Journal: iScience

Article Title: Modification of lysine-260 2-hydroxyisobutyrylation destabilizes ALDH1A1 expression to regulate bladder cancer progression

doi: 10.1016/j.isci.2023.108142

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-ALDH1A2 , Proteintech , Cat #: 13951; RRID:AB_2224033.

Techniques: Virus, Recombinant, Activity Assay, SYBR Green Assay, Software

Journal: International Journal of Molecular Sciences

Article Title: Inhibition of RhoA and Cdc42 by miR-133a Modulates Retinoic Acid Signalling during Early Development of Posterior Cardiac Tube Segment

doi: 10.3390/ijms23084179

Figure Lengend Snippet: Whole-mount ISH for miR-133a and whole mount IMH for RhoA, Cdc42 and Raldh2 during early chick cardiac development, from HH8–HH11 stages, in control embryos. Note miR-133a expression pattern at the level of the anterior region of the primitive endocardial tube (PET), being observable in the atrium (A) and ventricle (V) at later stages. Note the location of RhoA, Cdc42 and Raldh2 at the level of the PET posterior region, and subsequently in the atrium and inflow tract (IFT). The scheme illustrates the complementary location in cranial-caudal trend of miR-133a (blue) in comparison with RhoA, Cdc42 and Raldh2 (orange).

Article Snippet: Experimental and control (CFDA) embryos were subjected to whole mount IMH was performed as previously described by us [ , ], using RhoA Polyclonal Antibody Rabbit (1:300, Invitrogen, OSR00266W), ALDH1A2 Polyclonal Antibody Rabbit (1:100, Invitrogen PA5-22377), followed by Goat anti-rabbit IgG-HRP antibody (1:1000, Upstate, 12-348) and Cdc42 monoclonal antibody Mouse (1:50, Santa-Cruz, sc-8401), followed by a Goat anti-mouse IgG-HRP antibody (1:200, Jackson Inmuno Research 115-035-003).

Techniques: Expressing

Journal: International Journal of Molecular Sciences

Article Title: Inhibition of RhoA and Cdc42 by miR-133a Modulates Retinoic Acid Signalling during Early Development of Posterior Cardiac Tube Segment

doi: 10.3390/ijms23084179

Figure Lengend Snippet: Effect of miR-133a gain- and loss-of-function on posterior cardiac segment. Whole-mount IMH for RhoA, Cdc42 and Raldh2. and ISH for Tbx5 and AMHC1. Embryos microinjected with CFDA (control), premiR-133a or antimiR-133a, at the level of the posterior cardiac precursors of both primitive endocardial tubes, and visualisation of CFDA ( A ). Note that, at the atrium and inflow tract levels, RhoA ( B ), Cdc42 ( C ), Raldh2 ( D ), Tbx5 ( E ) and AMHC1 ( F ) are dramatically reduced, and an atrophic sino-atrial region in the heart tube after premiR-133a treatment is indicated by the red arrows, whereas they are markedly increased and expanded after miR-133a inhibition (blue arrows). RT-qPCR of RNA from dissected cardiac asa (left side) in embryos microinjected either with CFDA, premiR-133a or antimiR-133a. A high level of miR-133a leads to decreased RhoA, Cdc42, Raldh2, Tbx5 and AMHC1 transcripts, whereas miR-133a inhibition leads to increased transcripts. The standard deviations are from three independent experiments. Student’s t -test: * p < 0.05, ** p < 0.01, *** p < 0.005 with respect to control (CFDA) embryos.

Article Snippet: Experimental and control (CFDA) embryos were subjected to whole mount IMH was performed as previously described by us [ , ], using RhoA Polyclonal Antibody Rabbit (1:300, Invitrogen, OSR00266W), ALDH1A2 Polyclonal Antibody Rabbit (1:100, Invitrogen PA5-22377), followed by Goat anti-rabbit IgG-HRP antibody (1:1000, Upstate, 12-348) and Cdc42 monoclonal antibody Mouse (1:50, Santa-Cruz, sc-8401), followed by a Goat anti-mouse IgG-HRP antibody (1:200, Jackson Inmuno Research 115-035-003).

