDR1) siRNA duplexes (Santa Cruz Biotechnology) using Lipofectamine 3000 [ ].

Techniques: Transfection, Plasmid Preparation

Journal: Oncotarget

Article Title: Multiplicity of acquired cross-resistance in paclitaxel-resistant cancer cells is associated with feedback control of TUBB3 via FOXO3a-mediated ABCB1 regulation

doi: 10.18632/oncotarget.9118

Figure Lengend Snippet: ( A ) Schematic diagram of general strategy used for the generation of transient cross-resistance to 5-FU, DCT, or CIS in PacR phenotype cancer cells or ABCB1 -GFP transfected HEK293 cells. ( B ) Growth rate response of indicated cells (lower panel) to 5-FU treatment in a dose-dependent manner. Schematic schedule of treatment is also displayed (upper panel). Cell viability was determined using MTT assay. Data are represented as means ± SEM. ( C ) Characterization for maintained ABCB1 and ABCC1 mRNA expressions in developed A549-PacR/5-FU and PC-3-PacR/5-FU cells after indicated subsequent cell cultures. Passages of cells were maintained with 1 μM 5-FU final concentration. ( D – F ) Characterization for TUBB3 and FOXO3a mRNA expressions in indicated developed transient cross-resistance in PacR phenotype derived from A549 (D), PC-3 (E), and in developed transient 5-FU cross-resistance derived from HEK293 (F) cells. ( G and H ) Western blot analysis of A549, PC-3-PacR cells (G) and HEK293 cells transfected with either empty vector or ABCB1 -GFP (H) cells, all with developed transient cross-resistance to indicated drugs. Cells were assessed for expressions of indicated proteins after 24 hr cell culture. ( I ) Flow cytometric determination of verapamil-induced apoptosis in indicated cells. Cells were treated with or without 100 μM verapamil for 24 hr. Data are shown as bar graph represented as means ± SEM. ( J ) Western blot analysis of indicated cells for expressions of apoptotic markers Bax, Bcl-2, and p53. Cells were treated with or without 100 μM verapamil for 24 hr. ( K ) Intracellular ATP level assessment in indicated cells. Cells were treated with or without 100 μM verapamil for 24 hr and ATP levels were determined in 10 4 fraction of cells. RLU, relative luciferase units. Data are represented as means ± SEM. (L ) Determination of verapamil-induced inhibition of P-gp/ ABCB1 in indicated cells assessed through qRT-PCR (left), Western blotting (center) and confocal microscopy (right). Cells were treated with 60 μM verapamil for 24 hr. Confocal images shown were magnified to 80 μm.

Article Snippet: For ABCB1 silencing, cells were transfected with scramble siRNA (FITC-conjugate) or human-specific ABCB1 (MDR1) siRNA duplexes (Santa Cruz Biotechnology) using Lipofectamine 3000 [ ].

Techniques: Transfection, MTT Assay, Concentration Assay, Derivative Assay, Western Blot, Plasmid Preparation, Cell Culture, Luciferase, Inhibition, Quantitative RT-PCR, Confocal Microscopy

Journal: Oncotarget

Article Title: Multiplicity of acquired cross-resistance in paclitaxel-resistant cancer cells is associated with feedback control of TUBB3 via FOXO3a-mediated ABCB1 regulation

