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    Echelon Biosciences img treatments 3ac
    Img Treatments 3ac, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    3ac  (Echelon Biosciences)


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    Echelon Biosciences 3ac
    a , b iMGs were treated with vehicle (ethanol) or <t>3AC</t> (1.25 μM) for 6 h. a Cells were then lysed, RNA purified, and RNAseq performed, n = 6 per condition. Volcano plot of DEGs comparing vehicle and 3AC treatment conditions. b Cells were lysed in urea and TMT-MS performed. n = 4 per condition. Volcano plot of DEPs; For a , b differential expression was calculated using a linear modeling with empirical Bayesian statistical analysis using the limma package in R. All p -values are adjusted using the Benjamini Hochberg (BH) procedure. c Heatmap of expression of DEGs that showed concordant differential expression at the RNA and protein level. d Heatmap of relative expression of DEPs encoded by LOAD GWAS candidate genes between vehicle and 3AC-treated iMGs. e . Relative RNA and protein levels of CD33 and PTK2B between vehicle and 3AC-treated microglia, as quantified by RNAseq and TMT-MS. Mean ± SEM. Two-sided Welch’s t -test. f , g . A variety of microglial subtypes previously have been defined through single-cell sequencing, with specific subtypes implicated to be altered in AD brain and model systems – . Volcano plots comparing 3AC vs vehicle treatment for genes defining these subtypes within the transcriptomic and proteomic datasets are shown; significance determined by BH FDR < 0.05. h Heatmap of relative protein-levels of scavenger receptors in vehicle and 3AC-treated iMGs. i , j . Enriched GO terms of biological processes for the differentially expressed genes that are elevated with 3AC treatment (geneontology.com ); Fisher’s exact test, Bonferroni multiple comparison’s test, adj p -value as shown by size of circles. j Heatmap of relative expression between vehicle and 3AC treatment of DEGs associated with NFκB signaling and JAK-STAT signaling. k Relative abundance measures of RNA or protein levels of inflammasome related components in vehicle and 3AC-treated iMGs. Mean ± SEM. Two-sided Welch’s t -test. l Representative western blot of iMGs treated with either vehicle (ethanol) or 3AC (1.25 μM) for 6 h showing protein expression of INPP5D and inflammasome related proteins: CASP1, PYCARD/ASC, GSDMD and NLRP3, as well as GAPDH. Similar WB results obtain in 3 separate differentiations. Cell lines used for experiments and full datasets for can be found in Supplementary Data and , Supplementary Data . For all graphs, the number of biological replicates is represented by dots. For all panels: ** p < 0.01, *** p < 0.001, **** p < 0.0001; ns = p < 0.05. See also Supplementary Figs. and .
    3ac, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "INPP5D regulates inflammasome activation in human microglia"

    Article Title: INPP5D regulates inflammasome activation in human microglia

    Journal: Nature Communications

    doi: 10.1038/s41467-023-42819-w

    a , b iMGs were treated with vehicle (ethanol) or 3AC (1.25 μM) for 6 h. a Cells were then lysed, RNA purified, and RNAseq performed, n = 6 per condition. Volcano plot of DEGs comparing vehicle and 3AC treatment conditions. b Cells were lysed in urea and TMT-MS performed. n = 4 per condition. Volcano plot of DEPs; For a , b differential expression was calculated using a linear modeling with empirical Bayesian statistical analysis using the limma package in R. All p -values are adjusted using the Benjamini Hochberg (BH) procedure. c Heatmap of expression of DEGs that showed concordant differential expression at the RNA and protein level. d Heatmap of relative expression of DEPs encoded by LOAD GWAS candidate genes between vehicle and 3AC-treated iMGs. e . Relative RNA and protein levels of CD33 and PTK2B between vehicle and 3AC-treated microglia, as quantified by RNAseq and TMT-MS. Mean ± SEM. Two-sided Welch’s t -test. f , g . A variety of microglial subtypes previously have been defined through single-cell sequencing, with specific subtypes implicated to be altered in AD brain and model systems – . Volcano plots comparing 3AC vs vehicle treatment for genes defining these subtypes within the transcriptomic and proteomic datasets are shown; significance determined by BH FDR < 0.05. h Heatmap of relative protein-levels of scavenger receptors in vehicle and 3AC-treated iMGs. i , j . Enriched GO terms of biological processes for the differentially expressed genes that are elevated with 3AC treatment (geneontology.com ); Fisher’s exact test, Bonferroni multiple comparison’s test, adj p -value as shown by size of circles. j Heatmap of relative expression between vehicle and 3AC treatment of DEGs associated with NFκB signaling and JAK-STAT signaling. k Relative abundance measures of RNA or protein levels of inflammasome related components in vehicle and 3AC-treated iMGs. Mean ± SEM. Two-sided Welch’s t -test. l Representative western blot of iMGs treated with either vehicle (ethanol) or 3AC (1.25 μM) for 6 h showing protein expression of INPP5D and inflammasome related proteins: CASP1, PYCARD/ASC, GSDMD and NLRP3, as well as GAPDH. Similar WB results obtain in 3 separate differentiations. Cell lines used for experiments and full datasets for can be found in Supplementary Data and , Supplementary Data . For all graphs, the number of biological replicates is represented by dots. For all panels: ** p < 0.01, *** p < 0.001, **** p < 0.0001; ns = p < 0.05. See also Supplementary Figs. and .
    Figure Legend Snippet: a , b iMGs were treated with vehicle (ethanol) or 3AC (1.25 μM) for 6 h. a Cells were then lysed, RNA purified, and RNAseq performed, n = 6 per condition. Volcano plot of DEGs comparing vehicle and 3AC treatment conditions. b Cells were lysed in urea and TMT-MS performed. n = 4 per condition. Volcano plot of DEPs; For a , b differential expression was calculated using a linear modeling with empirical Bayesian statistical analysis using the limma package in R. All p -values are adjusted using the Benjamini Hochberg (BH) procedure. c Heatmap of expression of DEGs that showed concordant differential expression at the RNA and protein level. d Heatmap of relative expression of DEPs encoded by LOAD GWAS candidate genes between vehicle and 3AC-treated iMGs. e . Relative RNA and protein levels of CD33 and PTK2B between vehicle and 3AC-treated microglia, as quantified by RNAseq and TMT-MS. Mean ± SEM. Two-sided Welch’s t -test. f , g . A variety of microglial subtypes previously have been defined through single-cell sequencing, with specific subtypes implicated to be altered in AD brain and model systems – . Volcano plots comparing 3AC vs vehicle treatment for genes defining these subtypes within the transcriptomic and proteomic datasets are shown; significance determined by BH FDR < 0.05. h Heatmap of relative protein-levels of scavenger receptors in vehicle and 3AC-treated iMGs. i , j . Enriched GO terms of biological processes for the differentially expressed genes that are elevated with 3AC treatment (geneontology.com ); Fisher’s exact test, Bonferroni multiple comparison’s test, adj p -value as shown by size of circles. j Heatmap of relative expression between vehicle and 3AC treatment of DEGs associated with NFκB signaling and JAK-STAT signaling. k Relative abundance measures of RNA or protein levels of inflammasome related components in vehicle and 3AC-treated iMGs. Mean ± SEM. Two-sided Welch’s t -test. l Representative western blot of iMGs treated with either vehicle (ethanol) or 3AC (1.25 μM) for 6 h showing protein expression of INPP5D and inflammasome related proteins: CASP1, PYCARD/ASC, GSDMD and NLRP3, as well as GAPDH. Similar WB results obtain in 3 separate differentiations. Cell lines used for experiments and full datasets for can be found in Supplementary Data and , Supplementary Data . For all graphs, the number of biological replicates is represented by dots. For all panels: ** p < 0.01, *** p < 0.001, **** p < 0.0001; ns = p < 0.05. See also Supplementary Figs. and .

    Techniques Used: Purification, Expressing, Sequencing, Western Blot

    a Fold change of secreted cytokines from iMGs treated with LPS (100 ng/mL) or 3AC (1.25 μM) for 6 h across iMGs derived from four iPSC lines, assayed by ELISA. Fold change was calculated compared to vehicle-treated cells. b Levels of IL1B following 6 h treatment with vehicle, LPS (100 ng/mL), or 3AC (1.25 μM, 2.5 μM) treatment as measured by quantitative real-time PCR (qPCR). Data are normalized to GAPDH expression then values were normalized to vehicle treatment within each experiment. n = 3 differentiations with well-level data shown as dots. One-way ANOVA with Dunnett’s T3 multiple comparisons test. c Secreted levels of IL-1ß measured for the same experiments as ( b ) following 6 h treatment as measured by ELISA. IL-1ß levels were normalized to 1.25 μM 3AC-treated conditions within each experiment. n = 3 differentiations with well-level data shown as dots. d Representative western blot of protein expression in iMGs with biallelic loss-of-function mutations generated with CRISPR targeting. Loss of INPP5D protein observed repeatedly over 3 differentiations. e , f WT INPP5D iMGs and KO INPP5D iMGs were treated with 3AC for 6 h and the levels of IL-1ß and IL-18 were measured by ELISA. Multiple two-sided t -tests, Two-stage step-up (Benjamini, Krieger, and Yekutieli), ** q < 0.01; *** q < 0.005; **** q < 0.001; LLOD= lower limit of detection. n = 3 biological replicates. g , h Treatment of iMGs with either vehicle or 3AC (5 μM) with either VX-765 (25 μM) or its vehicle (DMSO). Levels of secreted IL-1ß and IL-18 measured via ELISA following treatments and normalized to 3AC-treated samples in each experiment. One-way ANOVA with Sidak’s multiple comparison test. 3 differentiations, n = 10 per condition. i , j iMGs were treated with either vehicle, primed with LPS (100 ng/mL) for 3 h and then treated with nigericin (10 μM) for 1 h, or treated with 3AC (1.25 μM or 5 μM) for 2 h. Cells were immunostained for ASC and IBA1, DNA is stained with DAPI (i) and imaged using confocal microscopy. Scale bars = 100 μm. j Quantification of ASC specks. Number of cells analyzed: n = 226 (vehicle); 151 (1.25 μM 3AC); 173 (5.0 μM 3AC) over three experiments and 10 images per condition. Kruskal–Wallis test to determine significance. k , l Treatment of iMGs with either vehicle or 3AC (5 μM) with either MCC950 (10 μM) or its vehicle (DMSO). Levels of secreted IL-1ß and IL-18 in the media were measured and normalized to 3AC-treated samples in each experiment. One-way ANOVA with Sidak’s multiple comparison test. n = 4 differentiations with well-level data shown as dots. m A summary figure of experiments performed to interrogate inflammasome activation, created using BioRender.com. For all graphs, data are presented as mean values ± SEM. For b , c , g , h , j , l : ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Cell lines used for experiments are detailed in Supplementary Data . See also Supplementary Figs. and .
    Figure Legend Snippet: a Fold change of secreted cytokines from iMGs treated with LPS (100 ng/mL) or 3AC (1.25 μM) for 6 h across iMGs derived from four iPSC lines, assayed by ELISA. Fold change was calculated compared to vehicle-treated cells. b Levels of IL1B following 6 h treatment with vehicle, LPS (100 ng/mL), or 3AC (1.25 μM, 2.5 μM) treatment as measured by quantitative real-time PCR (qPCR). Data are normalized to GAPDH expression then values were normalized to vehicle treatment within each experiment. n = 3 differentiations with well-level data shown as dots. One-way ANOVA with Dunnett’s T3 multiple comparisons test. c Secreted levels of IL-1ß measured for the same experiments as ( b ) following 6 h treatment as measured by ELISA. IL-1ß levels were normalized to 1.25 μM 3AC-treated conditions within each experiment. n = 3 differentiations with well-level data shown as dots. d Representative western blot of protein expression in iMGs with biallelic loss-of-function mutations generated with CRISPR targeting. Loss of INPP5D protein observed repeatedly over 3 differentiations. e , f WT INPP5D iMGs and KO INPP5D iMGs were treated with 3AC for 6 h and the levels of IL-1ß and IL-18 were measured by ELISA. Multiple two-sided t -tests, Two-stage step-up (Benjamini, Krieger, and Yekutieli), ** q < 0.01; *** q < 0.005; **** q < 0.001; LLOD= lower limit of detection. n = 3 biological replicates. g , h Treatment of iMGs with either vehicle or 3AC (5 μM) with either VX-765 (25 μM) or its vehicle (DMSO). Levels of secreted IL-1ß and IL-18 measured via ELISA following treatments and normalized to 3AC-treated samples in each experiment. One-way ANOVA with Sidak’s multiple comparison test. 3 differentiations, n = 10 per condition. i , j iMGs were treated with either vehicle, primed with LPS (100 ng/mL) for 3 h and then treated with nigericin (10 μM) for 1 h, or treated with 3AC (1.25 μM or 5 μM) for 2 h. Cells were immunostained for ASC and IBA1, DNA is stained with DAPI (i) and imaged using confocal microscopy. Scale bars = 100 μm. j Quantification of ASC specks. Number of cells analyzed: n = 226 (vehicle); 151 (1.25 μM 3AC); 173 (5.0 μM 3AC) over three experiments and 10 images per condition. Kruskal–Wallis test to determine significance. k , l Treatment of iMGs with either vehicle or 3AC (5 μM) with either MCC950 (10 μM) or its vehicle (DMSO). Levels of secreted IL-1ß and IL-18 in the media were measured and normalized to 3AC-treated samples in each experiment. One-way ANOVA with Sidak’s multiple comparison test. n = 4 differentiations with well-level data shown as dots. m A summary figure of experiments performed to interrogate inflammasome activation, created using BioRender.com. For all graphs, data are presented as mean values ± SEM. For b , c , g , h , j , l : ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Cell lines used for experiments are detailed in Supplementary Data . See also Supplementary Figs. and .

