acidifluor orange kit (GORYO Chemical)


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Acidifluor Orange Kit, supplied by GORYO Chemical, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/acidifluor orange kit/product/GORYO Chemical
Average 93 stars, based on 1 article reviews
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1) Product Images from "PNPO–PLP axis senses prolonged hypoxia in macrophages by regulating lysosomal activity"
Article Title: PNPO–PLP axis senses prolonged hypoxia in macrophages by regulating lysosomal activity
Journal: Nature Metabolism
doi: 10.1038/s42255-024-01053-4

Figure Legend Snippet: a , Representative AcidiFluor ORANGE staining for lysosomal acidification in BMDMs from three independent experiments. b , Immunoblot analysis detecting Lamp1 in BMDMs. Tubulin was detected as a loading control. A representative result from three independent experiments is shown. c , d , Lysosomal acidification in BMDMs generated from Hif1a mutant mice ( c ) and Hif2a mutant mice ( d ). Representative AcidiFluor ORANGE staining (left) and its quantification (right) from three independent experiments for each. e , A heatmap illustrating RNA-seq data of genes that belong to ChL class and assigned to ‘lysosome’ in the GO database, which is the same gene set shown in Extended Data Fig. . Norm BMDMs were treated with concanamycin A (ConA), bafilomycin A1 (Baf) or vehicle (DMSO) at 16 h before LPS stimulation. f , Expression of genes upregulated by ConA treatment in BMDMs ( n = 3 biologically independent samples). Error bars represent s.e.m. g , Scatter-plot showing a correlation of gene expression fold changes by prolonged hypoxia and lysosomal inhibition (ConA) in BMDMs. A horizontal axis indicates log 2 FC of ConA treatment versus DMSO and a vertical axis indicates log 2 FC of CHyp versus Norm (shown in Fig. ). The strength of the correlation was evaluated with a Pearson product-moment correlation coefficient. h , GSEA comparing the impacts of prolonged hypoxia with lysosomal inhibition. Gene sets were defined as upregulated (left) or downregulated (right) genes by more than fourfold (log 2 4) by ConA treatment. Changes in the LPS-induced transcriptome by prolonged hypoxia were analysed against the gene sets. ES, enrichment score. Scale bars, 50 μm ( a , c , d ). A two-way ANOVA was conducted to evaluate statistical significance ( c , d , f ).
Techniques Used: Staining, Western Blot, Control, Generated, Mutagenesis, RNA Sequencing Assay, Expressing, Inhibition

Figure Legend Snippet: a . Lysosomal acidification in BMDMs cultured from Vhl mutant mice. Scale bars correspond to 50 μm. Representative AcidiFluor ORANGE staining (top) and its quantification (bottom). Two-way ANOVA was conducted to evaluate statistical significance. A representative result from 3 independent experiments is shown. b . Gene expression in BMDMs after LPS stimulation in the presence or absence of ConA (n = 3). Error bars represent S.E.M. Two-way ANOVA was conducted to evaluate statistical significance. c, d . Scatter-plots showing correlations of gene expression fold changes (AUC ratios) by ConA and Baf (c) and by prolonged hypoxia and Baf (d) in BMDMs. For panel c, a horizontal axis indicates log 2 fold change of ConA treatment vs. DMSO, and a vertical axis indicates log 2 fold change of Baf treatment vs. DMSO. For panel d, a horizontal axis indicates log 2 fold change of Baf treatment vs. DMSO, and a vertical axis indicates log 2 fold change of CHyp vs. Norm (shown in Fig. ). The strength of each correlation was evaluated with Pearson product-moment correlation coefficient. Norm: BMDMs differentiated and stimulated with LPS under normoxia, CHyp: BMDMs differentiated and stimulated with LPS under 1% O 2 . e, f . Gene set enrichment analysis comparing the impacts of prolonged hypoxia with lysosomal inhibition (Baf). Gene sets were defined as upregulated (e) or downregulated (f) genes by more than 4-fold (log 2 4) by Baf treatment. Changes in the LPS-induced transcriptome by prolonged hypoxia were analysed against the gene sets. g-j . Gene set enrichment analysis comparing the impacts of lysosomal inhibition with prolonged hypoxia. Gene sets were defined as upregulated (g, i) or downregulated (h, j) genes by more than 4-fold (log 2 4) by prolonged hypoxia. Changes in the LPS-induced transcriptome by lysosomal inhibitors, ConA (g, h) and Baf (i, j), were analysed against the gene sets.
Techniques Used: Cell Culture, Mutagenesis, Staining, Expressing, Inhibition

Figure Legend Snippet: a . Relative amounts of cellular succinate and lactate in the BMDMs differentiated and stimulated with LPS under normoxia, 5% O 2 and 1% O 2 (n = 2). The metabolites were also measured on day 3 of differentiation. b . Schematic illustration indicating vitamin B6 metabolism and catalysing enzymes. PDXP: pyridoxal phosphatase, PDXK: pyridoxal kinase, PNPO: pyridoxine 5’-phosphate oxidase. c, d . Lysosomal acidification in PMA-treated U937 cells under different oxygen tension (c) and with or without pyridoxine in the culture medium (d). Scale bars correspond to 50 μm. Representative AcidiFluor ORANGE staining (left) and its quantification (right). Two-sided Student’s t -test was conducted to evaluate statistical significance. A representative result from 3 independent experiments is shown for each.
Techniques Used: Staining