doi: 10.18632/oncotarget.9118

Figure Lengend Snippet: ( A ) Characterization of ABCB1 and TUBB3 expressions at both protein (upper panel) and mRNA (lower panel) levels after transient transfections with indicated vectors or siRNA in A549-PacR and PC-3-PacR both with developed 5-FU transient cross-resistance. Cells were transfected with empty vector or FOXO3a-GFP and scrambled siRNA or ABCB1 siRNA for 48 hr. Data are represented as means ± SEM. ( B ) Protein expressions (upper panel) of ABCB1 and TUBB3 and mRNA levels of TUBB3 (lower panel) in cells same as in A after transient transfection with either scrambled siRNA or FOXO3a siRNA for 48 hr. Data are represented as means ± SEM. ( C ) Effect of proteasome inhibitor, MG132 (MG), on β III -tubulin distribution. Cells treated with or without 50 μM MG for 24 h were subjected to immunocytochemistry. Magnified images were zoomed at ×80. The cells were stained with TUBB3 antibody and DAPI. ( D ) Effect of MG on TUBB3 protein expressions in same cells as in A. Cells were transfected with either empty vector or FOXO3a-GFP for 48 hr and treated with or without 50 μM MG132 for 24 hr. Whole-cell lysates were assayed by Western blotting. ( E ) Detection of ubiquitinated TUBB3 protein in A549-PacR/5-FU cells. Cells were transfected with either scramble siRNA or FOXO3a-siRNA for 48 hr and cells were further transfected with an empty vector or myc-tagged ubiquitin-encoding vector for 24 hr. Immunoprecipitation was performed by TUBB3 antibody, then ubiquitinated proteins were detected by myc antibody assayed by Western blotting. ( F ) Methylation-specific PCR of ABCB1 -50GC and -110GC boxes in A549-PacR/5-FU cells. Primer sets are designed to amplify methylated (M) and unmethylated (U) alleles. A primer set encoding the whole GC region (T) was used as loading control. Cells were transiently transfected with either empty vector or FOXO3a-GFP for 48 hr and cells were treated with 10 μM PTX for 12 hr (left lane) or 24 hr (right lane). ( G ) Combined bisulphite restriction analysis of the Inr ABCB1 promoter region in A549-PacR/5-FU cells treated with or without 10 μM PTX, or 40 μM 5-FU for 24 hr or in combination for 24 or 48 hr as indicated. Figures represent the methylation percentages observed in the indicated drug-treated cells obtained from two separate independent experiments. ( H ) Effect of doxorubicin (Dox) on FOXO3a-induced regulation of Akt-related signals. Cells were transiently transfected with either empty vector or FOXO3a-GFP for 48 hr and cells were treated with or without 2 μM Dox for 8 hr. Protein expression levels were analyzed by Western blotting. ( I ) Methylated FOXO3a status in PC-3-PacR and –PacR/5-FU cells. Cells were transiently transfected with either empty vector or FOXO3a-GFP for 48 hr and cells were treated with or without 2 μM Dox for 8 hr. Cell lysates were taken and dimethylated proteins at arginine residues were immunoprecipitated using anti-human FOXO3a antibody followed by Western blotting using anti-dimethyl arginine antibody. Actin was used as input. ( J ) Average GFP-positive cells determination in the nucleus or cytoplasm of PC-3-PacR/5-FU cells. Cells were transiently transfected with FOXO3a-GFP for 48 hr followed by treatment with or without 2 μM Dox, 300 nM PTX, 120 μM 5-FU or their indicated combinations for 8 hr. ( K ) Lysates of PC-3-PacR/5-FU treated with or without 2 μM Dox for 8 hr were subjected to cytoplasmic or nuclear extraction followed by Western blotting. ( L ) Confocal microscopic analysis of PC-3-PacR/5-FU cells. Cells were transiently transfected with either empty vector or FOXO3a-GFP and cells were treated with or without 2 μM Dox for 8 hr and stained with FOXO3a antibody and DAPI. Images shown were magnified at 60 μm. ( M ) Characterization of ABCC1 expressions at both protein (upper panel) and gene transcript (lower panel) levels in both PC-3-PacR and –PacR/5-FU cells. Cells were transiently transfected with either empty vector or FOXO3a-GFP for 48 hr and cells were treated with or without 2 μM Dox for 8 hr.

Article Snippet: For ABCB1 silencing, cells were transfected with scramble siRNA (FITC-conjugate) or human-specific ABCB1 (MDR1) siRNA duplexes (Santa Cruz Biotechnology) using Lipofectamine 3000 [ ].