    Techniques Used: Derivative Assay, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Generated, CRISPR, Comparison, Staining, Confocal Microscopy, Activation Assay

    a–c Volcano plot ( a ) of adjusted p -values showing 71 DEPs from the 7868 quantified via TMT-MS (two-way t -test with Benjamini–Hochberg multiple comparisons test; FDR < 0.05. Heatmap ( b ) of relative expression levels of proteins involved in immune signaling that are differentially up and downregulated in INPP5D WT vs HET iMGs. Asterisks indicate proteins that also were differentially up or downregulated ( q < 0.05) with 3AC treatment (Fig. ). GO analysis ( c ) of DEPs; Fisher’s exact test, Bonferroni multiple comparison’s test, adj p -value as shown by size of circles. d , e Representative western blot and quantification across three differentiations of MRC1, PLA2G7, COLEC12, IBA1, and GAPDH in INPP5D WT and HET iMGs. n = 3 independent experiments, mean ± SEM; two-sided t -test. f Heatmap of TMT-MS data (z-score) for lysosomal DEPs in INPP5D HET vs WT iMGs. g Representative western blot of INPP5D iMGs treated with either vehicle (DMSO) or bafilomycin (baf, 100 nM) for either 24 or 6 h. Western blots are probed for INPP5D and LC3 with GAPDH as a loading control. h Quantification of autophagic flux (baf - veh of LC3-II/GAPDH) in iMGs with treatment of 24 h of baf. n = 6 wells vehicle, n = 6 wells of 6 h baf. Mean ± SEM, two-sided t -test with Welch’s correction. i , j Secreted IL-1ß and IL-18 measured by ELISA of media collected from INPP5D HET and WT iMGs. Media were concentrated tenfold in order to be above the limit of detection and normalized to WT mean. n = 14 wells WT, n = 21 wells HET. Mean ± SEM. Two-sided Mann–Whitney test. k , l Secreted IL-1ß and IL-18 measured by ELISA of media collected from INPP5D HET iMGs treated with either vehicle (DMSO) or MCC950 (10 μM) for 24 h. Media were concentrated tenfold for detection. n = 10 wells vehicle, n = 10 wells MCC950-treated. Mean ± SEM. Mann–Whitney test. For e , h – l * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. See also Supplementary Fig. , Supplementary Data and for full datasets.
    Figure Legend Snippet: a–c Volcano plot ( a ) of adjusted p -values showing 71 DEPs from the 7868 quantified via TMT-MS (two-way t -test with Benjamini–Hochberg multiple comparisons test; FDR < 0.05. Heatmap ( b ) of relative expression levels of proteins involved in immune signaling that are differentially up and downregulated in INPP5D WT vs HET iMGs. Asterisks indicate proteins that also were differentially up or downregulated ( q < 0.05) with 3AC treatment (Fig. ). GO analysis ( c ) of DEPs; Fisher’s exact test, Bonferroni multiple comparison’s test, adj p -value as shown by size of circles. d , e Representative western blot and quantification across three differentiations of MRC1, PLA2G7, COLEC12, IBA1, and GAPDH in INPP5D WT and HET iMGs. n = 3 independent experiments, mean ± SEM; two-sided t -test. f Heatmap of TMT-MS data (z-score) for lysosomal DEPs in INPP5D HET vs WT iMGs. g Representative western blot of INPP5D iMGs treated with either vehicle (DMSO) or bafilomycin (baf, 100 nM) for either 24 or 6 h. Western blots are probed for INPP5D and LC3 with GAPDH as a loading control. h Quantification of autophagic flux (baf - veh of LC3-II/GAPDH) in iMGs with treatment of 24 h of baf. n = 6 wells vehicle, n = 6 wells of 6 h baf. Mean ± SEM, two-sided t -test with Welch’s correction. i , j Secreted IL-1ß and IL-18 measured by ELISA of media collected from INPP5D HET and WT iMGs. Media were concentrated tenfold in order to be above the limit of detection and normalized to WT mean. n = 14 wells WT, n = 21 wells HET. Mean ± SEM. Two-sided Mann–Whitney test. k , l Secreted IL-1ß and IL-18 measured by ELISA of media collected from INPP5D HET iMGs treated with either vehicle (DMSO) or MCC950 (10 μM) for 24 h. Media were concentrated tenfold for detection. n = 10 wells vehicle, n = 10 wells MCC950-treated. Mean ± SEM. Mann–Whitney test. For e , h – l * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. See also Supplementary Fig. , Supplementary Data and for full datasets.

    Techniques Used: Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

    3ac  (Echelon Biosciences)


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    Echelon Biosciences 3ac
    a . iMGLs were treated with vehicle (ethanol) or <t>3AC</t> (1.25 uM) for 6 hours. Cells were then lysed, RNA purified, and RNAseq performed, n=6 per condition. Volcano plot of differentially expressed genes (DEGs) comparing vehicle and 3AC treatment conditions. 2,004 of 12,669 genes quantified were differentially expressed (Benjamini-Hochberg (BH), FDR<0.05) between vehicle and 3AC conditions. b . iMGLs were treated with vehicle (ethanol) or 3AC (1.25 uM) for 6 hours. Cells were then lysed in urea and mass spectrometry performed. n=4 per condition. Volcano plot of differentially expressed proteins (DEPs); 159 of 7,866 proteins were differentially expressed with 3AC treatment (BH FDR<0.05). c . Heat map of expression of DEGs that showed concordant differential expression at the RNA and protein level. d . Heatmap of relative expression of DEPs encoded by LOAD GWAS candidate genes between vehicle and 3AC treated iMGLs. e . Relative RNA and protein levels of CD33 and PTK2B between vehicle and 3AC treated microglia, as quantified by RNA sequencing and MS. Mean +/-SEM. Welch’s t-test. f-g . A variety of microglial subtypes previously have been defined through single-cell sequencing, with specific subtypes implicated to be altered in AD brain and model systems - . Volcano plots comparing 3AC vs vehicle treatment for genes defining these subtypes within the transcriptomic and proteomic data sets; significance determined by BH FDR<0.05. h . Heatmap of relative protein-levels of scavenger receptors in vehicle and 3AC treated iMGLs. i . Enriched gene ontology (GO) terms of biological processes for the differentially expressed genes that are elevated with 3AC treatment ( geneontology.com ). j . Heatmap of relative expression between vehicle and 3AC-treatment of DEGs associated with NFΚB signaling and JAK-STAT signaling. k . Relative RNA and protein levels of inflammasome related components—NLRP3, PYCARD, IL1B, CASP1—in vehicle and 3AC-treated iMGLs, graphed by z-score. Mean +/-SEM. Welch’s t-test. l . Western blot of iMGL treated with either vehicle (ethanol) or 3AC (1.25 uM) for 6 hours showing protein expression of INPP5D and inflammasome related proteins: CASP1, ASC, and NLRP3, as well as GAPDH. Cell lines used for experiments are detailed in Supplement Table 3 . For e and k: **p < 0.01, *** p < 0.001, **** p < 0.0001
    3ac, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/3ac/product/Echelon Biosciences
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    3ac - by Bioz Stars, 2024-03
    86/100 stars

    Images

    1) Product Images from "INPP5D/SHIP1 regulates inflammasome activation in human microglia"