Techniques: Inhibition, Quantitative RT-PCR

Journal: International Journal of Molecular Sciences

Article Title: Inhibition of RhoA and Cdc42 by miR-133a Modulates Retinoic Acid Signalling during Early Development of Posterior Cardiac Tube Segment

doi: 10.3390/ijms23084179

Figure Lengend Snippet: Whole-mount ISH for miR-133a, Tbx5 , AMHC1 and IMH for Raldh2. Embryos microinjected with CFDA (control), Retinoic acid (RA) or Citral, at the level of the posterior cardiac precursors into both primitive endocardial tubes, and visualisation of CFDA ( A ). The gain-of-function of RA leads to diminished miR-133a expression (red arrows) at the cardiac asa level ( B ), accompanied by increased protein levels of Raldh2 ( C ) and expanded expression of Tbx5 ( D ) and AMHC1 ( E ) in the heart tube and in the inflow tract (blue arrows). Note atrophic sino-atrial region with increased miR-133a expression (blue arrows) at cardiac asa level ( B ), and also (red arrows) diminished Raldh2 protein level ( C ), and decreased Tbx5 ( D ) and AMHC1 ( E ) expressions by RA synthesis inhibition. RT-qPCR of RNA from dissected cardiac asa (left side) in embryos microinjected either with CFDA, RA or Citral. The standard deviations are from three independent experiments. Student’s t -test: * p < 0.05, ** p < 0.01, *** p < 0.005 with respect to control (CFDA) embryos.

Article Snippet: Experimental and control (CFDA) embryos were subjected to whole mount IMH was performed as previously described by us [ , ], using RhoA Polyclonal Antibody Rabbit (1:300, Invitrogen, OSR00266W), ALDH1A2 Polyclonal Antibody Rabbit (1:100, Invitrogen PA5-22377), followed by Goat anti-rabbit IgG-HRP antibody (1:1000, Upstate, 12-348) and Cdc42 monoclonal antibody Mouse (1:50, Santa-Cruz, sc-8401), followed by a Goat anti-mouse IgG-HRP antibody (1:200, Jackson Inmuno Research 115-035-003).

Techniques: Expressing, Inhibition, Quantitative RT-PCR

Journal: International Journal of Molecular Sciences

Article Title: Inhibition of RhoA and Cdc42 by miR-133a Modulates Retinoic Acid Signalling during Early Development of Posterior Cardiac Tube Segment

doi: 10.3390/ijms23084179

Figure Lengend Snippet: Model proposed for the interplay between miR-133a and RA during posterior heart tube formation. Our model indicates that miR-133a downregulates RhoA and Cdc42. Consequently, Raldh2 downregulation is induced by miR-133a, via these Rho GTPases. RA synthesis is Raldh2-dependent. Thus, miR-133a modulates RA signalling via Raldh2 expression. Also, RA negatively modulates miR-133a expression during the early genetic programme of the sinoatrial region. Additionally, our model indicates that miR-133a modulates cell proliferation by acting on the cell cycle regulators p21 (via RhoA) and cyclin A (via Cdc42). We hypothesise that there is a negative feedback mechanism between miR-133a and RA signalling during early development of the posterior cardiac tube segment. A: anterior segment, P: posterior segment.

Article Snippet: Experimental and control (CFDA) embryos were subjected to whole mount IMH was performed as previously described by us [ , ], using RhoA Polyclonal Antibody Rabbit (1:300, Invitrogen, OSR00266W), ALDH1A2 Polyclonal Antibody Rabbit (1:100, Invitrogen PA5-22377), followed by Goat anti-rabbit IgG-HRP antibody (1:1000, Upstate, 12-348) and Cdc42 monoclonal antibody Mouse (1:50, Santa-Cruz, sc-8401), followed by a Goat anti-mouse IgG-HRP antibody (1:200, Jackson Inmuno Research 115-035-003).

