Journal: Cell reports

Article Title: Tracing Tumor Evolution in Sarcoma Reveals Clonal Origin of Advanced Metastasis

doi: 10.1016/j.celrep.2019.08.029

Figure Lengend Snippet: (A) Effect of stable overexpression of each candidate gene on the metastatic ability of KPCC cells after tail-vein injection. Each point represents the relative metastatic area in the lungs of an individual nude mouse. Data represented as mean ± SD (*p < 0.05, one-way ANOVA). (B) Metastatic burden following tail-vein injection of control or Aldh1a2 overexpressing cells in an independent set of animals quantified by relative area of metastatic lesions. Data represented as mean ± SD (**p < 0.01, Student’s t test). (C) Representative H&E images of lungs from mice injected with Aldh1a2-OE and empty-vector control cells (scale, 1.5 mm). (D) Confocal lung metastasis after competition between Aldh1a2-OE (labeled green) and empty-vector control cells (labeled red) injected at 1:1 ratio into the tail vein of nude mice (scale, 1.5 mm; inset scale, 100 μm). (E) Quantification of metastases area. Data represented as mean ± SD (***p < 0.001, Student’s t test). (F) Confocal microscopy of orthotopic competition between Aldh1a2-OE cells (labeled green) and control cells (labeled red) injected at 1:1 ratio into the muscle of the extremity (scale, 700 μm; inset scale, 100 μm). (G) Quantification of tumor tissue area for each fluorescent reporter in tumor sections (Student’s t test).

Article Snippet: Aldh1a2, Rabbit Polyclonal , ProteinTech , Cat#: 13951–1-AP; RRID:AB_2224033.

Techniques: Over Expression, Injection, Control, Plasmid Preparation, Labeling, Confocal Microscopy

Journal: Cell reports

Article Title: Tracing Tumor Evolution in Sarcoma Reveals Clonal Origin of Advanced Metastasis

doi: 10.1016/j.celrep.2019.08.029

Figure Lengend Snippet:

Article Snippet: Aldh1a2, Rabbit Polyclonal , ProteinTech , Cat#: 13951–1-AP; RRID:AB_2224033.

Techniques: Clone Assay, Virus, Plasmid Preparation, Software, Microscopy

Journal: HemaSphere

Article Title: Upregulation of ALDH1 as an adaptive epigenetic response to anthracyclines in acute myeloid leukemia

doi: 10.1002/hem3.70244

Figure Lengend Snippet: Elevated ALDH1A1 associates with chemotherapy response and poor prognosis in acute myeloid leukemia (AML) patients. (A) Schematic workflow of bioinformatics analyses. Created with BioRender ( https://BioRender.com/tyzpbfr ). (B) ALDH1A1 expression in AML patients stratified by ELN‐2022 prognostic categories. (C) ALDH1A1 expression according to response to induction chemotherapy. (D) ALDH1A1 and ALDH1A2 expression in de novo AML patients stratified by the presence or absence of diagnostic qualifiers linked to secondary AML. (E) Kaplan–Meier survival analysis showing reduced overall survival associated with high ALDH1A1 expression, most pronounced in relapsed/refractory AML patients. Numbers at risk are displayed below the x‐axis. Statistical significance was assessed using the log‐rank test. HR, hazard ratio; MDS, myelodysplastic syndrome; MDS/MPN, myelodysplastic/myeloproliferative neoplasm.

Article Snippet: The following primary antibodies were used: ALDH1A1 (Proteintech, Cat# 15910‐1‐AP; Manchester, UK), ALDH1A2 (Proteintech, Cat# 13951‐1‐AP), ALDH1A3 (Bio‐Techne, Cat# NBP2‐15339), JNK (Cell Signaling Technology, Cat# 9252; Saint‐Cyr‐L'École, France), c‐Jun (Cell Signaling Technology, Cat# 9165), Phospho‐c‐Jun Ser73 (S73) (Cell Signaling Technology, Cat# 3270), STAT3 (Cell Signaling Technology, Cat# 9139), Phospho‐STAT3 (Y705) (Cell Signaling technology, Cat# 9145) and Protein Simple loading control (Bio‐Techne, Cat# 042‐196).

Techniques: Expressing, Diagnostic Assay

Journal: HemaSphere

Article Title: Upregulation of ALDH1 as an adaptive epigenetic response to anthracyclines in acute myeloid leukemia