    Article Title: INPP5D/SHIP1 regulates inflammasome activation in human microglia

    Journal: bioRxiv

    doi: 10.1101/2023.02.25.530025

    a . iMGLs were treated with vehicle (ethanol) or 3AC (1.25 uM) for 6 hours. Cells were then lysed, RNA purified, and RNAseq performed, n=6 per condition. Volcano plot of differentially expressed genes (DEGs) comparing vehicle and 3AC treatment conditions. 2,004 of 12,669 genes quantified were differentially expressed (Benjamini-Hochberg (BH), FDR<0.05) between vehicle and 3AC conditions. b . iMGLs were treated with vehicle (ethanol) or 3AC (1.25 uM) for 6 hours. Cells were then lysed in urea and mass spectrometry performed. n=4 per condition. Volcano plot of differentially expressed proteins (DEPs); 159 of 7,866 proteins were differentially expressed with 3AC treatment (BH FDR<0.05). c . Heat map of expression of DEGs that showed concordant differential expression at the RNA and protein level. d . Heatmap of relative expression of DEPs encoded by LOAD GWAS candidate genes between vehicle and 3AC treated iMGLs. e . Relative RNA and protein levels of CD33 and PTK2B between vehicle and 3AC treated microglia, as quantified by RNA sequencing and MS. Mean +/-SEM. Welch’s t-test. f-g . A variety of microglial subtypes previously have been defined through single-cell sequencing, with specific subtypes implicated to be altered in AD brain and model systems - . Volcano plots comparing 3AC vs vehicle treatment for genes defining these subtypes within the transcriptomic and proteomic data sets; significance determined by BH FDR<0.05. h . Heatmap of relative protein-levels of scavenger receptors in vehicle and 3AC treated iMGLs. i . Enriched gene ontology (GO) terms of biological processes for the differentially expressed genes that are elevated with 3AC treatment ( geneontology.com ). j . Heatmap of relative expression between vehicle and 3AC-treatment of DEGs associated with NFΚB signaling and JAK-STAT signaling. k . Relative RNA and protein levels of inflammasome related components—NLRP3, PYCARD, IL1B, CASP1—in vehicle and 3AC-treated iMGLs, graphed by z-score. Mean +/-SEM. Welch’s t-test. l . Western blot of iMGL treated with either vehicle (ethanol) or 3AC (1.25 uM) for 6 hours showing protein expression of INPP5D and inflammasome related proteins: CASP1, ASC, and NLRP3, as well as GAPDH. Cell lines used for experiments are detailed in Supplement Table 3 . For e and k: **p < 0.01, *** p < 0.001, **** p < 0.0001
    Figure Legend Snippet: a . iMGLs were treated with vehicle (ethanol) or 3AC (1.25 uM) for 6 hours. Cells were then lysed, RNA purified, and RNAseq performed, n=6 per condition. Volcano plot of differentially expressed genes (DEGs) comparing vehicle and 3AC treatment conditions. 2,004 of 12,669 genes quantified were differentially expressed (Benjamini-Hochberg (BH), FDR<0.05) between vehicle and 3AC conditions. b . iMGLs were treated with vehicle (ethanol) or 3AC (1.25 uM) for 6 hours. Cells were then lysed in urea and mass spectrometry performed. n=4 per condition. Volcano plot of differentially expressed proteins (DEPs); 159 of 7,866 proteins were differentially expressed with 3AC treatment (BH FDR<0.05). c . Heat map of expression of DEGs that showed concordant differential expression at the RNA and protein level. d . Heatmap of relative expression of DEPs encoded by LOAD GWAS candidate genes between vehicle and 3AC treated iMGLs. e . Relative RNA and protein levels of CD33 and PTK2B between vehicle and 3AC treated microglia, as quantified by RNA sequencing and MS. Mean +/-SEM. Welch’s t-test. f-g . A variety of microglial subtypes previously have been defined through single-cell sequencing, with specific subtypes implicated to be altered in AD brain and model systems - . Volcano plots comparing 3AC vs vehicle treatment for genes defining these subtypes within the transcriptomic and proteomic data sets; significance determined by BH FDR<0.05. h . Heatmap of relative protein-levels of scavenger receptors in vehicle and 3AC treated iMGLs. i . Enriched gene ontology (GO) terms of biological processes for the differentially expressed genes that are elevated with 3AC treatment ( geneontology.com ). j . Heatmap of relative expression between vehicle and 3AC-treatment of DEGs associated with NFΚB signaling and JAK-STAT signaling. k . Relative RNA and protein levels of inflammasome related components—NLRP3, PYCARD, IL1B, CASP1—in vehicle and 3AC-treated iMGLs, graphed by z-score. Mean +/-SEM. Welch’s t-test. l . Western blot of iMGL treated with either vehicle (ethanol) or 3AC (1.25 uM) for 6 hours showing protein expression of INPP5D and inflammasome related proteins: CASP1, ASC, and NLRP3, as well as GAPDH. Cell lines used for experiments are detailed in Supplement Table 3 . For e and k: **p < 0.01, *** p < 0.001, **** p < 0.0001

    Techniques Used: Purification, Mass Spectrometry, Expressing, RNA Sequencing Assay, Sequencing, Western Blot

    a . Fold change of secreted cytokines from iMGLs treated with LPS (100 ng/mL) or 3AC (1.25 uM) for 6 hours across iMGLs derived from four iPSC lines, assayed on a multiplex pro-inflammatory MSD ELISA assay. Fold change was calculated compared to vehicle (ethanol) treated cells. b . Levels of IL1B following 6-hour treatment with vehicle (ethanol), LPS (100 ng/mL), or 3AC (1.25 uM, 2.5 uM) treatment as measured by quantitative real-time PCR (qPCR). Data are normalized to GAPDH expression then values were normalized to vehicle treatment within each experiment. 3 differentiations, n=3-4 per condition. Mean +/-SEM. One-way ANOVA with Dunnett’s T3 multiple comparisons test. c . Secreted levels of IL-1ß measured in the conditioned media for the same experiments as (b) following 6-hour treatment with vehicle (ethanol), LPS (100 ng/mL), or 3AC (1.25 uM, 2.5 uM) as measured by ELISA. IL-1ß levels were normalized to 1.25 uM 3AC treated conditions within each experiment. 3 differentiations, n=3-4 per condition. Mean +/-SEM. One-way ANOVA with Dunnett’s T3 multiple comparisons test. d . iMGLs treated with either vehicle (ethanol), primed with LPS (100 ng/mL) for 3 hours and then treated with nigericin (10 uM) for 1 hour, or treated with 3AC (1.25 uM or 5 uM) for 2 hours. Cells were immunostained for ASC and IBA1. DNA is stained with DAPI. Imaged using confocal microscopy. Scale bars = 100 um. e-f . Treatment of iMGLs with either vehicle (ethanol) or 3AC (5 uM) with either VX-765 (25 uM) or its vehicle (DMSO). Levels of secreted IL-1ß and IL-18 were measured using an MSD dual IL-1ß and IL-18 ELISA following treatments and normalized to 3AC-treated samples in each experiment. Mean +/-SEM. One-way ANOVA with Sidak’s multiple comparison test. 3 differentiations, n = 10 per condition. g-h . Treatment of iMGLs with either vehicle (ethanol) or 3AC (5 uM) with either MCC950 (10 uM) or its vehicle (DMSO). Levels of secreted IL-1ß and IL-18 in the conditioned media were measured using an MSD dual IL-1ß and IL-18 ELISA and normalized to 3AC-treated samples in each experiment. Mean +/-SEM. One-way ANOVA with Sidak’s multiple comparison test. 4 differentiations, n=5-9 per condition. i . A summary figure of experiments performed to interrogate inflammasome activation, created using BioRender.com. For b-c, e-h: ns = not significant, *p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 Cell lines used for experiments are detailed in Supplement Table 3 .
    Figure Legend Snippet: a . Fold change of secreted cytokines from iMGLs treated with LPS (100 ng/mL) or 3AC (1.25 uM) for 6 hours across iMGLs derived from four iPSC lines, assayed on a multiplex pro-inflammatory MSD ELISA assay. Fold change was calculated compared to vehicle (ethanol) treated cells. b . Levels of IL1B following 6-hour treatment with vehicle (ethanol), LPS (100 ng/mL), or 3AC (1.25 uM, 2.5 uM) treatment as measured by quantitative real-time PCR (qPCR). Data are normalized to GAPDH expression then values were normalized to vehicle treatment within each experiment. 3 differentiations, n=3-4 per condition. Mean +/-SEM. One-way ANOVA with Dunnett’s T3 multiple comparisons test. c . Secreted levels of IL-1ß measured in the conditioned media for the same experiments as (b) following 6-hour treatment with vehicle (ethanol), LPS (100 ng/mL), or 3AC (1.25 uM, 2.5 uM) as measured by ELISA. IL-1ß levels were normalized to 1.25 uM 3AC treated conditions within each experiment. 3 differentiations, n=3-4 per condition. Mean +/-SEM. One-way ANOVA with Dunnett’s T3 multiple comparisons test. d . iMGLs treated with either vehicle (ethanol), primed with LPS (100 ng/mL) for 3 hours and then treated with nigericin (10 uM) for 1 hour, or treated with 3AC (1.25 uM or 5 uM) for 2 hours. Cells were immunostained for ASC and IBA1. DNA is stained with DAPI. Imaged using confocal microscopy. Scale bars = 100 um. e-f . Treatment of iMGLs with either vehicle (ethanol) or 3AC (5 uM) with either VX-765 (25 uM) or its vehicle (DMSO). Levels of secreted IL-1ß and IL-18 were measured using an MSD dual IL-1ß and IL-18 ELISA following treatments and normalized to 3AC-treated samples in each experiment. Mean +/-SEM. One-way ANOVA with Sidak’s multiple comparison test. 3 differentiations, n = 10 per condition. g-h . Treatment of iMGLs with either vehicle (ethanol) or 3AC (5 uM) with either MCC950 (10 uM) or its vehicle (DMSO). Levels of secreted IL-1ß and IL-18 in the conditioned media were measured using an MSD dual IL-1ß and IL-18 ELISA and normalized to 3AC-treated samples in each experiment. Mean +/-SEM. One-way ANOVA with Sidak’s multiple comparison test. 4 differentiations, n=5-9 per condition. i . A summary figure of experiments performed to interrogate inflammasome activation, created using BioRender.com. For b-c, e-h: ns = not significant, *p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 Cell lines used for experiments are detailed in Supplement Table 3 .

    Techniques Used: Derivative Assay, Multiplex Assay, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Expressing, Staining, Confocal Microscopy, Activation Assay

    a . A table of data regarding the 8 iPSC lines utilized, and the measured fold change of IL-1ß and IL-18 after 3AC (1.25 uM 3AC, 6 hours) treatment. b-c . IL-1ß and IL-18 measured at various timepoints (0 hour, 2 hour, 6 hour, 24 hour) of BR33 iMGLs treated with increasing concentration of 3AC (Vehicle, 0.625 uM, 1.25 uM, 5 uM, 10 uM 3AC). n=3-4 wells per condition.
    Figure Legend Snippet: a . A table of data regarding the 8 iPSC lines utilized, and the measured fold change of IL-1ß and IL-18 after 3AC (1.25 uM 3AC, 6 hours) treatment. b-c . IL-1ß and IL-18 measured at various timepoints (0 hour, 2 hour, 6 hour, 24 hour) of BR33 iMGLs treated with increasing concentration of 3AC (Vehicle, 0.625 uM, 1.25 uM, 5 uM, 10 uM 3AC). n=3-4 wells per condition.

    Techniques Used: Concentration Assay

    a . iMGLs were treated with either vehicle (ethanol) or 3AC (5 uM) for 3 or 2 hours and harvested to collect protein. Caspase 1 and cleaved caspase 1 levels were assayed via western blotting. b-c . Treatment of iMGLs with either vehicle (ethanol) or 5 uM 3AC with either DMSO or Ac-YVAD-cmk (20 uM, 40 uM, 80 uM) pre-treatment for 1 hour. Levels of secreted IL-1ß and IL-18 were measured using an MSD ELISA. n = 3 wells per condition. Mean +/-SEM. One-way ANOVA with Sidak’s multiple comparison test. For b-c: ns = not significant, p>0.05, *p < 0.05, *** p < 0.001.
    Figure Legend Snippet: a . iMGLs were treated with either vehicle (ethanol) or 3AC (5 uM) for 3 or 2 hours and harvested to collect protein. Caspase 1 and cleaved caspase 1 levels were assayed via western blotting. b-c . Treatment of iMGLs with either vehicle (ethanol) or 5 uM 3AC with either DMSO or Ac-YVAD-cmk (20 uM, 40 uM, 80 uM) pre-treatment for 1 hour. Levels of secreted IL-1ß and IL-18 were measured using an MSD ELISA. n = 3 wells per condition. Mean +/-SEM. One-way ANOVA with Sidak’s multiple comparison test. For b-c: ns = not significant, p>0.05, *p < 0.05, *** p < 0.001.