Techniques: Expressing

Journal: Scientific Reports

Article Title: Global miRNA/proteomic analyses identify miRNAs at 14q32 and 3p21, which contribute to features of chronic iron-exposed fallopian tube epithelial cells

doi: 10.1038/s41598-021-85342-y

Figure Lengend Snippet: miR-138-5p overexpression partially regulates stem cell marker hTERT transcript levels, but does not alter another stem cell marker ALDH1A2. ( a ) Real-time PCR analysis of miR-138-5p after isolating total miRNAs from miR-138-5p transfected 250 nM FAC-treated cells relative to control transfected FAC-treated and Untreated FT194 cells, at days 122, 125 and 129 of FAC treatment (p = 35, 36, and 37) to validate the overexpression. ( b ) Western blotting was performed for cell lysates collected from miR-138-5p transfected and control transfected Untreated and FAC-treated FT194 cells to analyze ALDH1A2 levels at days 119, 122, and 126 of FAC treatment ( p = 35–37). White space between cropped blots delineate different antibody applications to the same blot. The full-length uncropped blots are displayed in the Supplementary Information File. ( c ) Western blotting was performed using cell lysates collected from control or miR-432-5p or miR-127-3p, transfected Untreated and FAC-treated FT194 cells with the indicated antibodies at days 123, 131, and 137 of FAC treatment ( p = 35, 37, and 38). White space between cropped blots delineate different antibody applications to the same blot. The full-length uncropped blots are displayed in the Supplementary Information File. ( d ) The predicted miR-138-5p binding site in the TERT sequence is shown. ( e ) Real-time PCR analysis of TERT in miR-138-5p transfected FAC-treated FT194 cells, relative to control transfected Untreated and FAC-treated FT194 cells after isolating miRNAs at days 119, 122, and 126 of FAC treatment ( p = 35–37). The data is the composite of three independent experiments.

Article Snippet: Western blotting was performed using the following Cell Signaling Technology (Danvers, MA, USA) primary antibodies: DNMT1 rabbit monoclonal (1:1000, #5032), EVI1 rabbit monoclonal (1:500, #2593), Pan-Actin rabbit polyclonal (1:1000, #4968), Acetyl Histone H3 (Lys9/Lys14) rabbit polyclonal (1:1000, #9677), ALDH1A2 rabbit polyclonal (1:1000, #83805), and CRYAB rabbit monoclonal (1:1000, #45844).

Techniques: Over Expression, Marker, Real-time Polymerase Chain Reaction, Transfection, Control, Western Blot, Binding Assay, Sequencing

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) Schematic of experiments for local recurrence (LR). Tumor-bearing KPCC mice were either amputated or focally irradiated. (B) Kaplan-Meier curve representing the percentage of mice and time to local recurrence following surgery. (C) Confocal microscopy images of locally recurrent tumor after surgery. GFP (green), YFP (yellow), RFP (red), CFP (cyan) (scale, 1 mm; inset scale, 100 μm). (D) Confocal microscopy images of recurrent tumor after irradiation (scale, 1 mm; inset scale, 100 μm). (E) Two polyclonal tumors were transplanted into multiple nude mice and stereotactic irradiation delayed tumor growth. The graph shows the effect of irradiation on tumor growth over time. Data represented as mean ± SD of relative tumor volume to starting tumor volume. (F) Confocal microscopy images of recurrent and control tumors after radiation therapy showed recurrence was driven by multiple clones. GFP(green), YFP (yellow), RFP (red), CFP (cyan), DAPI (blue) (scale, 50 μm).

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

Techniques: Irradiation, Confocal Microscopy, Control, Clone Assay

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) H&E (scale, 1 mm) and confocal images of monoclonal early metastatic lesions from a KPCC mouse (scale, 1 mm; inset scale, 100 μm). (B) H&E (scale, 1 mm) and confocal images of polyclonal early metastatic lesions from a KPCC mouse (scale, 1 mm; inset 1 scale, 50 μm; inset 2 scale, 100 μm). (C) Table indicating the number of tail-vein-injected mice that developed metastases for each sorted population and their totals (*p < 0.05, chi-square test comparing the number of mice with and without metastases between different clones). (D) H&E images of representative lung metastases from each sorted population (scale, 1 mm).

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

Techniques: Injection, Clone Assay

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: 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