doi: 10.1002/hem3.70244

Figure Lengend Snippet: Anthracyclines induce aldehyde dehydrogenase 1 (ALDH1) in acute myeloid leukemia (AML). (A) Schematic of the ALDH1A1 ‐eGFP knock‐in construct inserted into the terminal exon of the ALDH1A1 gene in K562 cells. (B) GFP fluorescence quantification in ALDH1A1 ‐eGFP knock‐in K562 cells following 24 h treatment with 1 μM of the indicated chemotherapeutic agents: BUS (busulfan), CAR (carmustine), CYC (cyclophosphamide), DAC (dacarbazine), IFO (ifosfamide), LOM (lomustine), OXA (oxaliplatin), TEM (temozolomide), AraC (cytarabine), and the anthracyclines DNR (daunorubicin), DOXO (doxorubicin), and EPI (epirubicin). Results are expressed as fold‐change (FC) in mean fluorescence intensity (MFI) relative to untreated control (UNT) cells (mean ± SD, n = 3), P < 0.0001 (****). (C) Heatmap showing gene expression changes of ALDH 1 isoforms ( ALDH1A1 , ALDH1A2 , and ALDH1A3 ) following treatment with 1 μM DNR, DOXO, EPI, or IDA (idarubicin) for 18 and 24 h, expressed as FC relative to untreated controls ( n = 3). Expression was normalized to housekeeping genes ( GAPDH , B2M , RPLP0 , and/or RPL13A1 ), selected based on gene stability in each cell line. (D) Protein expression levels of ALDH1 isoforms after 24‐h exposure to 1 μM of the indicated anthracyclines. Quantification tables below each blot show values normalized to the loading control and expressed as a percentage of the area under the curve (AUC) relative to non‐treated (NT; –) controls. (E) ALDH1‐specific enzymatic activity following 24‐h treatment with 1 μM DNR, P < 0.001 (***). (F) Reverse transcription quantitative polymerase chain reaction (RT‐qPCR) analysis of ALDH1A1 , ALDH1A2 , and ALDH1A3 mRNA expression in primary AML blast cells from patients ( N = 16) treated in vitro with 0.5 μM DNR for 24 h. Expression levels are presented as log₂ FC relative to untreated conditions for each individual patient sample. Each dot represents the median of three technical replicates per sample. Statistical differences between isoforms were assessed using the Wilcoxon signed‐rank test with FDR correction: ALDH1A1 versus ALDH1A2 , P = 0.051 (ns); ALDH1A1 versus ALDH1A3 , P = 0.024 (*); ALDH1A2 versus ALDH1A3 , P = 0.001 (**). UPN denotes Unique Patient Number.

Article Snippet: The following primary antibodies were used: ALDH1A1 (Proteintech, Cat# 15910‐1‐AP; Manchester, UK), ALDH1A2 (Proteintech, Cat# 13951‐1‐AP), ALDH1A3 (Bio‐Techne, Cat# NBP2‐15339), JNK (Cell Signaling Technology, Cat# 9252; Saint‐Cyr‐L'École, France), c‐Jun (Cell Signaling Technology, Cat# 9165), Phospho‐c‐Jun Ser73 (S73) (Cell Signaling Technology, Cat# 3270), STAT3 (Cell Signaling Technology, Cat# 9139), Phospho‐STAT3 (Y705) (Cell Signaling technology, Cat# 9145) and Protein Simple loading control (Bio‐Techne, Cat# 042‐196).

Techniques: Knock-In, Construct, Fluorescence, Control, Gene Expression, Expressing, Activity Assay, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, In Vitro

Journal: HemaSphere

Article Title: Upregulation of ALDH1 as an adaptive epigenetic response to anthracyclines in acute myeloid leukemia

doi: 10.1002/hem3.70244

Figure Lengend Snippet: Identification and validation of daunorubicin (DNR)‐induced enhancers associated with aldehyde dehydrogenase 1 (ALDH1) expression. (A) H3K27ac chromatin immunoprecipitation‐sequencing (ChIP‐seq) profiles at the ALDH1 loci in control (CTRL; black) and daunorubicin‐treated (DNR; blue) K562 cells following 24‐h exposure to 1 μM DNR. Topologically associating domains (TADs) and selected candidate DNR‐induced enhancers are indicated. The fold‐change increase in H3K27ac signal for each candidate enhancer is indicated, and the signal intensity scale is shown above each panel. (B) Relative luciferase activity in K562 cells transfected with constructs containing candidate enhancer regions (see also Supporting Information S8: Table ), measured after 24‐h treatment with 1 μM DNR. Results are expressed as fold‐change relative to the minimal promoter control (pSV40) and represent mean ± SD ( n = 3), P < 0.001 (***); P < 0.0001 (****). (C) UCSC Genome Browser (hg19) visualization of the ALDH1A1 ‐E3 and ALDH1A2 ‐E1 enhancer regions. Publicly available datasets—including H3K27ac ChIP‐seq (K562), ReMap transcription factor binding profiles, and DNase I hypersensitivity sites (ENCODE)—were used to define the indicated short fragments (A–C) with putative regulatory activity. (D) Luciferase assays comparing full‐length and shorter variants (A and B for ALDH1A1‐E3 ; A, B and B for ALDH1A2‐E1 ) showing relative luciferase activity following DNR treatment as described in (B). (E) Heatmap of H3K27ac ChIP‐seq signal within ±5 kb of enhancer peaks, ranked by fold‐change between untreated controls (CTRL) and DNR‐treated samples. (F) Venn diagram showing overlapped transcription factor binding sites (TBFS) among enhancers enriched in DNR‐treated samples and within the DNR‐responsive ALDH1A1‐E3 and ALDH1A2‐E1‐A enhancer regions. TBFS motifs that were selected for experimental validation by luciferase assays are highlighted. (G) Sequence logos representing transcription factor motif families enriched in ALDH1A1‐E3 and ALDH1A2‐E1‐A : FOSL1 , STAT3 , TEAD1 , and ETV . Associated Q ‐values are shown below each logo.