    Techniques Used: Western Blot, Enzyme-linked Immunosorbent Assay

    a . Table of CRISPR-Cas9 generated INPP5D heterozygous (HET) and monoclonally-selected wildtype (WT) lines. b . INPP5D RNA levels measured by qPCR, normalized to GAPDH levels. Mean +/-SEM. Mann-Whitney test. c . Western blot quantification of INPP5D protein levels in INPP5D WT and HET iMGLs normalized to GAPDH levels, Mean +/-SEM. Mann-Whitney test. d . Representative western blot of INPP5D, GAPDH, and IBA1 protein levels in INPP5D WT and HET iMGLs. e . INPP5D WT and HET iMGLs were lysed in urea and mass spectrometry performed, n=4 WT, n=4 HET. Relative protein levels of AIF1, P2RY12, CX3CR1, C1QA, as quantified by mass spectrometry. Mean +/-SEM. Unpaired t-test. f . Images of INPP5D WT and HET cells immunostained for IBA1 and P2RY12. DNA is stained with DAPI. Imaged using confocal microscopy. Scale bars = 200 um. g . Volcano plot of adjusted p-values showing 75 DEPs from the 7,868 quantified via TMT-MS (BH FDR<0.05). h . Heatmap of relative expression levels of proteins involved in immune signaling that are differentially up and down regulated (adj. p-value <0.05) in INPP5D WT vs HET iMGLs. Asterisks indicate proteins that also were differentially up or down regulated (q<0.05) with 3AC treatment . i . Representative western blot of MRC1, PLA2G7, COLEC12, IBA1, and GAPDH in INPP5D WT and HET iMGLs. j . Western blot quantification of MRC1, PLA2G7, and COLEC12 levels normalized to IBA1 levels. 3 differentiations. Mean +/-SEM; unpaired t-test. k-l . Secreted IL-1ß and IL-18 measured by ELISA of media collected from INPP5D HET and WT iMGLs. Media were concentrated 10-fold for detection and normalized to WT mean. n=14 wells WT, n=21 well HET. Mean +/-SEM. Mann-Whitney test. m-n . Secreted IL-1ß and IL-18 measured by ELISA of media collected from INPP5D HET iMGLs treated with either vehicle (DMSO) or MCC950 (10 uM) for 24 hours. Media were concentrated 10-fold for detection. n=10 wells vehicle, n=10 wells MCC950-treated. Mean +/-SEM. Mann-Whitney test. For b-c, e, j-n: ns = not significant, *p < 0.05, ** p < 0.01, *** p < 0.001, **** p< 0.0001
    Figure Legend Snippet: a . Table of CRISPR-Cas9 generated INPP5D heterozygous (HET) and monoclonally-selected wildtype (WT) lines. b . INPP5D RNA levels measured by qPCR, normalized to GAPDH levels. Mean +/-SEM. Mann-Whitney test. c . Western blot quantification of INPP5D protein levels in INPP5D WT and HET iMGLs normalized to GAPDH levels, Mean +/-SEM. Mann-Whitney test. d . Representative western blot of INPP5D, GAPDH, and IBA1 protein levels in INPP5D WT and HET iMGLs. e . INPP5D WT and HET iMGLs were lysed in urea and mass spectrometry performed, n=4 WT, n=4 HET. Relative protein levels of AIF1, P2RY12, CX3CR1, C1QA, as quantified by mass spectrometry. Mean +/-SEM. Unpaired t-test. f . Images of INPP5D WT and HET cells immunostained for IBA1 and P2RY12. DNA is stained with DAPI. Imaged using confocal microscopy. Scale bars = 200 um. g . Volcano plot of adjusted p-values showing 75 DEPs from the 7,868 quantified via TMT-MS (BH FDR<0.05). h . Heatmap of relative expression levels of proteins involved in immune signaling that are differentially up and down regulated (adj. p-value <0.05) in INPP5D WT vs HET iMGLs. Asterisks indicate proteins that also were differentially up or down regulated (q<0.05) with 3AC treatment . i . Representative western blot of MRC1, PLA2G7, COLEC12, IBA1, and GAPDH in INPP5D WT and HET iMGLs. j . Western blot quantification of MRC1, PLA2G7, and COLEC12 levels normalized to IBA1 levels. 3 differentiations. Mean +/-SEM; unpaired t-test. k-l . Secreted IL-1ß and IL-18 measured by ELISA of media collected from INPP5D HET and WT iMGLs. Media were concentrated 10-fold for detection and normalized to WT mean. n=14 wells WT, n=21 well HET. Mean +/-SEM. Mann-Whitney test. m-n . Secreted IL-1ß and IL-18 measured by ELISA of media collected from INPP5D HET iMGLs treated with either vehicle (DMSO) or MCC950 (10 uM) for 24 hours. Media were concentrated 10-fold for detection. n=10 wells vehicle, n=10 wells MCC950-treated. Mean +/-SEM. Mann-Whitney test. For b-c, e, j-n: ns = not significant, *p < 0.05, ** p < 0.01, *** p < 0.001, **** p< 0.0001

    Techniques Used: CRISPR, Generated, MANN-WHITNEY, Western Blot, Mass Spectrometry, Staining, Confocal Microscopy, Expressing, Enzyme-linked Immunosorbent Assay

    a . Heatmap of relative expression of all the differentially expressed proteins comparing INPP5D WT and HET iMGLs (BH FDR<0.05). b . Heatmap of relative expression of overlapping differentially expressed proteins between chronic decrease of INPP5D activity (WT and HET) and acute decrease of INPP5D activity (vehicle vs 3AC). Of note, many DEPs are in the opposite direction between acute and chronic INPP5D reduction, suggesting a potential feedback mechanism following 3AC treatment.
    Figure Legend Snippet: a . Heatmap of relative expression of all the differentially expressed proteins comparing INPP5D WT and HET iMGLs (BH FDR<0.05). b . Heatmap of relative expression of overlapping differentially expressed proteins between chronic decrease of INPP5D activity (WT and HET) and acute decrease of INPP5D activity (vehicle vs 3AC). Of note, many DEPs are in the opposite direction between acute and chronic INPP5D reduction, suggesting a potential feedback mechanism following 3AC treatment.

    Techniques Used: Expressing, Activity Assay

    3ac  (Echelon Biosciences)


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    Echelon Biosciences 3ac
    SHIP paralog-selective inhibitors are unable to prevent obesity, but dual treatment with SHIP1- and SHIP2-selective inhibitors reduces weight and body fat increases due to increased caloric intake (A, D, G) Percent body weight, (B, E, H) percent body fat and (C, F, I) percent lean mass in C57BL/6 mice during HFD consumption and simultaneous treatment with (A–C) <t>3AC</t> or vehicle (0.3% Klucel:saline), (D-F) AS1949490 or vehicle (5% DMSO:saline) or (G–I) dual treatment with both 3AC and AS1949490 (As19) or dual vehicle (0.3% Klucel:saline + 5% DMSO:saline). Mice were dosed with 3AC and/or AS1949490 or vehicle two times per week (on days 1 and 4 of each week at 26.5mg/kg for 3AC or 20mg/kg for AS1949490 via i.p. injection) for the 4-week duration of the study. (Body Fat and Lean mass were measured by DEXA imaging before initiation of the study and after 4 or 6 weeks on HFD with SHIPi or vehicle treatment (Mean ± SEM, 2-way repeated measures ANOVA with Bonferroni multiple comparison test in A, B, D, G, two-tailed t-test with Welch’s correction when needed for C, E, F, H, and I, ∗∗p < 0.01, ∗∗∗p < 0.001,∗∗∗∗p < 0.0001, pooled from two experiments with n = 5).
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    1) Product Images from "Obesity control by SHIP inhibition requires pan-paralog inhibition and an intact eosinophil compartment"

    Article Title: Obesity control by SHIP inhibition requires pan-paralog inhibition and an intact eosinophil compartment

    Journal: iScience

    doi: 10.1016/j.isci.2023.106071

    SHIP paralog-selective inhibitors are unable to prevent obesity, but dual treatment with SHIP1- and SHIP2-selective inhibitors reduces weight and body fat increases due to increased caloric intake (A, D, G) Percent body weight, (B, E, H) percent body fat and (C, F, I) percent lean mass in C57BL/6 mice during HFD consumption and simultaneous treatment with (A–C) 3AC or vehicle (0.3% Klucel:saline), (D-F) AS1949490 or vehicle (5% DMSO:saline) or (G–I) dual treatment with both 3AC and AS1949490 (As19) or dual vehicle (0.3% Klucel:saline + 5% DMSO:saline). Mice were dosed with 3AC and/or AS1949490 or vehicle two times per week (on days 1 and 4 of each week at 26.5mg/kg for 3AC or 20mg/kg for AS1949490 via i.p. injection) for the 4-week duration of the study. (Body Fat and Lean mass were measured by DEXA imaging before initiation of the study and after 4 or 6 weeks on HFD with SHIPi or vehicle treatment (Mean ± SEM, 2-way repeated measures ANOVA with Bonferroni multiple comparison test in A, B, D, G, two-tailed t-test with Welch’s correction when needed for C, E, F, H, and I, ∗∗p < 0.01, ∗∗∗p < 0.001,∗∗∗∗p < 0.0001, pooled from two experiments with n = 5).
    Figure Legend Snippet: SHIP paralog-selective inhibitors are unable to prevent obesity, but dual treatment with SHIP1- and SHIP2-selective inhibitors reduces weight and body fat increases due to increased caloric intake (A, D, G) Percent body weight, (B, E, H) percent body fat and (C, F, I) percent lean mass in C57BL/6 mice during HFD consumption and simultaneous treatment with (A–C) 3AC or vehicle (0.3% Klucel:saline), (D-F) AS1949490 or vehicle (5% DMSO:saline) or (G–I) dual treatment with both 3AC and AS1949490 (As19) or dual vehicle (0.3% Klucel:saline + 5% DMSO:saline). Mice were dosed with 3AC and/or AS1949490 or vehicle two times per week (on days 1 and 4 of each week at 26.5mg/kg for 3AC or 20mg/kg for AS1949490 via i.p. injection) for the 4-week duration of the study. (Body Fat and Lean mass were measured by DEXA imaging before initiation of the study and after 4 or 6 weeks on HFD with SHIPi or vehicle treatment (Mean ± SEM, 2-way repeated measures ANOVA with Bonferroni multiple comparison test in A, B, D, G, two-tailed t-test with Welch’s correction when needed for C, E, F, H, and I, ∗∗p < 0.01, ∗∗∗p < 0.001,∗∗∗∗p < 0.0001, pooled from two experiments with n = 5).