Figure Legend Snippet: a , Volcano plot showing metabolome comparison between CHyp BMDMs and Norm BMDMs. A horizontal dashed line indicates a significance threshold ( P = 0.01) and green vertical dashed lines indicate levels of twofold increase and decrease. The blue vertical dashed line indicates the level at which CHyp and Norm are equal. A two-sided Welch’s t -test was conducted for calculating P values. b , Relative amounts of pyridoxal, PLP and pyridoxine in BMDMs differentiated and stimulated with LPS under normoxia, 5% and 1% oxygen ( n = 2 biologically independent samples). The metabolites were also measured on day 3 of differentiation. c – g , Relative amount of PLP in Norm BMDM, AHyp BMDM and CHyp BMDM ( c ) and Norm BMDM and CHyp BMDM generated from Hif1a mutant mice ( d ), Hif2a mutant mice ( e ), Vhl mutant mice ( f ) and Norm BMDM and CHyp BMDM with or without pyridoxine in the culture medium ( g ) ( n = 3 biologically independent samples for each experiment). Error bars represent s.e.m. h , Lysosomal acidification in Norm BMDMs and CHyp BMDMs with or without pyridoxine in the culture medium. Scale bars, 50 μm. Representative AcidiFluor ORANGE staining (left) and its quantification (right) from three independent experiments. A two-way ANOVA ( d – h ) and one-way ANOVA ( c ) were conducted to evaluate statistical significance.
Techniques Used: Comparison, Generated, Mutagenesis, Staining

Figure Legend Snippet: a . PLP levels in HeLa cells in normoxia and hypoxia for 2 and 3 days (n = 4 biologically independent samples). b . Lysosomal acidification in HeLa cells. Representative AcidiFluor ORANGE staining (left) and its quantification (right). A representative result from 3 independent experiments is shown. c . Gene expression in HeLa cells with or without ARNT knockdown in normoxia and hypoxia for 16 hours (n = 3). d . PLP levels in HeLa cells with or without ARNT knockdown in normoxia and hypoxia for 2 days (n = 3). e . Lysosomal acidification in HeLa cells with or without ARNT knockdown. Representative AcidiFluor ORANGE staining (left) and its quantification (right) from 3 independent experiments. Scale bars correspond to 50 μm (b, e). Error bars represent S.E.M. (a, c, d). One-way ANOVA (a, b) and two-way ANOVA (c-e) were conducted to evaluate statistical significance.
Techniques Used: Staining, Expressing, Knockdown

Figure Legend Snippet: a , Immunoblot analysis detecting PNPO in U937 cells treated with PNPO siRNAs or control siRNA. Tubulin was detected as a loading control. A representative result from three independent experiments is shown. b , PLP increase in U937 cells pre-exposed to 1% oxygen. PLP quantity upon reoxygenation is plotted ( n = 3 biologically independent samples for each experiment). c , Immunoblot analysis detecting PNPO protein in BMDM generated from Pnpo mutant mice. A representative result from two independent experiments is shown. d , PLP levels in Pnpo -deficient Norm BMDMs ( n = 3 biologically independent samples). e , Lysosomal acidification in Pnpo -deficient Norm BMDMs. Scale bars, 50 μm. Representative AcidiFluor ORANGE staining (left) and its quantification (right) from three independent experiments. f , g , Gene expression in response to LPS in normoxia ( f ) and in response to acute and chronic hypoxia without LPS ( g ) in Pnpo -deficient BMDMs ( n = 4 biologically independent samples). Error bars represent s.e.m. ( b , d , f , g ). A two-sided Student’s t -test ( d , e ) was conducted to evaluate statistical significance.
Techniques Used: Western Blot, Control, Generated, Mutagenesis, Staining, Expressing

Figure Legend Snippet: a , Lysosomal acidification in BMDMs. Representative AcidiFluor ORANGE staining (left) and its quantification (right) from three independent experiments. CHyp + Pyridoxal, BMDMs differentiated under 1% oxygen in the presence of 50 μg ml −1 pyridoxal. b . Immunoblot analysis detecting Lamp1 in BMDMs. Tubulin was detected as a loading control. A representative result from three independent experiments is shown. c , Expression of representative lysosome-related genes in BMDMs ( n = 3 biologically independent samples). d , Immunoblot analysis detecting 2OG-dependent dioxygenases in BMDMs. Tubulin was detected as a loading control. A representative result from three independent experiments is shown. e , Relative Il6 expression in BMDMs ( n = 3 biologically independent samples). f , ELISA of IL-6 in the culture supernatant of BMDMs ( n = 4 biologically independent samples). g , Il6 expression in BMDM with or without PNPO overexpression ( n = 4 biologically independent samples). h , SSP4 staining to detect supersulfides in BMDM. Representative SSP4 staining (left) and quantification (right) from three independent experiments. i , Effects of GSSSG, a supersulfide donor, and GSSG on lysosomal acidification. Representative AcidiFluor ORANGE staining (left) and its quantification (right) from three independent experiments. Oxidized glutathione, GSSG, was added as a negative control. j , Il6 expression in BMDM treated with GSSSG or GSSG ( n = 3 biologically independent samples). Scale bars, 50 μm ( a , h , i ). Error bars represent s.e.m. ( c , e – g , j ). One-way ANOVA ( a , h ) and two-way ANOVA ( c , e – g , i , j ) were conducted to evaluate statistical significance.
Techniques Used: Staining, Western Blot, Control, Expressing, Enzyme-linked Immunosorbent Assay, Over Expression, Negative Control