Journal: Environmental Health Perspectives

Article Title: Mechanisms and Consequences of Variable TRPA1 Expression by Airway Epithelial Cells: Effects of TRPV1 Genotype and Environmental Agonists on Cellular Responses to Pollutants in Vitro and Asthma

doi: 10.1289/EHP11076

Figure Lengend Snippet: (A) Volcano plot of RNA sequencing data from NHBE cells with either the TRPV1 I585I/I or I585I/V genotypes. Summary data can be found in Excel Table S1. (B) TRPA1 , (C) TRPV1 , (D) NLRP2 , and (E) IGFBP2 mRNA expression in NHBE cells having either the TRPV1 I585I/I or I585I/V genotype ( n = 6 or 7 donors/genotype). Data (Rq) are the mean ± SD , relative to β 2 M mRNA and the average for target gene expression in NHBEs with the I585I/I genotype. * p ≤ 0.05 and ** p < 0.01 using a one-tailed unpaired Student’s t -test. Summary data can be found in Excel Table S2 (B,C) Excel Table S3 (D,E). Note: IGFBP2, insulin like growth factor binding protein 2; NHBE, normal human bronchial epithelial (cells); NLRP2, nucleotide-binding oligomerization domain, leucine rich repeat and pyrin domain containing 2; Rq, relative quantification; SD, standard deviation; TRPA1, transient receptor potential cation channel subfamily A member 1; TRPV1, transient receptor potential cation channel subfamily V member 1; β 2 M , β 2 -microglobulin .

Article Snippet: NHBEs immortalized with cyclin-dependent kinase 4 (CDK4) and telomerase reverse transcriptase (hTERT), or HBEC3-KT cells, were from ATCC (CRL-4051) and were grown in airway epithelial cell basal medium supplemented with bronchial epithelial cell growth kit, 30 μ g / mL Geneticin, and 250 ng / mL puromycin (ATCC).

Techniques: RNA Sequencing, Expressing, Targeted Gene Expression, One-tailed Test, Binding Assay, Quantitative Proteomics, Standard Deviation

Journal: Environmental Health Perspectives

Article Title: Mechanisms and Consequences of Variable TRPA1 Expression by Airway Epithelial Cells: Effects of TRPV1 Genotype and Environmental Agonists on Cellular Responses to Pollutants in Vitro and Asthma

doi: 10.1289/EHP11076

Figure Lengend Snippet: (A) TRPA1 and (B) TRPV1 mRNA expression in HBEC3-KT cells following 24-h treatment with nonivamide ( 10 μ M ) or LJO-328 ( 25 μ M ; white bars), and co-treatment with pine WSPM in media ( 20 μ g / cm 2 ; gray hashed bars). Data (Rq) are the mean ± SD for target gene mRNA expression relative to β 2 M mRNA and cells treated with media containing 0.2% DMSO only ( n = 3 – 6 ). Control and WSPM co-treated groups were analyzed independently comparing all treatment groups using one-way ANOVA and Tukey’s multiple comparisons test. * p ≤ 0.05 , *** p < 0.001 , and **** p < 0.0001 . #### Indicates significant difference ( p < 0.0001 ) from all WSPM co-treated groups using two-way ANOVA and a Bonferroni multiple comparisons test comparing the corresponding ± WSPM groups. Summary data can be found in Excel Table S4. Note: ANOVA, analysis of variance; DMSO, dimethyl sulfoxide; HBEC3-KT, telomerase reverse transcriptase and CDK4–immortalized normal human bronchial epithelial (cells); LJO-328, N -(4- tert -butylbenzyl)- N -(1-[3-fluoro-4-(methylsulfonylamino)phenyl]ethyl)thiourea; nonivamide, 2-mercaptoethanol, n -vanillylnonanamide; Rq, relative quantity; SD, standard deviation; TRPA1, transient receptor potential cation channel subfamily A member 1; TRPV1, transient receptor potential cation channel subfamily V member 1; WSPM, wood smoke particulate matter; β 2 M , β 2 -microglobulin .

Article Snippet: NHBEs immortalized with cyclin-dependent kinase 4 (CDK4) and telomerase reverse transcriptase (hTERT), or HBEC3-KT cells, were from ATCC (CRL-4051) and were grown in airway epithelial cell basal medium supplemented with bronchial epithelial cell growth kit, 30 μ g / mL Geneticin, and 250 ng / mL puromycin (ATCC).

Techniques: Expressing, Control, Reverse Transcription, Standard Deviation

Journal: Environmental Health Perspectives

Article Title: Mechanisms and Consequences of Variable TRPA1 Expression by Airway Epithelial Cells: Effects of TRPV1 Genotype and Environmental Agonists on Cellular Responses to Pollutants in Vitro and Asthma

doi: 10.1289/EHP11076

Figure Lengend Snippet: Temporal changes in (A) TRPA1 and (B) TRPV1 mRNA expression in HBEC3-KT cells following treatment with media containing 0.2% DMSO (open circles, solid lines) or pine WSPM in media ( 20 μ g / cm 2 ; closed circles, dashed lines). Data (Rq) are the mean ± SD for target gene mRNA expression relative to β 2 M mRNA and the control ( n = 3 ). * p ≤ 0.05 and **** p < 0.0001 using repeated measures two-way ANOVA comparing the control and WSPM-treated groups at each time and correction using Bonferroni’s multiple comparisons test. Summary data can be found in Excel Table S6. Note: ANOVA, analysis of variance; DMSO, dimethyl sulfoxide; HBEC3-KT, telomerase reverse transcriptase and CDK4–immortalized normal human bronchial epithelial (cells); Rq, relative quantification; SD, standard deviation; TRPA1, transient receptor potential cation channel subfamily A member 1; TRPV1, transient receptor potential cation channel subfamily V member 1; WSPM, wood smoke particulate matter; β 2 M , β 2 -microglobulin .

Article Snippet: NHBEs immortalized with cyclin-dependent kinase 4 (CDK4) and telomerase reverse transcriptase (hTERT), or HBEC3-KT cells, were from ATCC (CRL-4051) and were grown in airway epithelial cell basal medium supplemented with bronchial epithelial cell growth kit, 30 μ g / mL Geneticin, and 250 ng / mL puromycin (ATCC).

Techniques: Expressing, Control, Reverse Transcription, Quantitative Proteomics, Standard Deviation

Journal: Environmental Health Perspectives

Article Title: Mechanisms and Consequences of Variable TRPA1 Expression by Airway Epithelial Cells: Effects of TRPV1 Genotype and Environmental Agonists on Cellular Responses to Pollutants in Vitro and Asthma

doi: 10.1289/EHP11076

Figure Lengend Snippet: TRPA1 (white bars) and TRPV1 (gray hashed bars) mRNA expression in HBEC3-KTs transfected with scramble, GAPDH , and TRPV1 siRNA ( 500 pmol / mL ) 24 h posttransfection. Data (Rq) are the mean ± SD for target gene mRNA expression relative to β 2 M mRNA and the average for cells transfected with scramble siRNA ( n = 3 ). Data for TRPA1 and TRPV1 were analyzed independently comparing all three siRNAs using one-way ANOVA and Tukey’s multiple comparisons test. *** p < 0.001 and **** p < 0.0001 . Summary data can be found in Excel Table S7. Note: ANOVA, analysis of variance; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HBEC3-KT, telomerase reverse transcriptase and CDK4–immortalized normal human bronchial epithelial (cells); Rq, relative quantification; SD, standard deviation; siRNA, small interfering RNA; TRP, transient receptor potential; TRPA1, transient receptor potential cation channel subfamily A member 1; TRPV1, transient receptor potential cation channel subfamily V member 1; β 2 M , β 2 -microglobulin .

Article Snippet: NHBEs immortalized with cyclin-dependent kinase 4 (CDK4) and telomerase reverse transcriptase (hTERT), or HBEC3-KT cells, were from ATCC (CRL-4051) and were grown in airway epithelial cell basal medium supplemented with bronchial epithelial cell growth kit, 30 μ g / mL Geneticin, and 250 ng / mL puromycin (ATCC).

Techniques: Expressing, Transfection, Reverse Transcription, Quantitative Proteomics, Standard Deviation, Small Interfering RNA

Journal: Environmental Health Perspectives

Article Title: Mechanisms and Consequences of Variable TRPA1 Expression by Airway Epithelial Cells: Effects of TRPV1 Genotype and Environmental Agonists on Cellular Responses to Pollutants in Vitro and Asthma

doi: 10.1289/EHP11076

Figure Lengend Snippet: (A) TRPA1 mRNA expression in HBEC3-KT cells following 24-h treatment with media containing 0.2% DMSO or the NF- κ B inhibitor BMS-345541 (BMS; 10 μ M ). **** p < 0.0001 using a one-tailed unpaired Student’s t -test. (B) TRPA1 , (C) TRPV1 , and (D) IL8 mRNA expression in HBEC3-KT cells following 4- and 24-h treatment with TNF α ( 50 ng / mL ) or 0.01% BSA in media ( n = 3 ). Data (Rq) are the mean ± SD for target gene mRNA expression relative to β 2 M mRNA and the control. * p ≤ 0.05 , ** p < 0.01 , and **** p < 0.0001 using two-way ANOVA and Tukey’s multiple comparisons test comparing all treatments groups. (E) Calcium flux in HBEC3-KT cells treated for 24 h with either media containing 0.01% BSA and 0.2% DMSO, TNF α ( 50 ng / mL ), BMS-345541 ( 10 μ M ), or TNF α and BMS subsequently stimulated by the addition of AITC ( 150 μ M ). Data were normalized to ionomycin ( n = 5 ). Raw images are shown in Figure S6. **** p < 0.0001 using one-way ANOVA and Tukey’s multiple comparison test comparing all groups. Summary data can be found in Excel Table S8. Note: AITC, allyl isothiocyanate; ANOVA, analysis of variance; BSA, bovine serum albumin; DMSO, dimethyl sulfoxide; HBEC3-KT, telomerase reverse transcriptase and CDK4–immortalized normal human bronchial epithelial (cells); IL8, interleukin-8; NF- κ B , nuclear factor kappa light chain enhancer of activated B cells; Rq, relative quantification; SD, standard deviation; TNF α , tumor necrosis factor-alpha; TRPA1, transient receptor potential cation channel subfamily A member 1; TRPV1, transient receptor potential cation channel subfamily V member 1; β 2 M , β 2 -microglobulin .