    Techniques Used: Injection, Imaging, Two Tailed Test


    Figure Legend Snippet:

    Techniques Used: Recombinant, Lysis, Enzyme-linked Immunosorbent Assay, Software

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    Echelon Biosciences 3ac
    3ac, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Echelon Biosciences 3ac
    3ac, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    3ac, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    a Normalized RNAseq counts of INPP5D mRNA (encoding SHIP1) are shown for IgV H -mutated CLL cases (M-CLL; black squares; n = 94) as compared to IgV H -unmutated CLL samples (UM-CLL; clear squares; n = 95); samples obtained from the peripheral blood. Data are presented as individual values and mean values ± SD. Statistical significance was assessed by a two-tailed unpaired Student’s t -test. b Immunoblot of pSHIP1 Y1020 and global SHIP1 with beta-actin (ActB) as a loading control in MACS-isolated CD19 + peripheral blood B cells from healthy donors ( n = 5) as compared to CLL samples obtained from peripheral blood ( n = 4). Numbers indicated reflect the sample IDs of the CLL samples listed in Supplementary Table . c Direct phosphatase activity was determined using Malachite green assay after pulldown from 250 μg protein isolated from (left) H1299 cells lentivirally overexpressing wt SHIP1 or the AML-derived R673Q variant of SHIP1 (as positive and negative control, respectively) and (right) healthy donor peripheral blood B cells ( n = 5) or primary peripheral blood CLL cells ( n = 4). Data are presented as individual values and mean values ± SD. Statistical significance was assessed by a two-tailed unpaired Student’s t -test. The respective protein expression is shown in Supplementary Fig. . d MEC-1 cells were treated with the SHIP1 inhibitor (SHIP1i) <t>3AC</t> 5 μM (red line) or vehicle (dark gray line) for 3 min and subjected to intracellular pAKT staining; unstained control cells are indicated in light gray; representative FACS blot (left) and the summary of three independent experiments (right) is shown. Right: Data are presented as individual values for each experiment; statistical significance was assessed by a two-tailed paired Student’s t -test. e Cytotoxicity dose–response to increasing concentrations of the SHIP1 inhibitor 3AC in 28 primary CLL samples. The percentage of dead cells was determined by flow cytometry via DAPI staining. The percentage of specific cell death was calculated as follows: 100 × (% dead cells − % baseline dead cells)/(100% − % baseline dead cells). Data are presented as mean values ± SD. f Viability was determined upon 48 h treatment with 5 μM 3AC in vitro in CD19 + B cells from healthy donors (filled squares), the B-cell lymphoma lines BJAB (human Burkitt lymphoma), SUDHL6 (human diffuse, mixed small and large cell lymphoma line), Bal17 (murine B-cell lymphoma; filled diamonds), and 28 primary CLL samples (clear squares) as well as the CLL-derived cell lines MEC-1 (light gray square) and EHEB (dark gray square), and the specific cell death was calculated as described in e . Data are presented as individual values and mean values ± SD. Statistical significance was assessed by a two-tailed unpaired Student’s t -test. g MEC-1 cells were treated with vehicle (control), treated with the AKT inhibitor AZD-5363 (5 μM) alone, or in combination with 3AC (5 μM). Left: representative flow cytometry analysis of DAPI negative, viable cells upon the treatments. Right: the specific cell death was determined as described in e , measured in four independent experiments. Data are presented as individual values for each experiment and statistical significance was assessed by a two-tailed paired Student’s t -test. h Primary CLL cells derived from the peripheral blood of 7 donors were treated with vehicle (control), treated with the AKT inhibitor AZD-5363 (5 μM) alone or in combination with 3AC (5 μM) and the specific cell death was determined as described in e . Data are presented as individual values per CLL donor and statistical significance was assessed by a two-tailed paired Student’s t -test. Source data are provided as a Source Data file.
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    1) Product Images from "Targeted PI3K/AKT-hyperactivation induces cell death in chronic lymphocytic leukemia"

    Article Title: Targeted PI3K/AKT-hyperactivation induces cell death in chronic lymphocytic leukemia

    Journal: Nature Communications

    doi: 10.1038/s41467-021-23752-2

    a Normalized RNAseq counts of INPP5D mRNA (encoding SHIP1) are shown for IgV H -mutated CLL cases (M-CLL; black squares; n = 94) as compared to IgV H -unmutated CLL samples (UM-CLL; clear squares; n = 95); samples obtained from the peripheral blood. Data are presented as individual values and mean values ± SD. Statistical significance was assessed by a two-tailed unpaired Student’s t -test. b Immunoblot of pSHIP1 Y1020 and global SHIP1 with beta-actin (ActB) as a loading control in MACS-isolated CD19 + peripheral blood B cells from healthy donors ( n = 5) as compared to CLL samples obtained from peripheral blood ( n = 4). Numbers indicated reflect the sample IDs of the CLL samples listed in Supplementary Table . c Direct phosphatase activity was determined using Malachite green assay after pulldown from 250 μg protein isolated from (left) H1299 cells lentivirally overexpressing wt SHIP1 or the AML-derived R673Q variant of SHIP1 (as positive and negative control, respectively) and (right) healthy donor peripheral blood B cells ( n = 5) or primary peripheral blood CLL cells ( n = 4). Data are presented as individual values and mean values ± SD. Statistical significance was assessed by a two-tailed unpaired Student’s t -test. The respective protein expression is shown in Supplementary Fig. . d MEC-1 cells were treated with the SHIP1 inhibitor (SHIP1i) 3AC 5 μM (red line) or vehicle (dark gray line) for 3 min and subjected to intracellular pAKT staining; unstained control cells are indicated in light gray; representative FACS blot (left) and the summary of three independent experiments (right) is shown. Right: Data are presented as individual values for each experiment; statistical significance was assessed by a two-tailed paired Student’s t -test. e Cytotoxicity dose–response to increasing concentrations of the SHIP1 inhibitor 3AC in 28 primary CLL samples. The percentage of dead cells was determined by flow cytometry via DAPI staining. The percentage of specific cell death was calculated as follows: 100 × (% dead cells − % baseline dead cells)/(100% − % baseline dead cells). Data are presented as mean values ± SD. f Viability was determined upon 48 h treatment with 5 μM 3AC in vitro in CD19 + B cells from healthy donors (filled squares), the B-cell lymphoma lines BJAB (human Burkitt lymphoma), SUDHL6 (human diffuse, mixed small and large cell lymphoma line), Bal17 (murine B-cell lymphoma; filled diamonds), and 28 primary CLL samples (clear squares) as well as the CLL-derived cell lines MEC-1 (light gray square) and EHEB (dark gray square), and the specific cell death was calculated as described in e . Data are presented as individual values and mean values ± SD. Statistical significance was assessed by a two-tailed unpaired Student’s t -test. g MEC-1 cells were treated with vehicle (control), treated with the AKT inhibitor AZD-5363 (5 μM) alone, or in combination with 3AC (5 μM). Left: representative flow cytometry analysis of DAPI negative, viable cells upon the treatments. Right: the specific cell death was determined as described in e , measured in four independent experiments. Data are presented as individual values for each experiment and statistical significance was assessed by a two-tailed paired Student’s t -test. h Primary CLL cells derived from the peripheral blood of 7 donors were treated with vehicle (control), treated with the AKT inhibitor AZD-5363 (5 μM) alone or in combination with 3AC (5 μM) and the specific cell death was determined as described in e . Data are presented as individual values per CLL donor and statistical significance was assessed by a two-tailed paired Student’s t -test. Source data are provided as a Source Data file.
    Figure Legend Snippet: a Normalized RNAseq counts of INPP5D mRNA (encoding SHIP1) are shown for IgV H -mutated CLL cases (M-CLL; black squares; n = 94) as compared to IgV H -unmutated CLL samples (UM-CLL; clear squares; n = 95); samples obtained from the peripheral blood. Data are presented as individual values and mean values ± SD. Statistical significance was assessed by a two-tailed unpaired Student’s t -test. b Immunoblot of pSHIP1 Y1020 and global SHIP1 with beta-actin (ActB) as a loading control in MACS-isolated CD19 + peripheral blood B cells from healthy donors ( n = 5) as compared to CLL samples obtained from peripheral blood ( n = 4). Numbers indicated reflect the sample IDs of the CLL samples listed in Supplementary Table . c Direct phosphatase activity was determined using Malachite green assay after pulldown from 250 μg protein isolated from (left) H1299 cells lentivirally overexpressing wt SHIP1 or the AML-derived R673Q variant of SHIP1 (as positive and negative control, respectively) and (right) healthy donor peripheral blood B cells ( n = 5) or primary peripheral blood CLL cells ( n = 4). Data are presented as individual values and mean values ± SD. Statistical significance was assessed by a two-tailed unpaired Student’s t -test. The respective protein expression is shown in Supplementary Fig. . d MEC-1 cells were treated with the SHIP1 inhibitor (SHIP1i) 3AC 5 μM (red line) or vehicle (dark gray line) for 3 min and subjected to intracellular pAKT staining; unstained control cells are indicated in light gray; representative FACS blot (left) and the summary of three independent experiments (right) is shown. Right: Data are presented as individual values for each experiment; statistical significance was assessed by a two-tailed paired Student’s t -test. e Cytotoxicity dose–response to increasing concentrations of the SHIP1 inhibitor 3AC in 28 primary CLL samples. The percentage of dead cells was determined by flow cytometry via DAPI staining. The percentage of specific cell death was calculated as follows: 100 × (% dead cells − % baseline dead cells)/(100% − % baseline dead cells). Data are presented as mean values ± SD. f Viability was determined upon 48 h treatment with 5 μM 3AC in vitro in CD19 + B cells from healthy donors (filled squares), the B-cell lymphoma lines BJAB (human Burkitt lymphoma), SUDHL6 (human diffuse, mixed small and large cell lymphoma line), Bal17 (murine B-cell lymphoma; filled diamonds), and 28 primary CLL samples (clear squares) as well as the CLL-derived cell lines MEC-1 (light gray square) and EHEB (dark gray square), and the specific cell death was calculated as described in e . Data are presented as individual values and mean values ± SD. Statistical significance was assessed by a two-tailed unpaired Student’s t -test. g MEC-1 cells were treated with vehicle (control), treated with the AKT inhibitor AZD-5363 (5 μM) alone, or in combination with 3AC (5 μM). Left: representative flow cytometry analysis of DAPI negative, viable cells upon the treatments. Right: the specific cell death was determined as described in e , measured in four independent experiments. Data are presented as individual values for each experiment and statistical significance was assessed by a two-tailed paired Student’s t -test. h Primary CLL cells derived from the peripheral blood of 7 donors were treated with vehicle (control), treated with the AKT inhibitor AZD-5363 (5 μM) alone or in combination with 3AC (5 μM) and the specific cell death was determined as described in e . Data are presented as individual values per CLL donor and statistical significance was assessed by a two-tailed paired Student’s t -test. Source data are provided as a Source Data file.