Article Snippet: The following primary antibodies were used: ALDH1A1 (Proteintech, Cat# 15910‐1‐AP; Manchester, UK), ALDH1A2 (Proteintech, Cat# 13951‐1‐AP), ALDH1A3 (Bio‐Techne, Cat# NBP2‐15339), JNK (Cell Signaling Technology, Cat# 9252; Saint‐Cyr‐L'École, France), c‐Jun (Cell Signaling Technology, Cat# 9165), Phospho‐c‐Jun Ser73 (S73) (Cell Signaling Technology, Cat# 3270), STAT3 (Cell Signaling Technology, Cat# 9139), Phospho‐STAT3 (Y705) (Cell Signaling technology, Cat# 9145) and Protein Simple loading control (Bio‐Techne, Cat# 042‐196).

Techniques: Biomarker Discovery, Expressing, ChIP-sequencing, Control, Luciferase, Activity Assay, Transfection, Construct, Binding Assay, Sequencing

Journal: HemaSphere

Article Title: Upregulation of ALDH1 as an adaptive epigenetic response to anthracyclines in acute myeloid leukemia

doi: 10.1002/hem3.70244

Figure Lengend Snippet: Identification of transcription factors involved in enhancer‐mediated activation of ALDH1A1 and ALDH1A2 . (A) UCSC Genome Browser views of ALDH1A1 ‐E3 (top) and ALDH1A2 ‐E1A (bottom) enhancer regions, with predicted transcription factor binding sites based on JASPAR database motifs. Sites for FOS/JUN, STAT, TEAD, and ETV families are indicated; mutated sequences used for functional assays are shown. (B, C) Relative luciferase activity in K562 cells transfected with wild‐type or site‐specific mutant constructs of ALDH1A1 ‐E3 (B) and ALDH1A2 ‐E1‐A (C), following 24‐h treatment with 1 μM DNR. Results are expressed as fold‐change relative to the minimal promoter control (pSV40). Data represent mean ± SD ( n = 3); statistical significance assessed by one‐way analysis of variance (ANOVA); ****P < 0.0001. (D) Protein analysis by capillary electrophoresis (WES system) showing increased phosphorylation of STAT3 and JUN after treatment with DNR (1 μM) for 6, 18, and 24 h in K562 cells. (E) ALDH1A1 and ALDH1A2 mRNA levels in K562 cells measured by reverse transcription quantitative polymerase chain reaction (RT‐qPCR) after treatment with daunorubicin (DNR, 1 μM), JNK inhibitor (JNK‐IN‐8, 20 μM), or STAT3 inhibitor (TTI‐101, 15 μM) given alone, or in combination with DNR following preincubation with JNK‐IN‐8 or TTI‐101. Results at 24 h are expressed as fold‐change relative to untreated control cells (CTRL). Data represent mean ± SD ( n = 3); statistical significance was assessed by one‐way ANOVA; ***P < 0.001. (F) Resazurin‐based cytotoxicity assay in K562 cells treated with vehicle (CTRL), 1 μM DNR, or DNR in combination with JNK‐IN‐8 (10 μM) and TTI‐101 (5 μM) for 48 h. Data represent mean ± SD ( n = 3), representative of three independent experiments; statistical significance was assessed by one‐way ANOVA; ***P < 0.001.

Article Snippet: The following primary antibodies were used: ALDH1A1 (Proteintech, Cat# 15910‐1‐AP; Manchester, UK), ALDH1A2 (Proteintech, Cat# 13951‐1‐AP), ALDH1A3 (Bio‐Techne, Cat# NBP2‐15339), JNK (Cell Signaling Technology, Cat# 9252; Saint‐Cyr‐L'École, France), c‐Jun (Cell Signaling Technology, Cat# 9165), Phospho‐c‐Jun Ser73 (S73) (Cell Signaling Technology, Cat# 3270), STAT3 (Cell Signaling Technology, Cat# 9139), Phospho‐STAT3 (Y705) (Cell Signaling technology, Cat# 9145) and Protein Simple loading control (Bio‐Techne, Cat# 042‐196).

Techniques: Activation Assay, Binding Assay, Functional Assay, Luciferase, Activity Assay, Transfection, Mutagenesis, Construct, Control, Electrophoresis, Phospho-proteomics, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Cytotoxicity Assay