Article Snippet: NHBEs immortalized with cyclin-dependent kinase 4 (CDK4) and telomerase reverse transcriptase (hTERT), or HBEC3-KT cells, were from ATCC (CRL-4051) and were grown in airway epithelial cell basal medium supplemented with bronchial epithelial cell growth kit, 30 μ g / mL Geneticin, and 250 ng / mL puromycin (ATCC).

Techniques: Expressing, One-tailed Test, Control, Comparison, Reverse Transcription, Quantitative Proteomics, Standard Deviation

Journal: Environmental Health Perspectives

Article Title: Mechanisms and Consequences of Variable TRPA1 Expression by Airway Epithelial Cells: Effects of TRPV1 Genotype and Environmental Agonists on Cellular Responses to Pollutants in Vitro and Asthma

doi: 10.1289/EHP11076

Figure Lengend Snippet: (A) Quantification of nuclear pNF- κ B /p65 in HBEC3-KTs treated for 24 h with media, TNF α ( 50 ng / mL ), nonivamide ( 10 μ M ), or LJO-328 ( 25 μ M ). pNF- κ B /p65 intensity was normalized to p84, and NF- κ B (total) was normalized to β -actin prior to calculating the pNF- κ B /NF- κ B ratio. Data represents the mean ± SD ( n = 3 ). ** p < 0.01 using one-way ANOVA and Tukey’s multiple comparisons test comparing all groups. (B) Representative western blot image where, from left to right, are the molecular weight standard (MW), control (1), TNF α (2), nonivamide (3), and LJO-328 treatments (4). Raw western blot data are shown in Figure S7. Summary data can be found in Excel Table S9. Note: ANOVA, analysis of variance; HBEC3-KT, telomerase reverse transcriptase and CDK4–immortalized normal human bronchial epithelial (cells); LJO-328, N -(4- tert -butylbenzyl)- N -(1-[3-fluoro-4-(methylsulfonylamino)phenyl]ethyl)thiourea; nonivamide, 2-mercaptoethanol, n -vanillylnonanamide; NF- κ B , nuclear factor kappa light chain enhancer of activated B cells; p84, rabbit nuclear matrix protein; pNF- κ B , phospho-nuclear factor kappa light chain enhancer of activated B cells; SD, standard deviation; TNF α , tumor necrosis factor-alpha.

Article Snippet: NHBEs immortalized with cyclin-dependent kinase 4 (CDK4) and telomerase reverse transcriptase (hTERT), or HBEC3-KT cells, were from ATCC (CRL-4051) and were grown in airway epithelial cell basal medium supplemented with bronchial epithelial cell growth kit, 30 μ g / mL Geneticin, and 250 ng / mL puromycin (ATCC).

Techniques: Western Blot, Molecular Weight, Control, Reverse Transcription, Standard Deviation

Journal: Environmental Health Perspectives

Article Title: Mechanisms and Consequences of Variable TRPA1 Expression by Airway Epithelial Cells: Effects of TRPV1 Genotype and Environmental Agonists on Cellular Responses to Pollutants in Vitro and Asthma

doi: 10.1289/EHP11076

Figure Lengend Snippet: (A) TRPA1 , (B) TRPV1 , and (C) IL8 mRNA expression in HBEC3-KT cells following 4- or 12-h treatment with media containing 0.2% DMSO or the PKC activator and the TRPV1 sensitizer PMA. Data (Rq) are the mean ± SD for target gene mRNA expression relative to β 2 M mRNA and the control ( n = 3 ). *** p < 0.001 and **** p < 0.0001 using two-way ANOVA and Dunnett’s multiple comparison test comparing to the 4 and 12 h gene-specific control. (D) Effects of 12-h treatment with the PKC inhibitor Go6983 ( 10 μ M ) and p38 MAPK inhibitor PD169316 ( 10 μ M ) on TRPA1 mRNA expression in HBEC3-KT and SAECs compared with cells treated with media containing 0.2% DMSO. Data (Rq) are the mean ± SD for target gene mRNA expression relative to β 2 M mRNA and the control. **** p < 0.0001 using multiple t -tests comparing treatment vs. the respective cell type–specific control. (E) TRPV1 and IL8 mRNA expression in HBEC3-KT cells following 12-h treatment with media containing 0.2% DMSO or Go6983 ( 10 μ M ). * p ≤ 0.05 and **** p < 0.0001 using two-way ANOVA and Bonferroni’s multiple comparisons test to compare the gene-specific control and treatment group. (F) TRPV1-mediated calcium flux in HEK-293 cells stably overexpressing human TRPV1 with and without 12-h treatment with media containing 0.2% DMSO or the PKC inhibitor Go6983 ( 10 μ M ). Data are the mean ± SD for change in fluorescence relative to media-treated cells normalized to the maximum response (100%) and fit using the log[agonist] vs. normalized response-variable slope equation ( n = 3 ). ** p < 0.01 and **** p < 0.0001 using two-way ANOVA and a Bonferroni test. Raw data are graphed in Figure S8. Summary data can be found in Excel Table S10 (A–C) and Excel Table S11 (D–F). Note: ANOVA, analysis of variance; DMSO, dimethyl sulfoxide; HBEC3-KT, telomerase reverse transcriptase and CDK4–immortalized normal human bronchial epithelial (cells); IL8, interleukin-8; p38 MAPK, p38 mitogen-activated protein kinase; PD169316, a p38 MAPK inhibitor; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; Rq, relative quantification; SAECs, small airway epithelial cells; SD, standard deviation; TRPA1, transient receptor potential cation channel subfamily A member 1; TRPV1, transient receptor potential cation channel subfamily V member 1; β 2 M , β 2 -microglobulin .

Article Snippet: NHBEs immortalized with cyclin-dependent kinase 4 (CDK4) and telomerase reverse transcriptase (hTERT), or HBEC3-KT cells, were from ATCC (CRL-4051) and were grown in airway epithelial cell basal medium supplemented with bronchial epithelial cell growth kit, 30 μ g / mL Geneticin, and 250 ng / mL puromycin (ATCC).

Techniques: Expressing, Control, Comparison, Stable Transfection, Fluorescence, Reverse Transcription, Quantitative Proteomics, Standard Deviation

Journal: Environmental Health Perspectives

Article Title: Mechanisms and Consequences of Variable TRPA1 Expression by Airway Epithelial Cells: Effects of TRPV1 Genotype and Environmental Agonists on Cellular Responses to Pollutants in Vitro and Asthma

doi: 10.1289/EHP11076

Figure Lengend Snippet: (A) NLRP2 mRNA expression in HBEC3-KT cells following 24-h treatment with media containing 0.2% DMSO or the NF- κ B inhibitor BMS-345541 ( 10 μ M ). Data (Rq) are the mean ± SD for target gene mRNA expression relative to β 2 M mRNA and the control ( n = 3 ). **** p < 0.0001 using a one-tailed, unpaired Student’s t -test. (B) Representative western blot image for NLRP2 in HBEC3-KT cells transfected with 100 pmol / mL NLRP2 siRNA, where, from left to right, are the molecular weight standards (MW), scramble siRNA (1), GAPDH siRNA (2), and NLRP2 siRNA 1 and 2 transfected cell lysates (3 and 4). (C) Quantification of NLRP2 protein in siRNA-transfected HBEC3-KTs. Raw data are provided in Figure S9. Data are the mean ± SD of the ratio of NLRP2 to β -actin band density ( n = 3 ). * p ≤ 0.05 and ** p < 0.01 using one-way ANOVA and Tukey’s multiple comparison test comparing all groups. (D–G) NLRP2 , TRPA1 , TRPV1 , and IL8 mRNA expression in HBEC3-KTs 24 h after NLRP2 siRNA-2 ( 100 pmol ) transfection, and (H) IL8 mRNA expression following TRPV1 siRNA ( 500 pmol ) transfection compared with the respective control siRNA and GAPDH siRNA groups. Data (Rq) are the mean ± SD for target gene mRNA expression relative to β 2 M mRNA and the scramble control ( n = 3 ). ** p < 0.01 , *** p < 0.001 , and **** p < 0.0001 using one-way ANOVA and Tukey’s multiple comparison test comparing all groups. Summary data can be found in Excel Table S12. Note: ANOVA, analysis of variance; BMS, BMS-345541; DMSO, dimethyl sulfoxide; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HBEC3-KT, telomerase reverse transcriptase and CDK4–immortalized normal human bronchial epithelial (cells); IL8, interleukin-8; NF- κ B , nuclear factor kappa light chain enhancer of activated B cells; NLRP2, nucleotide-binding oligomerization domain, leucine rich repeat and pyrin domain containing 2; Rq, relative quantification; SD, standard deviation; siRNA, small interfering RNA; TRPA1, transient receptor potential cation channel subfamily A member 1; TRPV1, transient receptor potential cation channel subfamily V member 1; β 2 M , β 2 -microglobulin .