    Techniques Used: Two Tailed Test, Western Blot, Isolation, Activity Assay, Malachite Green Assay, Derivative Assay, Variant Assay, Negative Control, Expressing, Staining, Flow Cytometry, In Vitro

    a Representative FACS analysis of murine CLL cells (mCLL) in the peripheral blood prior to treatment initiation (d8). b Time course of mCLL content in the peripheral blood (PB), arrow indicates the SHIP1 inhibitor (SHIP1i) 3AC (clear squares) or vehicle (black squares) treatment initiation ( n = 7 individual animals per treatment group, representative for two independent experiments), with 14 total doses of 20 mg/kg (treatment schedule is shown in Supplementary Fig. ). Data are presented as mean values ± SD. Statistical significance was assessed by a two-tailed unpaired Student’s t -test for the indicated time points. c murine CLL cell engraftment in the spleen (SP), the bone marrow (BM) and in peritoneal cavity (PC) after treatment with 3AC or vehicle control is shown as determined by FACS analysis; n = 7 animals per treatment group, representative for two independent experiments. Data are presented as individual values and mean values ± SD. Statistical significance was assessed by a two-tailed unpaired Student’s t -test. d Representative FACS analysis of aggressive mCLL cells in the peripheral blood prior to treatment initiation (d10). e Time course of mCLL content in the peripheral blood, arrow indicates 3AC/vehicle treatment initiation ( n = 7 animals per treatment group; representative for two independent experiments; treatment schedule is shown in Supplementary Fig. ). Data are presented as mean values ± SD. Statistical significance was assessed by a two-tailed unpaired Student’s t -test for the indicated time points. f Murine CLL cell engraftment in the spleen (SP), the bone marrow (BM), and in peritoneal cavity (PC) after treatment with the SHIP1i 3AC or vehicle control is shown as determined by FACS analysis. Data are presented as individual values and mean values ± SD. Statistical significance was assessed by a two-tailed unpaired Student’s t -test. g Summary of human CLL (hCLL) contents in the peripheral blood prior to treatment initiation (d1 post injection), representative for two independent experiments. h – j Reduction of CLL cells in the peripheral blood ( h ), and spleen, bone marrow, and peritoneal cavity ( i ) of NSG mice treated with 3AC ( n = 6) as compared to vehicle control ( n = 6), representative for two independent experiments. The treatment schedule is shown in Supplementary Fig. and the gating strategy in Supplementary Fig. . Data are presented as individual values and mean values ± SD. Statistical significance was assessed by a two-tailed unpaired Student’s t -test. Representative result for two independent CLL donors injected in 12 NSG mice, respectively. j – k Immunohistochemistry (IHC) for human CD20 (hCD20) in spleens, scale bars represent 500 μm ( j ), and quantified with automated hCD20+ IHC analysis ( k ); box plots indicate median (middle line), 25th, 75th percentile (box) and minimum and maximum (whiskers) and the statistical analysis was performed by Mann–Whitney test. Automated detection of hCD20 is depicted in Supplementary Fig. . Significance values are depicted in the graph; (n.s.) not significant. Source data are provided as a Source Data file.
    Figure Legend Snippet: a Representative FACS analysis of murine CLL cells (mCLL) in the peripheral blood prior to treatment initiation (d8). b Time course of mCLL content in the peripheral blood (PB), arrow indicates the SHIP1 inhibitor (SHIP1i) 3AC (clear squares) or vehicle (black squares) treatment initiation ( n = 7 individual animals per treatment group, representative for two independent experiments), with 14 total doses of 20 mg/kg (treatment schedule is shown in Supplementary Fig. ). Data are presented as mean values ± SD. Statistical significance was assessed by a two-tailed unpaired Student’s t -test for the indicated time points. c murine CLL cell engraftment in the spleen (SP), the bone marrow (BM) and in peritoneal cavity (PC) after treatment with 3AC or vehicle control is shown as determined by FACS analysis; n = 7 animals per treatment group, representative for two independent experiments. Data are presented as individual values and mean values ± SD. Statistical significance was assessed by a two-tailed unpaired Student’s t -test. d Representative FACS analysis of aggressive mCLL cells in the peripheral blood prior to treatment initiation (d10). e Time course of mCLL content in the peripheral blood, arrow indicates 3AC/vehicle treatment initiation ( n = 7 animals per treatment group; representative for two independent experiments; treatment schedule is shown in Supplementary Fig. ). Data are presented as mean values ± SD. Statistical significance was assessed by a two-tailed unpaired Student’s t -test for the indicated time points. f Murine CLL cell engraftment in the spleen (SP), the bone marrow (BM), and in peritoneal cavity (PC) after treatment with the SHIP1i 3AC or vehicle control is shown as determined by FACS analysis. Data are presented as individual values and mean values ± SD. Statistical significance was assessed by a two-tailed unpaired Student’s t -test. g Summary of human CLL (hCLL) contents in the peripheral blood prior to treatment initiation (d1 post injection), representative for two independent experiments. h – j Reduction of CLL cells in the peripheral blood ( h ), and spleen, bone marrow, and peritoneal cavity ( i ) of NSG mice treated with 3AC ( n = 6) as compared to vehicle control ( n = 6), representative for two independent experiments. The treatment schedule is shown in Supplementary Fig. and the gating strategy in Supplementary Fig. . Data are presented as individual values and mean values ± SD. Statistical significance was assessed by a two-tailed unpaired Student’s t -test. Representative result for two independent CLL donors injected in 12 NSG mice, respectively. j – k Immunohistochemistry (IHC) for human CD20 (hCD20) in spleens, scale bars represent 500 μm ( j ), and quantified with automated hCD20+ IHC analysis ( k ); box plots indicate median (middle line), 25th, 75th percentile (box) and minimum and maximum (whiskers) and the statistical analysis was performed by Mann–Whitney test. Automated detection of hCD20 is depicted in Supplementary Fig. . Significance values are depicted in the graph; (n.s.) not significant. Source data are provided as a Source Data file.

    Techniques Used: Two Tailed Test, Injection, Immunohistochemistry, MANN-WHITNEY

    a Heatmap analysis of differentially regulated genes associated with oxidative phosphorylation in MEC-1 cells upon transduction with pMIG EV (left) or pMIG-myrAKT1 (right), representative for two independent experiments. Color scale indicates the Z score (red = up; blue = down). b Gene set enrichment analysis (GSEA) results for the association with “oxidative phosphorylation” is shown upon myrAKT expression, Enrichment Score (ES) 0.65666306; Normalized Enrichment Score (NES) 2.847461; Nominal p -value < 0.001; FDR q -value < 0.001; FWER p-value < 0.001; representative for two independent experiments. c Oxygen consumption rate (OCR) was measured in GFP-sorted MEC-1 cells upon transduction with pMIG EV or pMIG-myrAKT1, pooled analysis of three independent experiments. Data are presented as mean values ± SEM (left) and as individual values and mean values ± SD in the bar graph (right). Statistical significance was assessed by a two-tailed unpaired Student’s t -test. d OCR was measured in MEC-1 cells treated for 1 h with SHIP1 inhibitor (SHIP1i; 5 μM 3AC; clear squares) or control (black squares), pooled analysis of seven independent experiments. Data are presented as mean values ± SEM over time (left) and as individual values and mean values ± SD in the bar graph (right). Right: Statistical significance was assessed using mean values of three independent experiments by a two-tailed unpaired Student’s t -test. e OCR was measured in a healthy donor (HD) B cells ( n = 6; circles) and primary CLL cells ( n = 6, squares) upon 1 h treatment with SHIP1i (5 μM 3AC; clear) or control (filled); pooled data from two independent experiments. Data are presented as mean values ± SEM (left) and as individual values and mean values ± SD in the bar graph (right). Statistical significance was assessed by a two-tailed unpaired Student’s t -test. f Quantification of MFI upon ROS analysis using the CellROX orange dye in primary CLL cells untreated, or treated for 4 h with 5 μM 3AC is shown ( n = 8). Data are presented as individual values per CLL donor. Statistical significance was assessed by a two-tailed paired Student’s t -test. g MEC-1 cells were treated with MitoTEMPO (MT; filled squares), SHIP1i (3AC; clear squares) or the combination (clear circles) for 24 h treatment, measured in five independent experiments; the specific cell death was determined as described in Fig. . Data are presented as individual values and mean values ± SD. Statistical significance was assessed by a two-tailed unpaired Student’s t -test. h Primary CLL samples ( n = 3) were treated with MT (filled squares), SHIP1i (3AC; clear squares), or the combination (clear circles) for 24 h; the specific cell death was determined as described in Fig. . Data are presented as individual values and mean values ± SD. Statistical significance was assessed by a two-tailed unpaired Student’s t -test. Significance values are depicted in the graph; (n.s.) not significant. Source data are provided as a Source Data file.
    Figure Legend Snippet: a Heatmap analysis of differentially regulated genes associated with oxidative phosphorylation in MEC-1 cells upon transduction with pMIG EV (left) or pMIG-myrAKT1 (right), representative for two independent experiments. Color scale indicates the Z score (red = up; blue = down). b Gene set enrichment analysis (GSEA) results for the association with “oxidative phosphorylation” is shown upon myrAKT expression, Enrichment Score (ES) 0.65666306; Normalized Enrichment Score (NES) 2.847461; Nominal p -value < 0.001; FDR q -value < 0.001; FWER p-value < 0.001; representative for two independent experiments. c Oxygen consumption rate (OCR) was measured in GFP-sorted MEC-1 cells upon transduction with pMIG EV or pMIG-myrAKT1, pooled analysis of three independent experiments. Data are presented as mean values ± SEM (left) and as individual values and mean values ± SD in the bar graph (right). Statistical significance was assessed by a two-tailed unpaired Student’s t -test. d OCR was measured in MEC-1 cells treated for 1 h with SHIP1 inhibitor (SHIP1i; 5 μM 3AC; clear squares) or control (black squares), pooled analysis of seven independent experiments. Data are presented as mean values ± SEM over time (left) and as individual values and mean values ± SD in the bar graph (right). Right: Statistical significance was assessed using mean values of three independent experiments by a two-tailed unpaired Student’s t -test. e OCR was measured in a healthy donor (HD) B cells ( n = 6; circles) and primary CLL cells ( n = 6, squares) upon 1 h treatment with SHIP1i (5 μM 3AC; clear) or control (filled); pooled data from two independent experiments. Data are presented as mean values ± SEM (left) and as individual values and mean values ± SD in the bar graph (right). Statistical significance was assessed by a two-tailed unpaired Student’s t -test. f Quantification of MFI upon ROS analysis using the CellROX orange dye in primary CLL cells untreated, or treated for 4 h with 5 μM 3AC is shown ( n = 8). Data are presented as individual values per CLL donor. Statistical significance was assessed by a two-tailed paired Student’s t -test. g MEC-1 cells were treated with MitoTEMPO (MT; filled squares), SHIP1i (3AC; clear squares) or the combination (clear circles) for 24 h treatment, measured in five independent experiments; the specific cell death was determined as described in Fig. . Data are presented as individual values and mean values ± SD. Statistical significance was assessed by a two-tailed unpaired Student’s t -test. h Primary CLL samples ( n = 3) were treated with MT (filled squares), SHIP1i (3AC; clear squares), or the combination (clear circles) for 24 h; the specific cell death was determined as described in Fig. . Data are presented as individual values and mean values ± SD. Statistical significance was assessed by a two-tailed unpaired Student’s t -test. Significance values are depicted in the graph; (n.s.) not significant. Source data are provided as a Source Data file.