Techniques: Transfection, Plasmid Preparation, Immunocytochemistry, Staining, Western Blot, Ubiquitin Proteomics, Immunoprecipitation, Methylation, Control, Expressing, Extraction

Journal: Oncotarget

Article Title: Multiplicity of acquired cross-resistance in paclitaxel-resistant cancer cells is associated with feedback control of TUBB3 via FOXO3a-mediated ABCB1 regulation

doi: 10.18632/oncotarget.9118

Figure Lengend Snippet: ( A ) Schematic diagram of the drug conditioned media (CM)-exposure procedure used in various assays ( B – J ). (B and C) Characterization of FOXO3a-mediated multiple cross-drug resistance in A549-PacR cells (B). Cells were preincubated with indicated 1:1 combination ratio of CM from DMSO-, DCT-, 5-FU-treated cells. After 24 hr, cells were transiently transfected with indicated vectors or siRNA for 48 hr and assessed for colony formation (C). Established drug resistant colonies were stained and visualized by crystal violet or sapphire 700. (D and E) TUBB3 knockdown or FOXO3a overexpression (D) and overexpression of ABCB1 and FOXO3a effects on growth rate of A549-PacR/5-FU cells (E). A549-PacR/DCT cells were treated with indicated 5-FU concentrations for 24 h then CM was collected. Adherent A549 cells were exposed to the collected CM for 24 h followed by transient transfection with indicated vectors or siRNA for 48 hr. Cell viability was assessed by MTT assay. Data are represented as means ± SEM. (F) Confocal microscopic analysis of A549-PacR/5-FU cells. Cells were transiently transfected with indicated vectors or siRNA for 48 hr and stained with P-gp antibody and DAPI. Images shown were magnified to 80 μm. (G) Western blot analysis (left) and agarose gel electrophoresis of qPCR products of A549-PacR/5-FU cells. Cells were transiently transfected with indicated vectors or siRNA for 48 hr and assessed for expression studies. (H) ABCB1 drug efflux activity of A549-PacR/5-FU cells. Cells were transiently transfected with either empty vector or FOXO3a-GFP for 48 hr and cells were treated with or without 100 μM 5-FU or 120 μM DCT for 24 hr. Drug-treated cells were assayed for Rhodamine-123 (Rho-123) uptake (left) and untreated cells were assayed for ABCB1 ATPase activity (right). Data are represented as means ± SEM. (I) ABCB1 drug efflux activity of A549-PacR/5-FU cells. Cells were transiently transfected with either scrambled siRNA or TUBB3 siRNA for 48 hr followed by drug treatment and assayed as in H. Data are represented as means ± SEM. (J) Multidrug resistant colonies. Cells were transiently transfected with either scramble siRNA or TUBB3 siRNA for 48 hr and cells were treated with 30 μM 5-FU for 7 days. Resistant colonies were visualized by sapphire 700. ( K ) Schematic diagram of drug CM exposure and respective TUBB3 feedback after induced gene expression changes via transient gene transfection or siRNA gene silencing. Indicated gene or siRNA transfection schemes were used in L and M for P-gp functional assays. ( L ) Growth rate of A549-PacR/5-FU cells. Cells were pre-incubated with indicated drug-CM and cells were transiently transfected with indicated genes or siRNA for 48 hr. Cell viability was assessed by MTT assay. Data are represented as means ± SEM. ( M ) ABCB1 drug efflux activity A549-PacR/5-FU cells. Cells were pre-incubated with indicated drug-CM and cells were transiently transfected with indicated genes or siRNA for 48 hr. Cells were then treated with indicated drugs and concentration for 36 hr. Data are represented as means ± SEM.

Article Snippet: For ABCB1 silencing, cells were transfected with scramble siRNA (FITC-conjugate) or human-specific ABCB1 (MDR1) siRNA duplexes (Santa Cruz Biotechnology) using Lipofectamine 3000 [ ].

Techniques: Transfection, Staining, Knockdown, Over Expression, MTT Assay, Western Blot, Agarose Gel Electrophoresis, Expressing, Activity Assay, Plasmid Preparation, Gene Expression, Functional Assay, Incubation, Concentration Assay

Journal: Oncotarget

Article Title: Multiplicity of acquired cross-resistance in paclitaxel-resistant cancer cells is associated with feedback control of TUBB3 via FOXO3a-mediated ABCB1 regulation

doi: 10.18632/oncotarget.9118

Figure Lengend Snippet: ( A and B ) Cell cycle progression of A549-PacR and -PacR/5-FU cells. Cells were transiently transfected with either scramble siRNA or TUBB3 siRNA for 48 hr. Transfected cells were subjected to FACS analysis (A) and BrdU incorporation assay (B). G1/S ratio of cells was calculated and displayed. Data are represented as means ± SEM. ( C ) Cell cycle regulation in A549-PacR and -PacR/5-FU cells. Cells were transiently transfected with either scramble siRNA or TUBB3 siRNA for 48 hr. Cells were then subjected to Western blotting to detect cell cycle regulatory proteins. ( D ) Epithelial-to-mesenchymal transition (EMT) status of A549-PacR and -PacR/5-FU cells. Cells were transiently transfected with either scramble siRNA or TUBB3 siRNA for 48 hr. Classical EMT markers were immunoblotted. ( E ) Immunofluorescence images of Vimentin in A549-PacR/5-FU cells. Cells were transiently transfected with either scramble siRNA or TUBB3 siRNA for 48 hr. Cells were stained with Vimentin antibody and DAPI. Images shown were magnified at 30 μm. ( F ) Inhibition of AKT in A549-PacR/5-FU cells. Cells were transiently transfected with either scramble siRNA or TUBB3 siRNA for 48 hr and cells were treated with or without 10 μM of AKT inhibitor, LY294002, for 18 hr. Phosphorylation of AKT was assayed by Western blotting. ( G ) Intracellular ATP assessment of A549-PacR and -PacR/5-FU cells. Cells were transiently transfected with either scramble siRNA or TUBB3 siRNA for 48 hr and cells were treated with or without 10 μM LY294002 for 18 hr. ATP levels was assessed. Data are represented as means ± SEM. ( H ) AKT-dependent apoptosis in A549-PacR/5-FU cells. Cells were transiently transfected with either scramble siRNA or TUBB3 siRNA for 48 hr and cells were treated with or without 10 μM LY294002 for 18 hr. Cells were analyzed for annexin-V/FITC-PI by FACS. Data are represented as means ± SEM. ( I ) Motility and colony formation of A549-PacR/5-FU cells. Cells were transiently transfected with either scramble siRNA or TUBB3 siRNA for 48 hr and cells were treated with or without 10 μM LY294002 for 18 hr in culture subjected to migration, invasion, and colony formation assays. ( J ) Schematic diagram of in vivo tumor xenograft experiments. ( K ) Evaluation of subcutaneous tumors. Indicated cells were transiently transfected with either scramble siRNA or TUBB3 siRNA for 48 hr. Transfected cells (1 × 10 6 cells/flank) were subcutaneously injected into the flanks of nude mice. Shown representative images depict the actual tumors prior to surgery and measurement (upper) and H&E (lower) immunohistochemical analysis indicated xenograft tumors. Stained sections were photographed with an inverted phase-contrast microscope with 150× magnification. ( L ) Relative expressions of ABCB1 and TUBB3 in tumor transcripts of indicated xenografts. Total RNA was isolated and analyzed by quantitative reverse transcriptase-PCR using ABCB1-, or TUBB3-specific primers and normalized to GAPDH expression. Data are represented as means ± SEM. ( M ) Protein expressions of β III -tubulin and P-gp in A549-PacR/5-FU xenograft tumors. Tumor sample lysates were immunoblotted with the indicated antibodies. ( N ) Relative expressions of AKT and VEGF in tumor transcripts of A549-PacR/5-FU. RNA was processed as in L. Data are represented as means ± SEM.

Article Snippet: For ABCB1 silencing, cells were transfected with scramble siRNA (FITC-conjugate) or human-specific ABCB1 (MDR1) siRNA duplexes (Santa Cruz Biotechnology) using Lipofectamine 3000 [ ].

Techniques: Transfection, BrdU Incorporation Assay, Western Blot, Immunofluorescence, Staining, Inhibition, Phospho-proteomics, Migration, In Vivo, Injection, Immunohistochemical staining, Microscopy, Isolation, Reverse Transcription, Expressing

Journal: Oncotarget

Article Title: Multiplicity of acquired cross-resistance in paclitaxel-resistant cancer cells is associated with feedback control of TUBB3 via FOXO3a-mediated ABCB1 regulation

doi: 10.18632/oncotarget.9118

Figure Lengend Snippet: ( A ) Microtubule stability in parental, PacR, and developed PacR phenotype cells with multiple transient cross-resistance. Cells were grown in the presence of indicated PTX concentrations for 18 hr. Following cell lysis, pellet (P) and the supernatant (S) protein fractions were separated by centrifugation and resolved on adjacent lanes by electrophoresis. Transferred filters were probed with tubulin antibody. ( B ) Acetylated tubulin status of parental, PacR, and developed PacR phenotype cells with multiple transient cross-resistance. Cells were grown in the absence or presence 20 nM PTX for 18 hr, and the amount of acetylated tubulin was measured by immunoblotting using antibody specific to acetylated tubulin. ( C ) Mixing tubulin experiment. Indicated cells were harvested by adding hypotonic buffer with 50 μg/mL PTX for 15 min. Whole cell lysate from PacR phenotype was added to that of developed PacR/5-FU subline at different ratios as indicated, and incubated for an additional 10 min. The polymerized (Pol) and soluble (Sol) protein fractions were processed as described in Materials and Methods. ( D ) Schematic diagram of transient gene transfection and/or siRNA silencing with respective TUBB3 feedback results used in E and F in A549-PacR/5-FU cells. ( E ) Confirmation of TUBB3 feedback gene expressions after transient transfection and/or siRNA silencing of indicated genes with C as control (respective empty vector and/or scramble siRNA) and TS as the transfection scheme shown in D. ( F ) Microtubule stability of A549-PacR/5-FU cells through tubulin polymerization after indicated transient gene and/or siRNA transfections. Cells were grown in the presence of 60 nM PTX for 18 hr. The polymerized (Pol) and soluble (Sol) protein fractions were processed as in C. ( G ) Illustration of proposed mechanism behind the feedback response of TUBB3 to silencing of FOXO3a and ABCB1 in regulating microtubule stability in A549-PacR/5-FU cells.

Article Snippet: For ABCB1 silencing, cells were transfected with scramble siRNA (FITC-conjugate) or human-specific ABCB1 (MDR1) siRNA duplexes (Santa Cruz Biotechnology) using Lipofectamine 3000 [ ].

Techniques: Lysis, Centrifugation, Electrophoresis, Western Blot, Incubation, Transfection, Control, Plasmid Preparation

Journal: Oncotarget

Article Title: Multiplicity of acquired cross-resistance in paclitaxel-resistant cancer cells is associated with feedback control of TUBB3 via FOXO3a-mediated ABCB1 regulation

doi: 10.18632/oncotarget.9118

Figure Lengend Snippet: Multiplicity of acquired cross-resistance to structurally different drugs in cancer cells selected for taxane resistance is associated with feedback control of TUBB3 through FOXO3a-mediated ABCB1 regulation inducing hyperfunctional P-gp-associated drug efflux and escape from potential inhibition.

Article Snippet: For ABCB1 silencing, cells were transfected with scramble siRNA (FITC-conjugate) or human-specific ABCB1 (MDR1) siRNA duplexes (Santa Cruz Biotechnology) using Lipofectamine 3000 [ ].

Techniques: Control, Inhibition