Article Snippet: NHBEs immortalized with cyclin-dependent kinase 4 (CDK4) and telomerase reverse transcriptase (hTERT), or HBEC3-KT cells, were from ATCC (CRL-4051) and were grown in airway epithelial cell basal medium supplemented with bronchial epithelial cell growth kit, 30 μ g / mL Geneticin, and 250 ng / mL puromycin (ATCC).

Techniques: Expressing, Control, One-tailed Test, Western Blot, Transfection, Molecular Weight, Comparison, Reverse Transcription, Binding Assay, Quantitative Proteomics, Standard Deviation, Small Interfering RNA

Journal: Environmental Health Perspectives

Article Title: Mechanisms and Consequences of Variable TRPA1 Expression by Airway Epithelial Cells: Effects of TRPV1 Genotype and Environmental Agonists on Cellular Responses to Pollutants in Vitro and Asthma

doi: 10.1289/EHP11076

Figure Lengend Snippet: (A–C) DDIT3 and (D–F) IL8 mRNA expression in NHBE cells having either the TRPV1 I585I/I or I585I/V genotype 24 h after treatment with media containing either 0.2% DMSO, DEP (CFA 10 μ g / cm 2 ), or CFA ( 180 μ g / cm 2 ) treatment ( n ≥ 4 donors/genotype). (A,B,D,E) Data (Rq) are the mean ± SD for target gene mRNA expression normalized to β 2 M mRNA, analyzed using a paired one-tailed t -test. Fold change in (C) DDIT3 and (F) IL8 mRNA expression in DEP- and CFA-treated cells normalized to media-treated controls for each donor. * p ≤ 0.05 using multiple t -tests to compare genotype effects for each particle. Summary data can be found in Excel Table S13. Note: CFA, coal fly ash; DDIT3, DNA damage-inducible transcript-3; DEP, diesel exhaust particles; DMSO, dimethyl sulfoxide; IL8, interleukin-8; NHBE, normal human bronchial epithelial (cells); Rq, relative quantification; SD, standard deviation; TRPV1, transient receptor potential cation channel subfamily V member 1; β 2 M , β 2 -microglobulin .

Article Snippet: NHBEs immortalized with cyclin-dependent kinase 4 (CDK4) and telomerase reverse transcriptase (hTERT), or HBEC3-KT cells, were from ATCC (CRL-4051) and were grown in airway epithelial cell basal medium supplemented with bronchial epithelial cell growth kit, 30 μ g / mL Geneticin, and 250 ng / mL puromycin (ATCC).

Techniques: Expressing, One-tailed Test, Quantitative Proteomics, Standard Deviation

Journal: Environmental Health Perspectives

Article Title: Mechanisms and Consequences of Variable TRPA1 Expression by Airway Epithelial Cells: Effects of TRPV1 Genotype and Environmental Agonists on Cellular Responses to Pollutants in Vitro and Asthma

doi: 10.1289/EHP11076

Figure Lengend Snippet: Schematic summarizing the authors hypothesis for how TRPV1, NF- κ B , PKC, and p38 MAPK may regulate TRPA1, TRPV1, and NLRP2 expression in AECs. The summary is based on the cumulative results of this and other referenced studies. (A) TRPV1 I585I/I and (B) TRPV1 I585I/V. Note: AECs, airway epithelial cells; Ca 2 + , calcium ions; Go6983, a PKC inhibitor; NF- κ B , nuclear factor kappa light chain enhancer of activated B cells; NLRP2, nucleotide-binding oligomerization domain, leucine rich repeat and pyrin domain containing 2; MAPK, p38 mitogen-activated protein kinase; PD169316, a p38 MAPK inhibitor; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; TRPA1, transient receptor potential cation channel subfamily A member 1; TRPV1, transient receptor potential cation channel subfamily V member 1.

Article Snippet: NHBEs immortalized with cyclin-dependent kinase 4 (CDK4) and telomerase reverse transcriptase (hTERT), or HBEC3-KT cells, were from ATCC (CRL-4051) and were grown in airway epithelial cell basal medium supplemented with bronchial epithelial cell growth kit, 30 μ g / mL Geneticin, and 250 ng / mL puromycin (ATCC).

Techniques: Expressing, Binding Assay

Journal: Nature Communications

Article Title: Vitamin D supplementation ameliorates ductular reaction, liver inflammation and fibrosis in mice by upregulating TXNIP in ductular cells

doi: 10.1038/s41467-025-59724-z

Figure Lengend Snippet: A The expression levels of Pcna, Casp3, Tnf , and Tgfb1 in cholangiocytes. Female C57BL/6 mice were fed DDC diet for 4 weeks with vehicle or 1,25(OH) 2 D 3 (5 µg/kg, 2x week) treatment and primary cholangiocytes were isolated ( n = 4/group). B Relative mRNA levels of Vdr . Female C57BL/6 mice were treated with 1,25(OH) 2 D 3 (5 µg/kg, 2x week) for 4 weeks, and primary cholangiocytes, HSCs, liver macrophages, and LSECs were isolated ( n = 3/group). C Hepatic mRNA levels of Txnip . Female C57BL/6 mice were treated with 1,25(OH) 2 D 3 (5 µg/kg, 2x week) for 4 weeks ( n = 3/group). D Relative mRNA levels of Txnip . Female C57BL/6 mice were treated with 1,25(OH) 2 D 3 (5 µg/kg, 2x week) for 4 weeks, and primary hepatocytes, cholangiocytes, HSCs, liver macrophages, and LSECs were isolated ( n = 3/group). E qPCR analysis. BMOL cells were pre-incubated with control or Vdr siRNA for 24 h and treated with 1,25(OH) 2 D 3 (10 µM) for 24 h ( n = 4 biological replicates). F Luciferase assay. BMOL cells were pre-incubated with control or Vdr siRNA for 24 h and treated with 1,25(OH) 2 D 3 (10 µM) for 6 h ( n = 4 biological replicates). Values represent means ± SDs. n.s., not significant. Significance determined by one-way ANOVA followed by Tukey’s multiple comparisons test ( A , E , F ) and unpaired two-tailed t test ( B , C , D ). Source data are provided as a Source Data file.

Article Snippet: The corresponding cDNA was synthesized using a ReverTra Ace® qPCR RT Kit (Toyobo, Osaka, Japan).

Techniques: Expressing, Isolation, Incubation, Control, Luciferase, Two Tailed Test

Journal: Nature Communications

Article Title: Vitamin D supplementation ameliorates ductular reaction, liver inflammation and fibrosis in mice by upregulating TXNIP in ductular cells

doi: 10.1038/s41467-025-59724-z

Figure Lengend Snippet: Female Txnip fl/fl and Txnip ΔSox9 mice were fed DDC diet for 4 weeks. A Representative H&E staining ( n = 4/group). B IHC staining for pan-CK. Right: Numbers of pan-CK + cells ( n = 4/group). C Relative hepatic mRNA levels of Krt19 , Krt7 , and Sox9 ( Txnip fl/fl , n = 4; Txnip ΔSox9 , n = 3). D IF staining for F4/80 and Sirius-red staining. Right: The F4/80 + cell numbers and Sirius red-positive areas ( n = 4/group). Magnification: 200X. E , F qPCR analysis of genes involved in inflammation ( E ) and fibrosis ( F ) ( Txnip fl/fl , n = 4; Txnip ΔSox9 , n = 3). Values represent means ± SDs. n.s., not significant. Significance determined by unpaired two-tailed t test. Source data are provided as a Source Data file.

Article Snippet: The corresponding cDNA was synthesized using a ReverTra Ace® qPCR RT Kit (Toyobo, Osaka, Japan).

Techniques: Staining, Immunohistochemistry, Two Tailed Test

Journal: Nature Communications

Article Title: Vitamin D supplementation ameliorates ductular reaction, liver inflammation and fibrosis in mice by upregulating TXNIP in ductular cells

doi: 10.1038/s41467-025-59724-z

Figure Lengend Snippet: Principal component analysis ( A ) and volcano map ( B ) for control and Txnip -overexpressing (OE) BMOL cells ( n = 3/group). C Top 20 enriched gene ontologies from DAVID analysis. D Heatmap of representative differentially expressed genes in control and Txnip OE BMOL cells. E Flow cytometric analysis of shRNA control and shRNA Txnip BMOL cells ( n = 3 biological replicates). F Effects of 1,25(OH) 2 D 3 on cell proliferation and apoptosis in shRNA control and shRNA Txnip BMOL cells, as assessed by Western blot analysis ( n = 3 biological replicates). G IF staining of pan-CK/BrdU and pan-CK/caspase-3 in liver tissues. Txnip fl/fl and Txnip ΔSox9 mice were DDC-fed for 4 weeks. Right: Percentages of pan-CK + BrdU + and pan-CK + caspase-3 + cells ( n = 4/group). Original magnifications: 400X and 800X. ( H ) qPCR analysis of cholangiocytes isolated from DDC-fed Txnip fl/fl and Txnip ΔSox9 mice ( n = 4/group). Values represent means ± SDs. n.s., not significant. Significance determined by unpaired two-tailed t test. Source data are provided as a Source Data file.

Article Snippet: The corresponding cDNA was synthesized using a ReverTra Ace® qPCR RT Kit (Toyobo, Osaka, Japan).

Techniques: Control, shRNA, Western Blot, Staining, Isolation, Two Tailed Test

Journal: Nature Communications

Article Title: Vitamin D supplementation ameliorates ductular reaction, liver inflammation and fibrosis in mice by upregulating TXNIP in ductular cells

doi: 10.1038/s41467-025-59724-z

Figure Lengend Snippet: A Representative images of IF staining for F4/80/pan-CK and α-SMA/pan-CK in DDC-fed mice. F4/80 + liver macrophages and α-SMA + HSCs were observed near pan-CK + cells (arrows) near portal regions (1) and in parenchymal regions (2). Original magnifications: 400X and 800X. B qPCR analysis. Primary KCs isolated from C57BL/6 mice were co-cultured with shRNA control or shRNA Txnip BMOL cells for 24 h ( n = 4 biological replicates). C mRNA levels of cytokines in shRNA control and shRNA Txnip BMOL cells ( n = 3 biological replicates). D Relative mRNA expression levels of pro-inflammatory genes in cholangiocytes isolated from DDC-fed Txnip fl/fl and Txnip ΔSox9 mice ( n = 4/group). E Levels of TNF-α in culture media of shRNA control or shRNA Txnip BMOL cells treated with or without TNF-α neutralizing antibody (5 µg/ml) for 24 h ( n = 4 biological replicates). F Expression of pro-inflammatory genes in KCs isolated from C57BL/6 mice and co-cultured with shRNA control or shRNA Txnip BMOL cells plus TNF-α neutralizing antibody ( n = 4 biological replicates). G Relative mRNA expression levels of fibrosis-related genes. HSCs isolated from C57BL/6 mice were co-cultured with shRNA control or shRNA Txnip BMOL cells ( n = 3 biological replicates). H Relative mRNA expression levels of Tgfb1 in shRNA control and shRNA Txnip BMOL cells ( n = 3 biological replicates). I qPCR analysis of fibrosis-related genes in cholangiocytes from DDC-fed Txnip fl/fl and Txnip ΔSox9 mice ( n = 4/group). J Levels of TGF-β in culture media from shRNA control or shRNA Txnip BMOL cells treated with or without TGF-β neutralizing antibody (2 µg/ml) for 24 hours ( n = 3 biological replicates). ( K ) Expression of fibrosis-related genes in primary HSCs co-cultured with shRNA control or shRNA Txnip BMOL cells with or without TGF-β neutralizing antibody ( n = 3 biological replicates). Values represent means ± SDs. n.s., not significant. Significance determined by unpaired two-tailed t test. Source data are provided as a Source Data file.

Article Snippet: The corresponding cDNA was synthesized using a ReverTra Ace® qPCR RT Kit (Toyobo, Osaka, Japan).

Techniques: Staining, Isolation, Cell Culture, shRNA, Control, Expressing, Two Tailed Test

Journal: Nature communications

Article Title: Genome-wide compendium and functional assessment of in vivo heart enhancers.

doi: 10.1038/ncomms12923

Figure Lengend Snippet: Figure 4 | Cardiac enhancers are required for normal expression of heart disease-associated genes. Knockout analysis of enhancers mm77 (a,c,e,g,h) and mm771 (b,d,f,i,j). (a,b) Representative transgenic reporter assay results. Heart activity is indicated by red arrows. Numbers indicate reproducibility over total transgenic embryos. Gene models not drawn to scale. LOF: loss-of-function, GOF: gain-of-function. (c,d) Chromosome-wide mRNA expression changes in E11.5 whole heart. Points indicate individual genes, with red indicating statistically significantly differences after FDR correction (Po0.01 using an FDRo5%, see the ‘Methods’ section for details). Dashed grey line indicates position of enhancer. Mb: megabases. (e,f) Normalized mRNA levels measured by quantitative RT-PCR in E11.5 hearts. Boxplots indicate median and quartile values for each data set; points indicate outliers. (g–j) Representative western blot images (g,i) and normalized protein levels (h,j) in wild-type ( þ ) and homozygous null ( ) samples. Gapdh was used as a loading control, MW, molecular weight. (g,i). Bars indicate group means, and points represent biological replicates (h,j). For (e,f,h,j): values were normalized to the wild-type mean, and P values were calculated using a one-tailed t-test.

Article Snippet: RNA sequencing libraries were made using the TruSeq Stranded Total RNA with Ribo-Zero Human/Mouse/Rat kit (Illumina) or the TruSeq Stranded mRNA Sample Prep Kit (Illumina) according to manufacturer instructions.

Techniques: Expressing, Knock-Out, Transgenic Assay, Reporter Assay, Activity Assay, Quantitative RT-PCR, Western Blot, Control, Molecular Weight, One-tailed Test

Journal: Pharmacological Reports

Article Title: Compromised diabetic heart function is not affected by miR-378a upregulation upon hyperglycemia

doi: 10.1007/s43440-023-00535-8

Figure Lengend Snippet: miR-378a is upregulated in hyperglycemic murine heart. A – I Heart lysates and J cardiac histological sections were obtained from either control or STZ-treated miR-378a+/+ and miR-378a−/− mice. The expression of A miR-378a-3p and B miR-378a-5p is increased in heart lysates of miR-378a+/+ mice 8 weeks upon STZ treatment, LNA miRCURY qPCR ( n = 4–8). mRNA level of C Igf1r , D Mapk1 and E Pik3ca (qPCR, n = 4–8). F – I Densitometric analysis of changes of IGF-1R pathway in response to STZ. F Combined analysis of IGF-1R and downstream mediators. Relative to GAPDH reference protein. The protein level of G IGF-1R (rel. to GAPDH), H pERK1/2 (rel. to total ERK1/2), and I p-AKT (rel. to total AKT), (western blot, densitometric analysis of five independent western blot repetitions, n = 3–4) upon STZ treatment. J IGF-1R expression in different cardiac cell types (CMs, SMCs, ECs; shown with arrows) (IF; ⍺-smooth muscle actin (⍺-SMA)/IGF-1R—green, laminin—purple, Cx43—red). Representative histological sections (×63 magnification, scale bar: 10 µm). Data are presented as mean ± SEM. # p < 0.05, ## p < 0.001, ### p < 0.0001—two-way ANOVA variation; * p < 0.05, ** p < 0.01 *** p < 0.001 by two-way ANOVA with Tukey’s post hoc test; $ p < 0.05, $$ p < 0.01 by unpaired two-tailed Student’s t test. Igf1r (IGF-1R) insulin-like growth factor 1 receptor, Mapk1 (ERK2) mitogen-activated protein kinase 1, Pik3ca PI3 kinase catalytic subunit alpha, (p)AKT (phosphorylated) protein kinase B, (p)ERK1/2 (phosphorylated) extracellular signal-regulated kinase 1/2

Article Snippet: Total RNA was used for reverse transcription (RT) of either mRNA (500 ng) or miRNA (10 ng) performed with RevertAid reverse transcriptase (200 U/μL, Thermo Fisher Scientific, cat. no. EP0442) and miRCURY LNA RT kit (Qiagen, cat. no. 339340), respectively, carried out in ProFlex PCR System (ThermoFisher Scientific).

Techniques: Control, Expressing, Western Blot, Two Tailed Test

Journal: Pharmacological Reports

Article Title: Compromised diabetic heart function is not affected by miR-378a upregulation upon hyperglycemia

doi: 10.1007/s43440-023-00535-8

Figure Lengend Snippet: High glucose increases miR-378a expression and stimulates pro-hypertrophic pathways in hiPSC-CMs. miR-378a+/+ hiPSCs and miR-378a−/− hiPSCs were differentiated to CMs and treated with glucose (GLU) for 48 h. Mannitol (55 mM) was used as a vehicle. The expression of A miR-378a-3p and -5p (LNA miRCURY qPCR, n = 4) and B IGF1R mRNA level (qPCR, n = 5). C – F Densitometric analysis of changes of IGF-1R pathway in response to glucose. C Combined analysis of IGF-1R and downstream mediators. Relative to GAPDH reference protein. D Protein level of IGF-1R (rel. to GAPDH), E pERK1/2 (rel. to total ERK1/2) and F p-AKT (rel. to total AKT), (western blot, densitometric analysis, n = 2–4). G Quantitative analysis of cell area ( n = 15/27/8/43) and representative pictures of hypertrophic growth of miR-378a−/− hiPSC-CMs under control conditions and 55 mM glucose (HG) (IF; ⍺-actinin—green), representative images (×200 magnification). Data are presented as mean ± SEM. # p < 0.05—two-way ANOVA variation; * p < 0.05, *** p < 0.001 by two-way ANOVA with Tukey’s post hoc test; $ p < 0.05 by unpaired two-tailed Student’s t test. GLU glucose, HG high glucose, hiPSC-CMs human induced pluripotent stem cell-derived cardiomyocytes, IGF1R (IGF-1R) insulin-like growth factor 1 receptor, (p)AKT (phosphorylated) protein kinase B, (p)ERK1/2 (phosphorylated) extracellular signal-regulated kinase 1/2

Article Snippet: Total RNA was used for reverse transcription (RT) of either mRNA (500 ng) or miRNA (10 ng) performed with RevertAid reverse transcriptase (200 U/μL, Thermo Fisher Scientific, cat. no. EP0442) and miRCURY LNA RT kit (Qiagen, cat. no. 339340), respectively, carried out in ProFlex PCR System (ThermoFisher Scientific).

Techniques: Expressing, Western Blot, Control, Two Tailed Test, Derivative Assay

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Growth Differentiation Factor 7 Prevents Sepsis-Induced Acute Lung Injury in Mice

doi: 10.1155/2022/3676444

Figure Lengend Snippet: GDF7 alleviates LPS-induced ALI and pulmonary dysfunction in mice. (a) Mice were intratracheally injected with LPS (5 mg/kg in 50 μ L saline) to generate sepsis-induced ALI, and serum GDF7 levels were detected using an ELISA kit after 12 h ( n = 6). (b) The levels of Gdf7 mRNA in lung tissues with or without LPS instillation were detected using quantitative real-time PCR ( n = 6). (c) The levels of GDF7 protein in lung tissues with or without LPS instillation were detected using western blot ( n = 6). (d-e) Mice were subcutaneously injected with rmGDF7 (25 μ g per mouse), and serum and lung GDF7 levels in mice were detected using an ELISA kit after 24 h ( n = 6). (f) Mice were subcutaneously injected with rmGDF7 (25 μ g per mouse), and 24 h later, ALI mice were intratracheally injected with LPS (5 mg/kg in 50 μ L saline) to generate sepsis-induced ALI. After 12 h, fresh lungs were harvested for the analysis of LDH activity using a commercial kit ( n = 6). (g) Lung wet to dry ratio ( n = 6). (h) Total proteins in BALF ( n = 6). (i) Arterial blood gas analysis results ( n = 6). (j) Respiratory function was detected by Buxco, including pulmonary ventilation, lung compliance, and airway resistance ( n = 6). (k) Mice were subcutaneously injected with rmGDF7 (25 μ g per mouse), and 24 h later, ALI mice were intratracheally injected with a lethal dose of LPS (25 mg/kg). The survival rate was monitored every 12 h post-LPS treatment ( n = 20). All data were presented as mean ± SD, and ∗ P < 0.05 was regarded to be statistically significant.

Article Snippet: Lactate Dehydrogenase (LDH) Assay Kit (#ab102526), Mouse Myeloperoxidase (MPO) ELISA Kit (#ab155458), Lipid Peroxidation (MDA) Assay Kit (#ab118970), Lipid Peroxidation (4-HNE) Assay Kit (#ab238538), Superoxide Dismutase (SOD) Activity Assay Kit (#ab65354), and Glutathione (GSH) Assay Kit (#ab239727) were purchased from Abcam (Cambridge, UK).

Techniques: Injection, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Western Blot, Activity Assay

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Growth Differentiation Factor 7 Prevents Sepsis-Induced Acute Lung Injury in Mice

doi: 10.1155/2022/3676444

Figure Lengend Snippet: GDF7 attenuates LPS-induced ALI through activating AMPK in vivo. (a) Mice were subcutaneously injected with rmGDF7 (25 μ g per mouse), and 24 h later, ALI mice were intratracheally injected with LPS (5 mg/kg in 50 μ L saline) to generate sepsis-induced ALI. After 12 h, lung tissues were harvested for the analysis of the AMPK pathway using western blot ( n = 6). (b) To inhibit AMPK, ALI mice were intraperitoneally injected with CpC (20 mg/kg) at 2 h pre- and 2 h post-rmGDF7 injection, and then IL-6 and TNF- α levels in lung tissues were detected ( n = 6). (c) ROS content in lung tissues ( n = 6). (d) MDA and 4-HNE levels in lung tissues ( n = 6). (e) LDH activity in lung tissues ( n = 6). (f) Lung wet to dry ratio ( n = 6). (g) Arterial blood gas analysis results ( n = 6). (h) Respiratory function was detected by Buxco, including pulmonary ventilation, lung compliance, and airway resistance ( n = 6). All data were presented as mean ± SD, and ∗ P < 0.05 was regarded to be statistically significant.

Article Snippet: Lactate Dehydrogenase (LDH) Assay Kit (#ab102526), Mouse Myeloperoxidase (MPO) ELISA Kit (#ab155458), Lipid Peroxidation (MDA) Assay Kit (#ab118970), Lipid Peroxidation (4-HNE) Assay Kit (#ab238538), Superoxide Dismutase (SOD) Activity Assay Kit (#ab65354), and Glutathione (GSH) Assay Kit (#ab239727) were purchased from Abcam (Cambridge, UK).

Techniques: In Vivo, Injection, Western Blot, Activity Assay

Journal: eLife

Article Title: UBTD1 regulates ceramide balance and endolysosomal positioning to coordinate EGFR signaling

doi: 10.7554/eLife.68348

Figure Lengend Snippet: ( A–L ) DU145 cells were transfected for 48 hr with the indicated siRNA (control, siCTRLpool or siCTRLsingle1; control, siCTRL; UBTD1: siUBTD1pool or siUBTD1single1 or single2). ( A,C,H ) Western blotting images of phospho-kinases spotted on the Proteome Profiler Human Phospho-Kinase Array in complete media (A) or under EGF stimulation (C, 50 ng/ml, 10 min) or serum-starved ( H ). Phospho-EGFR double spots are marked in red rectangles. ( B ) Immunoblot and quantification (n = 3 independent experiments) of p-EGFR (Y1068 or Y1086). p-EGFR levels were quantified by calculating the ratio between p-EGFR and EGFR, both normalized to loading control signal. Immunoblot of UBTD1 shows the level of siRNA depletion. ( D, E ) Immunoblot and quantification (n = 3 independent experiments) of pSTAT3, p-ERK, and p-AKT. p-STAT3, p-ERK, and p-AKT levels were quantified by calculating the ratio between phospho-protein and total-protein, both normalized to loading control signal. Immunoblot of UBTD1 shows the level of siRNA depletion. ( F ) Representative wide-field immunofluorescence images (left) and quantification (right) of pSTAT3 nuclear translocation corresponding to nuclei/cytoplasm mean intensity ratio of pSTAT3. ( G ) mRNA quantification of STAT3 target genes (bcl2, mmp2, mmp9 and hif2). ( I ) Quantification of EGFR mRNA level. ( J ) EGF secretion measured by ELISA. ( K ) mRNA quantification of EGFR ligands. ( L ) EGFR ligands secretion measured by ELISA. Scale bar = 10 µm. n ≥ 3 independent experiments; ns = non-significant; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; ( F, I, J ) two-tailed t-test; ( B,C,E,G, K,L ) two-way ANOVA and Bonferroni’s multiple comparisons test; data are mean ± s.e.m. Figure 1—figure supplement 1—source data 1. Uncropped western blot for .

Article Snippet: Relative phosphorylation levels of 43 kinases and two related proteins were assessed using the Proteome Profiler Human Phospho-Kinase Array Kit (R and D Systems), according to the manufacturer’s instructions.

Techniques: Transfection, Control, Western Blot, Immunofluorescence, Translocation Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: iScience

Article Title: SPI1-KLF1/LYL1 axis regulates lineage commitment during endothelial-to-hematopoietic transition from human pluripotent stem cells

doi: 10.1016/j.isci.2024.110409

Figure Lengend Snippet: Multi-omics analysis reveals potential regulatory roles of SPI1 in EHT (A) Heatmap of 575 expressed TFs during the differentiation of Pluripotent Stem Cells (PSC) into Hematopoietic Progenitor Cells (HPC). Four expression clusters (TC1-TC4) are identified and are denoted by color and number. (B) Line graph of variable accessible chromatin elements during PSC to HPC differentiation, again with four identified clusters (C1-C4). (C) Enriched TF motifs in peaks of clusters C1-C4. (D) Heatmap showing variations in TF activity among the top 50 active transcription factors during differentiation. (E) Pearson correlation analysis of TF activity and gene expression for five key TFs. (F) Visualization of ATAC-seq footprint for motifs of the five representative TFs across different developmental stages. See also Figure S2 and .

Article Snippet: CD34 MicroBeads, human , Miltenyi Biotec , Cat# 130-046-702.

Techniques: Biomarker Discovery, Expressing, Activity Assay, Gene Expression

Journal: iScience

Article Title: SPI1-KLF1/LYL1 axis regulates lineage commitment during endothelial-to-hematopoietic transition from human pluripotent stem cells

doi: 10.1016/j.isci.2024.110409

Figure Lengend Snippet: Knockdown of SPI1 at the in vitro -generated HECs stage severely impairs HPC generation (A) Schematic illustration of the in vitro differentiation approach. SPI1 siRNA or control siRNA were introduced on D4 of EHT, with evaluations from D5 to D8. (B) Flow cytometric sorting strategy for HECs treated with siRNA. (C) qPCR results showing SPI1 expression from D5 to D8, using two distinct siRNA sequences targeting SPI1 . (D) Western blot analysis of PU.1 expression in siCtrl and siSPI1_1/2 groups from D5 to D8, with GAPDH as the internal normalization control. (E) Representative flow cytometry density plots showing CD34 + CD43 + cells among total cells in siCtrl and siSPI1_1/2 groups, from D5 to D8. (F) Quantification of suspended CD34 + CD43 + HPCs from siCtrl and siSPI1_1/2 groups, from D5 to D8. (G) Microscopy images of cells on D8 for siCtrl and siSPI1 conditions, scale bar = 100 μm. (H) Representative flow cytometry density plots of CD34 + CD43 + HPCs in suspended cells for siCtrl and siSPI1_1/2 groups on D8. Data are from n = 3 independent experiments, and statistical significance was determined using two-tailed unpaired Student’s t-test. Data are presented as means ± SEM. Significance levels: ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. See also Figure S4 , Table S1 and Table S2 .

Article Snippet: CD34 MicroBeads, human , Miltenyi Biotec , Cat# 130-046-702.

Techniques: Knockdown, In Vitro, Generated, Control, Expressing, Western Blot, Flow Cytometry, Microscopy, Two Tailed Test

Journal: iScience

Article Title: SPI1-KLF1/LYL1 axis regulates lineage commitment during endothelial-to-hematopoietic transition from human pluripotent stem cells

doi: 10.1016/j.isci.2024.110409

Figure Lengend Snippet: Knockdown of SPI1 during EHT does not alter HPC proliferation or apoptosis but affects differentiation potential (A) Flow cytometry sorting strategy for D8 suspended CD34 + CD43 + HPCs (FAM +/− ). (B and C) Representative flow cytometry density plots (B) and frequency plot (C) for cell cycle, G0/G1 (Edu − Hoechst low ), S (Edu + Hoechst low/high ) and G2/M (Edu − Hoechst high ) on D8 suspended CD34 + CD43 + HPCs (FAM +/− ) in siCtrl and siSPI1_1/2 groups. (D and E) Representative flow cytometry density plots (D) and frequency plot (E) of early apoptosis (Annexin V + DAPI − ) and late apoptosis (Annexin V + DAPI + ) on D8 suspended CD34 + CD43 + HPCs (FAM +/− ) in siCtrl and siSPI1_1/2 groups. Significance determined by two-tailed unpaired Student’s t-test. (F) Colony Forming Unit (CFU) assay of FAM − CD34 + CD43 + HPCs generated on D8 under siCtrl and siSPI1_1/2 conditions. Unless otherwise stated, data are from n = 3 independent experiments, and statistical significance was determined using two-way ANOVA. Data are presented as means ± SEM. Significance levels: ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ns, no significance. See also Figure S5 .

Article Snippet: CD34 MicroBeads, human , Miltenyi Biotec , Cat# 130-046-702.

Techniques: Knockdown, Flow Cytometry, Two Tailed Test, Colony-forming Unit Assay, Generated

Journal: iScience

Article Title: SPI1-KLF1/LYL1 axis regulates lineage commitment during endothelial-to-hematopoietic transition from human pluripotent stem cells

doi: 10.1016/j.isci.2024.110409

Figure Lengend Snippet: Transcriptional regulatory network suggests LYL1 and KLF1 as potential downstream targets of SPI1 (A) Heatmap displaying reordered normalized trans -regulation score (TRS) at the HPC stage. Identified modules from nonnegative matrix factorization (NMF) are represented in different colors. (B) Boxplots showing gene expression levels in different modules from A, comparing Endothelial Progenitor Cells (EPC) and HPC. p values, two-tailed paired Student’s t test. (C) All SPI1 Target Genes (TGs) are ordered descendingly according to their TRS, with a focus on the top 10 transcription factors (TFs) within these TGs. (D) Violin plots showing expression levels of the top 10 TFs from SPI1 TGs in both in vivo AGM hematopoiesis (left) and the in vitro system (right). (E) qPCR results showing the expression of the top 10 transcription factors from SPI1 target genes in D8 CD34 + CD43 + HPCs, excluding non-expressed genes SNAI3 and RUNX3 , in siCtrl and siSPI1_1/2 groups. Data are from n = 3 independent experiments, with significance assessed using two-tailed unpaired Student’s t test. (F) Enrichment analysis of Gene Ontology (GO) terms and KEGG signaling pathways of SPI1 ’s TGs that have p -value <0.001. Data are presented as means ± SEM. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ns, no significance.

Article Snippet: CD34 MicroBeads, human , Miltenyi Biotec , Cat# 130-046-702.

Techniques: Gene Expression, Two Tailed Test, Expressing, In Vivo, In Vitro, Protein-Protein interactions

Journal: iScience

Article Title: SPI1-KLF1/LYL1 axis regulates lineage commitment during endothelial-to-hematopoietic transition from human pluripotent stem cells

doi: 10.1016/j.isci.2024.110409

Figure Lengend Snippet: Overexpression of LYL1 and KLF1 can rescue SPI1 knockdown phenotypes (A) Co-transfection strategy of SPI1 siRNA with KLF1 / LYL1 overexpression during EHT to assess D8 HPC differentiation potential. (B) Western blot analysis of KLF1/LYL1 from D6 differentiated cells in the siCtrl+OE-Ctrl, siSPI1, and siSPI1+OE-KLF1/LYL1 groups, normalized to GAPDH. (C and D) Flow cytometry analysis of suspended CD34 + CD43 + HPCs on D8: (C) Representative density plot and (D) quantification in siCtrl+OE-Ctrl, siSPI1, siSPI1+OE-KLF1 and siSPI1+OE-LYL1 groups. n = 3 independent experiments. (E) Colony-forming units (CFU) assay on D8 suspended CD34 + CD43 + HPCs across siCtrl+OE-Ctrl, siSPI1, siSPI1+OE-KLF1 and siSPI1+OE-LYL1 groups, with significance assessed using two-way ANOVA. (F) Schematic description of lymphoid differentiation. (G) Representative flow cytometry density plots of the distribution of CD45 -/mid/+ CD5 + CD7 + cells under the normal conditions at D29. (H–I) Analysis of CD45 + CD5 + CD7 + cells: (H) Representative flow cytometry density plots (H) and frequency plot (I) of CD45 + CD5 + CD7 + cells in siCtrl+OE-Ctrl, siSPI1, siSPI1+OE-KLF1 and siSPI1+OE-LYL1 groups. Unless otherwise stated, data are from n = 3 independent experiments, and statistical significance was determined using two-tailed unpaired Student’s t-test. Data are presented as means ± SEM. Significance levels: ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ns, no significance. See also Figures S6 and .

Article Snippet: CD34 MicroBeads, human , Miltenyi Biotec , Cat# 130-046-702.

Techniques: Over Expression, Knockdown, Cotransfection, Western Blot, Flow Cytometry, Colony-forming Unit Assay, Two Tailed Test

Journal: iScience

Article Title: SPI1-KLF1/LYL1 axis regulates lineage commitment during endothelial-to-hematopoietic transition from human pluripotent stem cells

doi: 10.1016/j.isci.2024.110409

Figure Lengend Snippet:

Article Snippet: CD34 MicroBeads, human , Miltenyi Biotec , Cat# 130-046-702.

Techniques: Recombinant, Transfection, Plasmid Preparation, Reverse Transcription, In Vivo, In Vitro, Biomarker Discovery, Software

Journal: Cancer discovery

Article Title: Active CREB1 promotes a malignant TGFβ2 autocrine loop in glioblastoma.

doi: 10.1158/2159-8290.CD-14-0275

Figure Lengend Snippet: Figure 2. TGFβ induces TGFβ2 expression in GBM and non-GBM cell lines. A, qRT-PCR of TGFB 1 , TGFB 2 , and TGFB 3 in LN229 cells treated with TGFβ1 for 3 hours. GAPDH mRNA levels were used as an internal normalization control. ***, P < 0.005, using the Student t test; data, mean ± SD. B, qRT-PCR of TGFB 2 in LN229 cells treated with TGFβ1, TGFβ2, and TGFβ3 for 3 hours. GAPDH mRNA levels were used as an internal normalization control. *, P < 0.05, using the Student t test; data, mean ± SD. C, secreted TGFβ2 protein levels determined by ELISA in culture supernatant from LN229 cells treated with TGFβ for 72 hours. ***, P < 0.005, using the Student t test; data, mean ± SD. D, immunoblot analysis and qRT-PCR of TGF b 2 in LN229 cells treated with TGFβ1 and/or the TβRI inhibitor (TβRI inh.) LY-2109761 for 3 hours. GAPDH mRNA levels were used as an internal normalization control. *, P < 0.05, using the Student t test; data, mean ± SD. E, qRT-PCR of TGFB 2 in GBM and non-GBM cell lines treated with TGFβ for 3 hours. GAPDH mRNA levels were used as an internal normalization control. ***, P < 0.005, using the Student t test; data, mean ± SD.

Article Snippet: For the quantitative determination of TGFβ2 protein levels secreted to the media, we used the Human TGFβ2 Quantikine ELISA Kit (R&D Systems) following the manufacturer’s specifi cations.

Techniques: Expressing, Quantitative RT-PCR, Control, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Cancer discovery

Article Title: Active CREB1 promotes a malignant TGFβ2 autocrine loop in glioblastoma.

doi: 10.1158/2159-8290.CD-14-0275

Figure Lengend Snippet: Figure 3. CREB1 regulates the autocrine induction of TGFβ2 by TGFβ. A, nucleotide sequence of the proximal region of the TGFB 2 promoter. The SBEs and CREB1 site (CRE) are indicated relative to the transcription start site. ClustalW sequence alignment for 3 animal species [ Homo sapiens ( H.s .), Pan troglodytes ( P.t. ), and Mus musculus ( M.m .)] shows the conservation of the binding sites. B, qRT-PCR of TGFB 2 and CREB1 in LN229 cells expressing an shRNA targeting CREB1 treated with TGFβ for 3 hours. GAPDH mRNA levels were used as an internal normalization control. ***, P < 0.005, using the Student t test; data, mean ± SD. C, qRT-PCR of TGFB 2 and CREB1 in LN229 cells expressing an siRNA targeting CREB1 treated with TGFβ for 3 hours. GAPDH mRNA levels were used as an internal normalization control. ***, P < 0.005, using the Student t test; data, mean ± SD. D, immunoblot analysis and qRT-PCR of TGFβ2 in LN229 cells expressing ICER treated with TGFβ for 3 hours. GAPDH mRNA levels were used as an internal normalization control. ***, P < 0.005, using the Student t test; data, mean ± SD. The molecular weights are shown.