    Techniques Used: Transduction, Expressing, Two Tailed Test

    a – d Primary CLL samples were treated with the SHIP1 inhibitor (SHIP1i) 3AC or the combination of 3AC with the Caspase inhibitor (PanCaspi) Emricasan ( a n = 7), the Caspase-8 inhibitor (Casp8i) Z-IETD ( b n = 8), the RIP1 inhibitor (RIP1i) NEC1s ( c n = 5) or the RIP3 inhibitor (RIP3i) GSK-843 ( d n = 7) and the specific cell death was determined as described in Fig. . For the combination treatment, the viability of the single treatments of the cell death inhibitors was used for determining the baseline cell death to calculate the specific cell death. Viability is depicted in Supplementary Fig. ; Data are presented as individual values and statistical significance was assessed by a two-tailed paired Student’s t -test. e Calreticulin (CALR) exposure to the outer membrane is determined after 4 h of 3AC treatment on primary CLL samples ( n = 8) as determined by flow cytometric analysis. Data are presented as individual values and statistical significance was assessed by a two-tailed paired Student’s t -test. f Supernatant of four primary CLL samples was collected after 48 h and HMGB1 levels determined by immunoblot. A representative loading control (unspecific band at 15 kDa) is shown for one CLL sample. g Dose-dependent accumulation of extracellular ATP levels in the supernatants of primary CLL samples ( n = 6) was determined after 4 h of treatment with the SHIP1 inhibitor 3AC. Data are presented as individual values and mean values ± SD. Statistical significance was assessed by an ordinary one-way ANOVA test. h Supernatant of four primary CLL samples was collected after 48 h with the SHIP1/2 inhibitor K118 1, 2, and 3AC 5 μM and in combination with the MLKL inhibitor (MLKLi) NSA (2 μM), and HMGB1 levels determined by immunoblot. A representative loading control is shown for one CLL sample. i Supernatants of puromycin-selected (shRNA expressing, d6) or GFP-sorted (EV, myrAKT expressing, d5) MEC-1 cells were analyzed for HMGB1 content. An unspecific band appearing at 15 kD served as a loading control. Representative example for two independent experiments. j Fold change to control of extracellular ATP levels in the supernatants of MEC-1 cells after SHIP1 knockdown (shSHIP1 KD1 (black squares) and KD2 (clear squares) vs. shScrambled, d6 post selection) and after myrAKT overexpression (gray squares; myrAKT vs. pMIG, d5 post sorting), individual values and mean value of two independent experiments are shown. k Cellular consequences of transient SHIP1 inhibition in CLL are illustrated (top panel) as compared to steady-state status (bottom panel), this figure was created using Servier Medical Art templates ( https://smart.servier.com ). Source data are provided as a Source Data file.
    Figure Legend Snippet: a – d Primary CLL samples were treated with the SHIP1 inhibitor (SHIP1i) 3AC or the combination of 3AC with the Caspase inhibitor (PanCaspi) Emricasan ( a n = 7), the Caspase-8 inhibitor (Casp8i) Z-IETD ( b n = 8), the RIP1 inhibitor (RIP1i) NEC1s ( c n = 5) or the RIP3 inhibitor (RIP3i) GSK-843 ( d n = 7) and the specific cell death was determined as described in Fig. . For the combination treatment, the viability of the single treatments of the cell death inhibitors was used for determining the baseline cell death to calculate the specific cell death. Viability is depicted in Supplementary Fig. ; Data are presented as individual values and statistical significance was assessed by a two-tailed paired Student’s t -test. e Calreticulin (CALR) exposure to the outer membrane is determined after 4 h of 3AC treatment on primary CLL samples ( n = 8) as determined by flow cytometric analysis. Data are presented as individual values and statistical significance was assessed by a two-tailed paired Student’s t -test. f Supernatant of four primary CLL samples was collected after 48 h and HMGB1 levels determined by immunoblot. A representative loading control (unspecific band at 15 kDa) is shown for one CLL sample. g Dose-dependent accumulation of extracellular ATP levels in the supernatants of primary CLL samples ( n = 6) was determined after 4 h of treatment with the SHIP1 inhibitor 3AC. Data are presented as individual values and mean values ± SD. Statistical significance was assessed by an ordinary one-way ANOVA test. h Supernatant of four primary CLL samples was collected after 48 h with the SHIP1/2 inhibitor K118 1, 2, and 3AC 5 μM and in combination with the MLKL inhibitor (MLKLi) NSA (2 μM), and HMGB1 levels determined by immunoblot. A representative loading control is shown for one CLL sample. i Supernatants of puromycin-selected (shRNA expressing, d6) or GFP-sorted (EV, myrAKT expressing, d5) MEC-1 cells were analyzed for HMGB1 content. An unspecific band appearing at 15 kD served as a loading control. Representative example for two independent experiments. j Fold change to control of extracellular ATP levels in the supernatants of MEC-1 cells after SHIP1 knockdown (shSHIP1 KD1 (black squares) and KD2 (clear squares) vs. shScrambled, d6 post selection) and after myrAKT overexpression (gray squares; myrAKT vs. pMIG, d5 post sorting), individual values and mean value of two independent experiments are shown. k Cellular consequences of transient SHIP1 inhibition in CLL are illustrated (top panel) as compared to steady-state status (bottom panel), this figure was created using Servier Medical Art templates ( https://smart.servier.com ). Source data are provided as a Source Data file.

    Techniques Used: Two Tailed Test, Western Blot, shRNA, Expressing, Selection, Over Expression, Inhibition

    3ac  (Echelon Biosciences)


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    a , b iMGs were treated with vehicle (ethanol) or <t>3AC</t> (1.25 μM) for 6 h. a Cells were then lysed, RNA purified, and RNAseq performed, n = 6 per condition. Volcano plot of DEGs comparing vehicle and 3AC treatment conditions. b Cells were lysed in urea and TMT-MS performed. n = 4 per condition. Volcano plot of DEPs; For a , b differential expression was calculated using a linear modeling with empirical Bayesian statistical analysis using the limma package in R. All p -values are adjusted using the Benjamini Hochberg (BH) procedure. c Heatmap of expression of DEGs that showed concordant differential expression at the RNA and protein level. d Heatmap of relative expression of DEPs encoded by LOAD GWAS candidate genes between vehicle and 3AC-treated iMGs. e . Relative RNA and protein levels of CD33 and PTK2B between vehicle and 3AC-treated microglia, as quantified by RNAseq and TMT-MS. Mean ± SEM. Two-sided Welch’s t -test. f , g . A variety of microglial subtypes previously have been defined through single-cell sequencing, with specific subtypes implicated to be altered in AD brain and model systems – . Volcano plots comparing 3AC vs vehicle treatment for genes defining these subtypes within the transcriptomic and proteomic datasets are shown; significance determined by BH FDR < 0.05. h Heatmap of relative protein-levels of scavenger receptors in vehicle and 3AC-treated iMGs. i , j . Enriched GO terms of biological processes for the differentially expressed genes that are elevated with 3AC treatment (geneontology.com ); Fisher’s exact test, Bonferroni multiple comparison’s test, adj p -value as shown by size of circles. j Heatmap of relative expression between vehicle and 3AC treatment of DEGs associated with NFκB signaling and JAK-STAT signaling. k Relative abundance measures of RNA or protein levels of inflammasome related components in vehicle and 3AC-treated iMGs. Mean ± SEM. Two-sided Welch’s t -test. l Representative western blot of iMGs treated with either vehicle (ethanol) or 3AC (1.25 μM) for 6 h showing protein expression of INPP5D and inflammasome related proteins: CASP1, PYCARD/ASC, GSDMD and NLRP3, as well as GAPDH. Similar WB results obtain in 3 separate differentiations. Cell lines used for experiments and full datasets for can be found in Supplementary Data and , Supplementary Data . For all graphs, the number of biological replicates is represented by dots. For all panels: ** p < 0.01, *** p < 0.001, **** p < 0.0001; ns = p < 0.05. See also Supplementary Figs. and .
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    a , b iMGs were treated with vehicle (ethanol) or 3AC (1.25 μM) for 6 h. a Cells were then lysed, RNA purified, and RNAseq performed, n = 6 per condition. Volcano plot of DEGs comparing vehicle and 3AC treatment conditions. b Cells were lysed in urea and TMT-MS performed. n = 4 per condition. Volcano plot of DEPs; For a , b differential expression was calculated using a linear modeling with empirical Bayesian statistical analysis using the limma package in R. All p -values are adjusted using the Benjamini Hochberg (BH) procedure. c Heatmap of expression of DEGs that showed concordant differential expression at the RNA and protein level. d Heatmap of relative expression of DEPs encoded by LOAD GWAS candidate genes between vehicle and 3AC-treated iMGs. e . Relative RNA and protein levels of CD33 and PTK2B between vehicle and 3AC-treated microglia, as quantified by RNAseq and TMT-MS. Mean ± SEM. Two-sided Welch’s t -test. f , g . A variety of microglial subtypes previously have been defined through single-cell sequencing, with specific subtypes implicated to be altered in AD brain and model systems – . Volcano plots comparing 3AC vs vehicle treatment for genes defining these subtypes within the transcriptomic and proteomic datasets are shown; significance determined by BH FDR < 0.05. h Heatmap of relative protein-levels of scavenger receptors in vehicle and 3AC-treated iMGs. i , j . Enriched GO terms of biological processes for the differentially expressed genes that are elevated with 3AC treatment (geneontology.com ); Fisher’s exact test, Bonferroni multiple comparison’s test, adj p -value as shown by size of circles. j Heatmap of relative expression between vehicle and 3AC treatment of DEGs associated with NFκB signaling and JAK-STAT signaling. k Relative abundance measures of RNA or protein levels of inflammasome related components in vehicle and 3AC-treated iMGs. Mean ± SEM. Two-sided Welch’s t -test. l Representative western blot of iMGs treated with either vehicle (ethanol) or 3AC (1.25 μM) for 6 h showing protein expression of INPP5D and inflammasome related proteins: CASP1, PYCARD/ASC, GSDMD and NLRP3, as well as GAPDH. Similar WB results obtain in 3 separate differentiations. Cell lines used for experiments and full datasets for can be found in Supplementary Data and , Supplementary Data . For all graphs, the number of biological replicates is represented by dots. For all panels: ** p < 0.01, *** p < 0.001, **** p < 0.0001; ns = p < 0.05. See also Supplementary Figs. and .

    Journal: Nature Communications

    Article Title: INPP5D regulates inflammasome activation in human microglia

    doi: 10.1038/s41467-023-42819-w

    Figure Lengend Snippet: a , b iMGs were treated with vehicle (ethanol) or 3AC (1.25 μM) for 6 h. a Cells were then lysed, RNA purified, and RNAseq performed, n = 6 per condition. Volcano plot of DEGs comparing vehicle and 3AC treatment conditions. b Cells were lysed in urea and TMT-MS performed. n = 4 per condition. Volcano plot of DEPs; For a , b differential expression was calculated using a linear modeling with empirical Bayesian statistical analysis using the limma package in R. All p -values are adjusted using the Benjamini Hochberg (BH) procedure. c Heatmap of expression of DEGs that showed concordant differential expression at the RNA and protein level. d Heatmap of relative expression of DEPs encoded by LOAD GWAS candidate genes between vehicle and 3AC-treated iMGs. e . Relative RNA and protein levels of CD33 and PTK2B between vehicle and 3AC-treated microglia, as quantified by RNAseq and TMT-MS. Mean ± SEM. Two-sided Welch’s t -test. f , g . A variety of microglial subtypes previously have been defined through single-cell sequencing, with specific subtypes implicated to be altered in AD brain and model systems – . Volcano plots comparing 3AC vs vehicle treatment for genes defining these subtypes within the transcriptomic and proteomic datasets are shown; significance determined by BH FDR < 0.05. h Heatmap of relative protein-levels of scavenger receptors in vehicle and 3AC-treated iMGs. i , j . Enriched GO terms of biological processes for the differentially expressed genes that are elevated with 3AC treatment (geneontology.com ); Fisher’s exact test, Bonferroni multiple comparison’s test, adj p -value as shown by size of circles. j Heatmap of relative expression between vehicle and 3AC treatment of DEGs associated with NFκB signaling and JAK-STAT signaling. k Relative abundance measures of RNA or protein levels of inflammasome related components in vehicle and 3AC-treated iMGs. Mean ± SEM. Two-sided Welch’s t -test. l Representative western blot of iMGs treated with either vehicle (ethanol) or 3AC (1.25 μM) for 6 h showing protein expression of INPP5D and inflammasome related proteins: CASP1, PYCARD/ASC, GSDMD and NLRP3, as well as GAPDH. Similar WB results obtain in 3 separate differentiations. Cell lines used for experiments and full datasets for can be found in Supplementary Data and , Supplementary Data . For all graphs, the number of biological replicates is represented by dots. For all panels: ** p < 0.01, *** p < 0.001, **** p < 0.0001; ns = p < 0.05. See also Supplementary Figs. and .

