aldha1a1  (Proteintech)

Journal: iScience

Article Title: Development of retinoid nuclear receptor pathway antagonists through targeting aldehyde dehydrogenase 1A3

doi: 10.1016/j.isci.2025.113675

Figure Lengend Snippet: ALDH1A3 is expressed by tumor cells to generate retinoic acid (A) Cancer cell lines were treated with atRA (10 μM) or DMSO (0.1%) for 24 h followed by RNA extraction and RT-qPCR for STRA6 , a marker of retinoid signaling. STRA6 mRNA was first normalized to GAPDH mRNA ( n = 3 biological replicates) and then to a universal human reference RNA. Student’s t test. Data are represented as mean ± SEM. (B) The Aldefluor assay was conducted on each cell line in the presence of 1 mM DEAB, as the negative control gate, or 1% DMSO. Data representative of n > 8 independent experiments. (C) RNA samples from DMSO-treated cells in (A) were assessed by RT-qPCR for ALDH1A1 and ALDH1A3 mRNA, which were first normalized to GAPDH mRNA ( n = 3 biological replicates) and then to a universal human reference RNA. Data are represented as mean ± SEM. (D) Human ALDH1A1, ALDH1A2, and ALDH1A3 were ectopically expressed in MCF7 cells as assessed by western blot. (E) Phase-contrast imaging of MCF7 cells stably expressing either ALDH1A1, ALDH1A2, ALDH1A3, or vector control after 10 passages. Image representative of >10 image fields across three separate stable cell derivations. Scale bar represents 150 μm. (F) RT-qPCR analysis of STRA6 mRNA in MCF7 cell lines with ectopic expression of each ALDH1A isoform compared to vector control cells. n = 4 biological replicates, Student’s t test. Data are represented as mean ± SEM. (G and H) The SCP28 breast cancer cell line was stably transduced with each ALDH1A isoform and implanted in the mammary fat pad of Nu/Nu mice. Tumor progression was followed by weekly caliper measurement (H) or tumor mass measurement at endpoint (G). n = 10 (ALDH1A1 and ALDH1A3), 11(ALDH1A2), and 9 (vector) mice/group. Student’s t test. Data are represented as mean ± SEM. (I) SCP28 cells were stably transduced with a retinoid-responsive bioluminescence reporter followed by stable transduction of plasmids expressing ALDH1A2 , ALDH1A3 , or empty vector. Weekly intravital imaging was performed to assess in vivo retinoid activation. n = 12 (ALDH1A2), 11 (ALDH1A3), and 5 (vector) mice/group. Data are represented as mean ± SEM. (J) Representative luciferase images from (I).

Article Snippet: ALDH1A1 , ProteinTech , Cat# 15910-1-AP; RRID: AB_2305276.

Techniques: RNA Extraction, Quantitative RT-PCR, Marker, Negative Control, Western Blot, Imaging, Stable Transfection, Expressing, Plasmid Preparation, Control, Transduction, Mass Measurement, In Vivo, Activation Assay, Luciferase

Journal: iScience

Article Title: Development of retinoid nuclear receptor pathway antagonists through targeting aldehyde dehydrogenase 1A3

doi: 10.1016/j.isci.2025.113675

Figure Lengend Snippet: ALDH1A3 is expressed by tumor cells to generate retinoic acid (A) Cancer cell lines were treated with atRA (10 μM) or DMSO (0.1%) for 24 h followed by RNA extraction and RT-qPCR for STRA6 , a marker of retinoid signaling. STRA6 mRNA was first normalized to GAPDH mRNA ( n = 3 biological replicates) and then to a universal human reference RNA. Student’s t test. Data are represented as mean ± SEM. (B) The Aldefluor assay was conducted on each cell line in the presence of 1 mM DEAB, as the negative control gate, or 1% DMSO. Data representative of n > 8 independent experiments. (C) RNA samples from DMSO-treated cells in (A) were assessed by RT-qPCR for ALDH1A1 and ALDH1A3 mRNA, which were first normalized to GAPDH mRNA ( n = 3 biological replicates) and then to a universal human reference RNA. Data are represented as mean ± SEM. (D) Human ALDH1A1, ALDH1A2, and ALDH1A3 were ectopically expressed in MCF7 cells as assessed by western blot. (E) Phase-contrast imaging of MCF7 cells stably expressing either ALDH1A1, ALDH1A2, ALDH1A3, or vector control after 10 passages. Image representative of >10 image fields across three separate stable cell derivations. Scale bar represents 150 μm. (F) RT-qPCR analysis of STRA6 mRNA in MCF7 cell lines with ectopic expression of each ALDH1A isoform compared to vector control cells. n = 4 biological replicates, Student’s t test. Data are represented as mean ± SEM. (G and H) The SCP28 breast cancer cell line was stably transduced with each ALDH1A isoform and implanted in the mammary fat pad of Nu/Nu mice. Tumor progression was followed by weekly caliper measurement (H) or tumor mass measurement at endpoint (G). n = 10 (ALDH1A1 and ALDH1A3), 11(ALDH1A2), and 9 (vector) mice/group. Student’s t test. Data are represented as mean ± SEM. (I) SCP28 cells were stably transduced with a retinoid-responsive bioluminescence reporter followed by stable transduction of plasmids expressing ALDH1A2 , ALDH1A3 , or empty vector. Weekly intravital imaging was performed to assess in vivo retinoid activation. n = 12 (ALDH1A2), 11 (ALDH1A3), and 5 (vector) mice/group. Data are represented as mean ± SEM. (J) Representative luciferase images from (I).

Article Snippet: Membranes were blocked in 5% milk in TBS-T, probed with antibodies for ALDH1A1 (ProteinTech), ALDH1A2 (Abcam), ALDH1A3 (Abcam) or β-actin (Santa Cruz), and imaged on the Licor Odyssey CLX system using Licor-supplied IRDye680 and IRDye800 secondary antibodies.

Techniques: RNA Extraction, Quantitative RT-PCR, Marker, Negative Control, Western Blot, Imaging, Stable Transfection, Expressing, Plasmid Preparation, Control, Transduction, Mass Measurement, In Vivo, Activation Assay, Luciferase