Article Snippet: For the quantitative determination of TGFβ2 protein levels secreted to the media, we used the Human TGFβ2 Quantikine ELISA Kit (R&D Systems) following the manufacturer’s specifi cations.

Techniques: Sequencing, Binding Assay, Quantitative RT-PCR, Expressing, shRNA, Control, Western Blot

Journal: Cancer discovery

Article Title: Active CREB1 promotes a malignant TGFβ2 autocrine loop in glioblastoma.

doi: 10.1158/2159-8290.CD-14-0275

Figure Lengend Snippet: Figure 5. PI3K and RSK regulate the TGFβ- mediated induction of TGFβ2 through CREB1. A, immunoblot analysis and qRT-PCR of TGFB2 in LN229 cells treated with TGFβ for 3 hours and the PI3K inhibitor (inh) LY-294002 for 24 hours. GAPDH mRNA levels were used as an internal normalization control. ***, P < 0.005, using the Student t test; data, mean ± SD. B, immunoblot analysis and qRT-PCR of TGFB2 in LN229 cells treated with increasing amounts of the RSK inhibitor BI-D1870 for 24 hours and TGFβ for 3 hours. GAPDH mRNA levels were used as an internal normalization control. ***, P < 0.005, using the Student t test; data, mean ± SD. C, secreted TGFβ2 protein levels determined by ELISA in LN229 cells treated with TGFβ for 48 hours and the PI3K inhibitor for 72 hours. *, P < 0.05, using the Student t test; data, mean ± SD. D, secreted TGFβ2 protein levels determined by ELISA in LN229 cells treated with the RSK inhibitor BI-D1870 for 72 hours and TGFβ for 48 hours. *, P < 0.05, using the Student t test; data, mean ± SD.

Article Snippet: For the quantitative determination of TGFβ2 protein levels secreted to the media, we used the Human TGFβ2 Quantikine ELISA Kit (R&D Systems) following the manufacturer’s specifi cations.

Techniques: Western Blot, Quantitative RT-PCR, Control, Enzyme-linked Immunosorbent Assay

Journal: Cancer discovery

Article Title: Active CREB1 promotes a malignant TGFβ2 autocrine loop in glioblastoma.

doi: 10.1158/2159-8290.CD-14-0275

Figure Lengend Snippet: Figure 6. TGFβ2 correlates with CREB1 expression in GBM patient tumors. A and B, graphs showing the correlation between CREB1 and TGFB 1 (B) or TGFB 2 (A) mRNA levels in patient GBM tumor samples. Data obtained from the REMBRANDT database. A Spearman test was used, and the correlation coeffi cient (ρ) and the two-tailed P value are shown. C, graph showing the correlation between p-CREB1 and TGFβ2 protein levels in tissue microarrays (TMA) from patient GBM samples. Not all spots were evaluable in all stainings. A Spearman test was used, and the correlation coeffi cient (ρ) and the two- tailed signifi cance are shown. Representative images from the TMAs are shown; scale bar, 50 μm. D, Kaplan–Meier curves showing the OS of patients with TGFB2 mRNA levels upregulated ≥3-fold and CREB1 mRNA levels upregulated ≥2-fold. Statistical signifi cance was assessed by the log-rank test. Data obtained from the REMBRANDT database.

Article Snippet: For the quantitative determination of TGFβ2 protein levels secreted to the media, we used the Human TGFβ2 Quantikine ELISA Kit (R&D Systems) following the manufacturer’s specifi cations.

Techniques: Expressing, Two Tailed Test

Journal: Cancer discovery

Article Title: Active CREB1 promotes a malignant TGFβ2 autocrine loop in glioblastoma.

doi: 10.1158/2159-8290.CD-14-0275

Figure Lengend Snippet: Figure 7. CREB1 regulates TGFβ2 expression in PDX models. A, scheme showing the experimental procedure. B, IHC of p-CREB1 and TGFβ2 from mouse tumors 60 days after inoculation with neurospheres expressing shRNAs targeting CREB1 and control shRNAs; scale bar, 50 μm (C). Kaplan–Meier survival curves from mice in B. D, the TGFβ2 malignant autocrine loop. In GBM, TGFβ collaborates with the PI3K and RSK pathways through a CREB1– SMAD3 transcriptional complex to induce TGFβ2 expression. This leads to the generation of an autocrine loop and accumulation of TGFβ2 in the tumor, hyperactivation of TGFβ, and tumor progression.

Article Snippet: For the quantitative determination of TGFβ2 protein levels secreted to the media, we used the Human TGFβ2 Quantikine ELISA Kit (R&D Systems) following the manufacturer’s specifi cations.

Techniques: Expressing, Paraffin-embedded Immunohistochemistry, Control

Journal: Nutrients

Article Title: Biomarkers of Broccoli Consumption: Implications for Glutathione Metabolism and Liver Health

doi: 10.3390/nu12092514

Figure Lengend Snippet: Broccoli feeding increases protein expression of Nqo1 and Nrf2 in rats after 14 days of broccoli feeding ( A ) Timeline of study. Rats were fed a diet with or without 10% freeze-dried broccoli ( n = 4/feeding group). ( B ) The protein expression of Nqo1 and Nrf2 in liver tissue was detected with Western blot. β-actin served as a loading control, used to normalize Nqo1 and Nrf2 protein expression and calculate data for C,D. ( C ) Quantification of Western blot results. Unpaired t -test analysis of Nqo1( C ) and Nrf2 ( D ) protein expression. p < 0.05 as compared with the control group, all data and standard deviation are plotted.

Article Snippet: Membranes were blocked (Blocking Buffer, Odyssey ® , Li-Cor, Lincoln, NE, USA) and target proteins were probed with the following primary antibodies: β-actin (Sigma SAB1305546) 1:10,000 dilution, NAD(P)H dehydrogenase [quinone] 1 (NQO1) (Abcam Cat# ab2346, RRID:AB_302995) 1:500 dilution, nuclear factor (erythroid-derived 2)-like 2 (Nrf2) (R&D SYSTEM MAB3925) 1:500 dilution.

Techniques: Expressing, Western Blot, Standard Deviation

Journal: Disease Models & Mechanisms

Article Title: Evaluation of human dermal fibroblasts directly reprogrammed to adipocyte-like cells as a metabolic disease model

doi: 10.1242/dmm.030981

Figure Lengend Snippet: Characterisation of direct reprogrammed adipocyte-like cells. (A) Quantitative RT-PCR analysis of expression of marker genes of white adipocytes ( FABP4 , ADIPOQ , CEBPA and SLC2A4 ) at four different differentiation time points. (B) Western blot analysis of key proteins in adipocyte-like cells during direct reprogramming up to day 18, using antibodies as indicated. (C) Adiponectin secretion from direct reprogrammed adipocyte-like cells in 48-h culture medium was determined with DELFIA. (D) Downregulation of PPARγ2 and then INSR expression after removal of DOX from the culture medium. (E) Delipidation was also observed in reprogrammed adipocyte-like cells. Images were taken 10 days after DOX withdrawal (left) or with DOX included in the culture medium throughout (right). Scale bars: 100 µm. (F) Glucose uptake assay. (G) Lipolysis assay of direct reprogrammed adipocyte-like cells treated with isoprotenerol and/or IBMX. Data are mean±s.e.m. from three independent experiments (*** P <0.001, Student's t -test).

Article Snippet: Blots were blocked in TBST (50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 0.1% Tween 20) containing 5% milk or BSA (Sigma-Aldrich) and probed overnight at 4°C with the following antibodies: anti-PPARγ (sc-7196, Santa Cruz Biotechnology, 1:500 dilution), anti-adiponectin (ab13881, Abcam, 1:1000 dilution), anti-Glut4 (ab654, Abcam, 1:4000 dilution), anti-C/EBPa (#2295, Cell Signaling Technology, 1:1000 dilution), anti-insulin receptor β (sc-711, Santa Cruz Biotechnology, 1:500 dilution), anti-IGF1 receptor (sc-713, Santa Cruz Biotechnology, 1:500 dilution), anti-calnexin (ab75801, Abcam, 1:10,000 dilution), anti-aP2 (sc-18661, Santa Cruz Biotechnology, 1:5000 dilution), anti-lamin A/C (sc-20681, Santa Cruz Biotechnology, 1:500 dilution), anti-perilipin (#3467s Cell Signaling Technology, 1:1000 dilution) and anti-lamin R482W (SAB4200422, Sigma-Aldrich, 1:5000 dilution).

Techniques: Quantitative RT-PCR, Expressing, Marker, Western Blot

Journal: Disease Models & Mechanisms

Article Title: Evaluation of human dermal fibroblasts directly reprogrammed to adipocyte-like cells as a metabolic disease model

doi: 10.1242/dmm.030981

Figure Lengend Snippet: Direct reprogramming of dermal fibroblasts derived from patients with metabolic diseases caused by mutations on PPARγ, insulin receptor, lamin and BSCL2. (A) Oil Red O staining showing that the four patient cell lines (SIR1-SIR4) were all successfully converted into adipocyte-like cells using our direct reprogramming protocol, as schematised in Fig. 1 . Scale bars: 200 µm. (B) Quantitative RT-PCR analysis of the expression of white adipocyte marker genes. (C) Western blot analysis of key proteins in undifferentiated control cells (DOX−) and reprogrammed adipocyte-like cells (DOX+). Sequencing confirmation of mutations on PPARG and BSCL2 genes is shown for SIR1 and SIR4, respectively. (D) Adiponectin secretion from direct reprogrammed adipocyte-like cells in 48-h culture medium was determined with DELFIA. Data are mean±s.e.m. from three independent experiments (*** P <0.001, Student's t -test).

Article Snippet: Blots were blocked in TBST (50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 0.1% Tween 20) containing 5% milk or BSA (Sigma-Aldrich) and probed overnight at 4°C with the following antibodies: anti-PPARγ (sc-7196, Santa Cruz Biotechnology, 1:500 dilution), anti-adiponectin (ab13881, Abcam, 1:1000 dilution), anti-Glut4 (ab654, Abcam, 1:4000 dilution), anti-C/EBPa (#2295, Cell Signaling Technology, 1:1000 dilution), anti-insulin receptor β (sc-711, Santa Cruz Biotechnology, 1:500 dilution), anti-IGF1 receptor (sc-713, Santa Cruz Biotechnology, 1:500 dilution), anti-calnexin (ab75801, Abcam, 1:10,000 dilution), anti-aP2 (sc-18661, Santa Cruz Biotechnology, 1:5000 dilution), anti-lamin A/C (sc-20681, Santa Cruz Biotechnology, 1:500 dilution), anti-perilipin (#3467s Cell Signaling Technology, 1:1000 dilution) and anti-lamin R482W (SAB4200422, Sigma-Aldrich, 1:5000 dilution).

Techniques: Derivative Assay, Staining, Quantitative RT-PCR, Expressing, Marker, Western Blot, Sequencing

Journal: The American journal of pathology

Article Title: Prolactin-induced prostate tumorigenesis links sustained Stat5 signaling with the amplification of basal/stem cells and emergence of putative luminal progenitors.

doi: 10.1016/j.ajpath.2014.07.020

Figure Lengend Snippet: Figure 2 Activation of canonical PRLR signaling pathways in WT and Pb-PRL ventral, lateral, and dorsal prostate lobes. A: Representative Western blot images per genotype. B: Quantification of p-Stat5 blot results normalized to total Stat5 levels. C: Results of semiquantitative AlphaScreen SureFire assay for p-Stat5 levels in WT and Pb-PRL mice. DeG: Quantification of results from Western blot analysis for p-Stat3, p-Akt, p-Erk1/2 (normalized to total Stat3, Akt, and Erk1/2 levels, respectively) and PRL levels (normalized to total Erk1/2 levels). H: Correlation between mean counts of basal cell clusters and mean levels of p-Stat5 measured by semiquantitative AlphaScreen SureFire assay in each prostate lobe. WT and Pb-PRL mouse values are represented with white and black symbols, respectively (circles, ventral lobes; squares, lateral lobes; triangles, dorsal lobes). Data for all bar graphs are expressed as means SD. n Z 6 mice per genotype (B); n Z 5 WT mice (C); n Z 12 Pb-PRL mice (C); n Z 6 mice per genotype (DeG). *P < 0.001 versus WT; yP < 0.01, yyP < 0.001 versus ventral lobe; zP < 0.05, zzP < 0.01, zzzP < 0.001 versus lateral lobe by two-way repeated measures ANOVA and Bonferroni’s multiple comparisons test (BeG). Pearson correlation test, P Z 0.002; r2 Z 0.93 (H).

Article Snippet: AlphaScreen SureFire p-Stat5 Assay An AlphaScreen SureFire Stat5 (p-Tyr694/699) Assay Kit (PerkinElmer, Waltham, MA) was used to detect p-Stat5 in a semiquantitative manner.

Techniques: Activation Assay, Protein-Protein interactions, Western Blot, Amplified Luminescent Proximity Homogenous Assay

Journal: The American journal of pathology

Article Title: Prolactin-induced prostate tumorigenesis links sustained Stat5 signaling with the amplification of basal/stem cells and emergence of putative luminal progenitors.

doi: 10.1016/j.ajpath.2014.07.020

Figure Lengend Snippet: Figure 3 Response to acute PRL stimulation in prostate lobes of WT mice and PRLR levels in prostate lobes of WT and Pb-PRL mice. A: Representative image of results from Western blot analysis for Stat5 activation after subcutaneous injection of vehicle or 50 mg of PRL. B: Quantification of p-Stat5 blotting results normalized to total Stat5 levels. C: Results of semiquantitative AlphaScreen SureFire assay for p-Stat5 levels. DeF: mRNA expression levels of long L (D) and short3 S3 (E) PRLR isoforms and S3/L ratios (F) in dorsal and ventral lobes of WT and Pb-PRL mice. All data are represented relative to WT ventral expression levels after normalization to cyclophilin A. Data are expressed as means SD. n Z 4 mice per group (B); n Z 3 mice per group (C); n Z 5 mice per group (DeF). *P < 0.05, **P < 0.01, ***P < 0.001 versus vehicle treatment (B and C); yP < 0.01, yyP < 0.001 versus WT; zP < 0.01 versus ventral lobe by two-way repeated measures ANOVA and Bonferroni’s multiple comparisons test (DeF). D, dorsal; L, lateral; V, ventral.

Article Snippet: AlphaScreen SureFire p-Stat5 Assay An AlphaScreen SureFire Stat5 (p-Tyr694/699) Assay Kit (PerkinElmer, Waltham, MA) was used to detect p-Stat5 in a semiquantitative manner.

Techniques: Western Blot, Activation Assay, Injection, Amplified Luminescent Proximity Homogenous Assay, Expressing

Journal: The American journal of pathology

Article Title: Prolactin-induced prostate tumorigenesis links sustained Stat5 signaling with the amplification of basal/stem cells and emergence of putative luminal progenitors.

doi: 10.1016/j.ajpath.2014.07.020

Figure Lengend Snippet: Figure 5 Analysis of basal cell clusters in WT and Pb-PRL mice. AeC: Frequency distributions for staining scores in basal cell cluster areas of each ge- notype: high versus low nuclear Stat5 (A) and Sca-1 (B) staining, and yes versus no for the presence of double positive CK-5þCK-8þ cells (C). Representative images of basal cell cluster areas that showed high versus low or yes versus no scores for each staining are shown on the right of each graph; together with corresponding CK-5/CK-8 IF images. DeF: Distribution of staining results detected in basal cell cluster areas of Pb-PRL mice for Sca-1 when double positive CK- 5þCK-8þ cells were present (yes) versus absent (no) (D), for Ki-67 when Sca-1 was high versus low (E), and when double CK-5þCK-8þ cells were yes versus no (F). GeH: Representative images of serial slides for two basal cell cluster areas displaying both presence of double positive CK-5þCK-8þ cells (IF staining) and high Sca-1 and Ki-67 scores (IHC staining). Basal cell cluster areas analyzed are encircled by dashed lines; arrows point to one or more double positive CK-5þCK-8þ cells. Sample sizes (n) are indicated on each graph. *P < 0.05, **P < 0.01, and ***P < 0.001 by two-tailed Fischer’s exact test (AeF). Scale bar: 25 mm (AeC, G, and H). Original magnification: 40 (AeC, G, and H, IHC images); 10 (AeC, G, and H, IF images).

Article Snippet: AlphaScreen SureFire p-Stat5 Assay An AlphaScreen SureFire Stat5 (p-Tyr694/699) Assay Kit (PerkinElmer, Waltham, MA) was used to detect p-Stat5 in a semiquantitative manner.

Techniques: Staining, Immunohistochemistry, Two Tailed Test

Journal: The American journal of pathology

Article Title: Prolactin-induced prostate tumorigenesis links sustained Stat5 signaling with the amplification of basal/stem cells and emergence of putative luminal progenitors.

doi: 10.1016/j.ajpath.2014.07.020

Figure Lengend Snippet: Figure 8 Proposed model of prostate epithelial hierarchy according to our results from Pb-PRL mice. PRL induces the amplification of basal, intermediate, and luminal cells, including a subpopulation of Sca-1þ luminal cells that we propose to be progenitors. The amplification of the basal compartment involves basal progenitors and might include stem cells as well (dashed pink line). Amplified intermediate cells originated from basal progenitors rapidly differentiate into luminal Sca-1þ progenitors (lightning-shaped arrows symbolize the rapidity of this process). In turn, these luminal progenitors give rise to mature luminal cells. Our results from sorted prostate subpopulations also indicated the existence of Sca-1 intermediate cells. We propose that these cells originate from Sca-1þ intermediate cells and that they subsequently differentiate into luminal cells (orange dashed arrows). All intermediate and luminal cells can respond to PRL stimulation with p-Stat5 and translocation into the nucleus (purple stars). Finally, the existence of terminally differentiated basal cells originating from basal progenitors was neither established nor denied by our results (black dashed arrow). CK-5þ cells are green; CK-8þ cells are red; double CK-5þCK-8þ intermediate cells are yellow. The membrane marker Sca-1 is represented as blue circles on the membrane of Sca-1þ cells.

Article Snippet: AlphaScreen SureFire p-Stat5 Assay An AlphaScreen SureFire Stat5 (p-Tyr694/699) Assay Kit (PerkinElmer, Waltham, MA) was used to detect p-Stat5 in a semiquantitative manner.

Techniques: Translocation Assay, Membrane, Marker