    Article Snippet: 3AC (Echelon Biosciences) was reconstituted in 100% ethanol as per manufacturer instructions.

    Techniques: Purification, Expressing, Sequencing, Western Blot

    a Fold change of secreted cytokines from iMGs treated with LPS (100 ng/mL) or 3AC (1.25 μM) for 6 h across iMGs derived from four iPSC lines, assayed by ELISA. Fold change was calculated compared to vehicle-treated cells. b Levels of IL1B following 6 h treatment with vehicle, LPS (100 ng/mL), or 3AC (1.25 μM, 2.5 μM) treatment as measured by quantitative real-time PCR (qPCR). Data are normalized to GAPDH expression then values were normalized to vehicle treatment within each experiment. n = 3 differentiations with well-level data shown as dots. One-way ANOVA with Dunnett’s T3 multiple comparisons test. c Secreted levels of IL-1ß measured for the same experiments as ( b ) following 6 h treatment as measured by ELISA. IL-1ß levels were normalized to 1.25 μM 3AC-treated conditions within each experiment. n = 3 differentiations with well-level data shown as dots. d Representative western blot of protein expression in iMGs with biallelic loss-of-function mutations generated with CRISPR targeting. Loss of INPP5D protein observed repeatedly over 3 differentiations. e , f WT INPP5D iMGs and KO INPP5D iMGs were treated with 3AC for 6 h and the levels of IL-1ß and IL-18 were measured by ELISA. Multiple two-sided t -tests, Two-stage step-up (Benjamini, Krieger, and Yekutieli), ** q < 0.01; *** q < 0.005; **** q < 0.001; LLOD= lower limit of detection. n = 3 biological replicates. g , h Treatment of iMGs with either vehicle or 3AC (5 μM) with either VX-765 (25 μM) or its vehicle (DMSO). Levels of secreted IL-1ß and IL-18 measured via ELISA following treatments and normalized to 3AC-treated samples in each experiment. One-way ANOVA with Sidak’s multiple comparison test. 3 differentiations, n = 10 per condition. i , j iMGs were treated with either vehicle, primed with LPS (100 ng/mL) for 3 h and then treated with nigericin (10 μM) for 1 h, or treated with 3AC (1.25 μM or 5 μM) for 2 h. Cells were immunostained for ASC and IBA1, DNA is stained with DAPI (i) and imaged using confocal microscopy. Scale bars = 100 μm. j Quantification of ASC specks. Number of cells analyzed: n = 226 (vehicle); 151 (1.25 μM 3AC); 173 (5.0 μM 3AC) over three experiments and 10 images per condition. Kruskal–Wallis test to determine significance. k , l Treatment of iMGs with either vehicle or 3AC (5 μM) with either MCC950 (10 μM) or its vehicle (DMSO). Levels of secreted IL-1ß and IL-18 in the media were measured and normalized to 3AC-treated samples in each experiment. One-way ANOVA with Sidak’s multiple comparison test. n = 4 differentiations with well-level data shown as dots. m A summary figure of experiments performed to interrogate inflammasome activation, created using BioRender.com. For all graphs, data are presented as mean values ± SEM. For b , c , g , h , j , l : ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Cell lines used for experiments are detailed in Supplementary Data . See also Supplementary Figs. and .

    Journal: Nature Communications

    Article Title: INPP5D regulates inflammasome activation in human microglia

    doi: 10.1038/s41467-023-42819-w

    Figure Lengend Snippet: a Fold change of secreted cytokines from iMGs treated with LPS (100 ng/mL) or 3AC (1.25 μM) for 6 h across iMGs derived from four iPSC lines, assayed by ELISA. Fold change was calculated compared to vehicle-treated cells. b Levels of IL1B following 6 h treatment with vehicle, LPS (100 ng/mL), or 3AC (1.25 μM, 2.5 μM) treatment as measured by quantitative real-time PCR (qPCR). Data are normalized to GAPDH expression then values were normalized to vehicle treatment within each experiment. n = 3 differentiations with well-level data shown as dots. One-way ANOVA with Dunnett’s T3 multiple comparisons test. c Secreted levels of IL-1ß measured for the same experiments as ( b ) following 6 h treatment as measured by ELISA. IL-1ß levels were normalized to 1.25 μM 3AC-treated conditions within each experiment. n = 3 differentiations with well-level data shown as dots. d Representative western blot of protein expression in iMGs with biallelic loss-of-function mutations generated with CRISPR targeting. Loss of INPP5D protein observed repeatedly over 3 differentiations. e , f WT INPP5D iMGs and KO INPP5D iMGs were treated with 3AC for 6 h and the levels of IL-1ß and IL-18 were measured by ELISA. Multiple two-sided t -tests, Two-stage step-up (Benjamini, Krieger, and Yekutieli), ** q < 0.01; *** q < 0.005; **** q < 0.001; LLOD= lower limit of detection. n = 3 biological replicates. g , h Treatment of iMGs with either vehicle or 3AC (5 μM) with either VX-765 (25 μM) or its vehicle (DMSO). Levels of secreted IL-1ß and IL-18 measured via ELISA following treatments and normalized to 3AC-treated samples in each experiment. One-way ANOVA with Sidak’s multiple comparison test. 3 differentiations, n = 10 per condition. i , j iMGs were treated with either vehicle, primed with LPS (100 ng/mL) for 3 h and then treated with nigericin (10 μM) for 1 h, or treated with 3AC (1.25 μM or 5 μM) for 2 h. Cells were immunostained for ASC and IBA1, DNA is stained with DAPI (i) and imaged using confocal microscopy. Scale bars = 100 μm. j Quantification of ASC specks. Number of cells analyzed: n = 226 (vehicle); 151 (1.25 μM 3AC); 173 (5.0 μM 3AC) over three experiments and 10 images per condition. Kruskal–Wallis test to determine significance. k , l Treatment of iMGs with either vehicle or 3AC (5 μM) with either MCC950 (10 μM) or its vehicle (DMSO). Levels of secreted IL-1ß and IL-18 in the media were measured and normalized to 3AC-treated samples in each experiment. One-way ANOVA with Sidak’s multiple comparison test. n = 4 differentiations with well-level data shown as dots. m A summary figure of experiments performed to interrogate inflammasome activation, created using BioRender.com. For all graphs, data are presented as mean values ± SEM. For b , c , g , h , j , l : ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Cell lines used for experiments are detailed in Supplementary Data . See also Supplementary Figs. and .

    Article Snippet: 3AC (Echelon Biosciences) was reconstituted in 100% ethanol as per manufacturer instructions.

    Techniques: Derivative Assay, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Generated, CRISPR, Comparison, Staining, Confocal Microscopy, Activation Assay

    a–c Volcano plot ( a ) of adjusted p -values showing 71 DEPs from the 7868 quantified via TMT-MS (two-way t -test with Benjamini–Hochberg multiple comparisons test; FDR < 0.05. Heatmap ( b ) of relative expression levels of proteins involved in immune signaling that are differentially up and downregulated in INPP5D WT vs HET iMGs. Asterisks indicate proteins that also were differentially up or downregulated ( q < 0.05) with 3AC treatment (Fig. ). GO analysis ( c ) of DEPs; Fisher’s exact test, Bonferroni multiple comparison’s test, adj p -value as shown by size of circles. d , e Representative western blot and quantification across three differentiations of MRC1, PLA2G7, COLEC12, IBA1, and GAPDH in INPP5D WT and HET iMGs. n = 3 independent experiments, mean ± SEM; two-sided t -test. f Heatmap of TMT-MS data (z-score) for lysosomal DEPs in INPP5D HET vs WT iMGs. g Representative western blot of INPP5D iMGs treated with either vehicle (DMSO) or bafilomycin (baf, 100 nM) for either 24 or 6 h. Western blots are probed for INPP5D and LC3 with GAPDH as a loading control. h Quantification of autophagic flux (baf - veh of LC3-II/GAPDH) in iMGs with treatment of 24 h of baf. n = 6 wells vehicle, n = 6 wells of 6 h baf. Mean ± SEM, two-sided t -test with Welch’s correction. i , j Secreted IL-1ß and IL-18 measured by ELISA of media collected from INPP5D HET and WT iMGs. Media were concentrated tenfold in order to be above the limit of detection and normalized to WT mean. n = 14 wells WT, n = 21 wells HET. Mean ± SEM. Two-sided Mann–Whitney test. k , l Secreted IL-1ß and IL-18 measured by ELISA of media collected from INPP5D HET iMGs treated with either vehicle (DMSO) or MCC950 (10 μM) for 24 h. Media were concentrated tenfold for detection. n = 10 wells vehicle, n = 10 wells MCC950-treated. Mean ± SEM. Mann–Whitney test. For e , h – l * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. See also Supplementary Fig. , Supplementary Data and for full datasets.

    Journal: Nature Communications

    Article Title: INPP5D regulates inflammasome activation in human microglia

    doi: 10.1038/s41467-023-42819-w

    Figure Lengend Snippet: a–c Volcano plot ( a ) of adjusted p -values showing 71 DEPs from the 7868 quantified via TMT-MS (two-way t -test with Benjamini–Hochberg multiple comparisons test; FDR < 0.05. Heatmap ( b ) of relative expression levels of proteins involved in immune signaling that are differentially up and downregulated in INPP5D WT vs HET iMGs. Asterisks indicate proteins that also were differentially up or downregulated ( q < 0.05) with 3AC treatment (Fig. ). GO analysis ( c ) of DEPs; Fisher’s exact test, Bonferroni multiple comparison’s test, adj p -value as shown by size of circles. d , e Representative western blot and quantification across three differentiations of MRC1, PLA2G7, COLEC12, IBA1, and GAPDH in INPP5D WT and HET iMGs. n = 3 independent experiments, mean ± SEM; two-sided t -test. f Heatmap of TMT-MS data (z-score) for lysosomal DEPs in INPP5D HET vs WT iMGs. g Representative western blot of INPP5D iMGs treated with either vehicle (DMSO) or bafilomycin (baf, 100 nM) for either 24 or 6 h. Western blots are probed for INPP5D and LC3 with GAPDH as a loading control. h Quantification of autophagic flux (baf - veh of LC3-II/GAPDH) in iMGs with treatment of 24 h of baf. n = 6 wells vehicle, n = 6 wells of 6 h baf. Mean ± SEM, two-sided t -test with Welch’s correction. i , j Secreted IL-1ß and IL-18 measured by ELISA of media collected from INPP5D HET and WT iMGs. Media were concentrated tenfold in order to be above the limit of detection and normalized to WT mean. n = 14 wells WT, n = 21 wells HET. Mean ± SEM. Two-sided Mann–Whitney test. k , l Secreted IL-1ß and IL-18 measured by ELISA of media collected from INPP5D HET iMGs treated with either vehicle (DMSO) or MCC950 (10 μM) for 24 h. Media were concentrated tenfold for detection. n = 10 wells vehicle, n = 10 wells MCC950-treated. Mean ± SEM. Mann–Whitney test. For e , h – l * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. See also Supplementary Fig. , Supplementary Data and for full datasets.

    Article Snippet: 3AC (Echelon Biosciences) was reconstituted in 100% ethanol as per manufacturer instructions.

    Techniques: Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY