Journal: Nature Communications

Article Title: Cathepsin D deficiency in mammary epithelium transiently stalls breast cancer by interference with mTORC1 signaling

doi: 10.1038/s41467-020-18935-2

Figure Lengend Snippet: a Pictures of Ctsd +/+ and Ctsd − /− PyMT cells cultured for 7–10 days in 10% FCS or 1% FCS medium. Bars, 50 μm. Representative of 5 independent experiments. b Staining for acidic β-galactosidase in 10% FCS and 1% FCS Ctsd +/+ and Ctsd − /− PyMT cells. Bars, 50 μm. Representative of 3 independent experiments. c Quantification of label-retaining cells among 10% FCS and 1% FCS Ctsd +/+ and Ctsd − /− PyMT cells ( n = 7 independent experiments; two-sided two-sample t -test). d Amount of Annexin-V - 7-AAD − (living) cells among 1% FCS Ctsd +/+ and Ctsd − /− PyMT cells treated with Navitoclax, relative to DMSO control and Ctsd +/+ ( n = 3 independent experiments; two-sided one-sample t -test). e Quantification of IL-6 by alphaLISA in cell-conditioned media from 10% FCS and 1% FCS Ctsd +/+ and Ctsd − /− PyMT cells ( n = 2 independent experiments). f Relative Cdkn2a/p16 and Cdkn1a/p21 expression determined by RT-PCR in 10% FCS and 1% FCS Ctsd +/+ and Ctsd − /− PyMT cells ( n = 3 independent experiments; two-sided two-sample t -test). Bar charts show all data points with mean + SD and p -value. Source data are provided as a Source Data file.

Article Snippet: CREB, phosphorylated CREB and IL-6 levels in cells and cell-conditioned media were measured with the AlphaLISA® SureFire® UltraTM CREB Total Assay Kit (ALSU-TCREB-A500), AlphaLISA® SureFire® UltraTM p-CREB (Ser133) Assay Kit (ALSU-PCREB-A500), and the AlphaLISA Mouse Interleukin 6 (mIL6) Kit (AL504 C) from PerkinElmer, respectively.

Techniques: Cell Culture, Staining, Control, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Nature Communications

Article Title: Cathepsin D deficiency in mammary epithelium transiently stalls breast cancer by interference with mTORC1 signaling

doi: 10.1038/s41467-020-18935-2

Figure Lengend Snippet: a Phospho-kinase antibody arrays with lysates from Ctsd +/+ and Ctsd − /− PyMT cells cultured for 7–10 days (1% FCS) or for ≥8 weeks (1% FCS LT) in 1% FCS medium. Changes in phosphorylation are plotted as log2 fold change (FC) for indicated comparisons ( n = 2 independent experiments). b , c Transcriptome analysis of 1% FCS and 1% FCS LT Ctsd +/+ and Ctsd − /− PyMT cells ( n = 3 independent experiments). b Change in expression of significantly regulated genes of the CREB gene set for indicated comparisons. Red dashed lines, borders for gene up- and downregulation (|log2 FC | > 1). c Clustering analysis of the log2 FC of CREB genes with significant upregulation in Ctsd − /− 1% FCS LT versus 1% FCS PyMT cells for indicated comparisons shown as heatmap. Source data are provided as a Source Data file.

Article Snippet: CREB, phosphorylated CREB and IL-6 levels in cells and cell-conditioned media were measured with the AlphaLISA® SureFire® UltraTM CREB Total Assay Kit (ALSU-TCREB-A500), AlphaLISA® SureFire® UltraTM p-CREB (Ser133) Assay Kit (ALSU-PCREB-A500), and the AlphaLISA Mouse Interleukin 6 (mIL6) Kit (AL504 C) from PerkinElmer, respectively.

Techniques: Cell Culture, Phospho-proteomics, Expressing

Journal: Glia

Article Title: Deletion of Alzheimer's disease-associated CD33 results in an inflammatory human microglia phenotype.

doi: 10.1002/glia.23968

Figure Lengend Snippet: FIGURE 3 Activation of SYK and ERK1/2 signaling pathways as a consequence of CD33 knockout in THP1 macrophages and human-induced pluripotent stem cell-derived microglia (iPSdMiG). (a) Western blot showing phosphorylated ERK1/2 (pERK1/2, top) and total ERK1/2 (tERK1/2, bottom) in wild type (WT) and CD33−/−macrophages. One representative image of five independent experiments is shown. (b) Quantification of pERK1/2 to tERK1/2 ratio in WT (black) and CD33−/−THP1 macrophages (dark gray). Knockout of CD33 resulted in an increase in pERK1/2 in THP1 macrophages. FcγRI antibody treatment tended to increase pERK1/2 levels slightly in WT THP1 macrophages. CD33 antibody treatment following FcγRI antibody treatment tended to decrease the FcγRI antibody-driven increase in pERK1/2 to tERK1/2 levels only in WT THP1 macrophages. Data are shown as mean + SEM (n = 3–5). *p ≤.05 determined by two-way analysis of variance (ANOVA) followed by Bonferroni post hoc test. (c) Western blot showing phosphorylated SYK (pSYK, top) and total SYK (tSYK, bottom) in WT and CD33−/−macrophages. One representative image of three independent experiments is shown. (d) Quantification of pSYK to tSYK ratio in WT (black) and CD33−/−THP1 macrophages (dark gray). Knockout of CD33 resulted in an increase in pSYK in THP1 macrophages. FcγRI antibody treatment tended to increase pSYK levels slightly and co-treatment with a CD33 antibody tended to counteract this increase, both only in WT THP1 macrophages. Data are shown as mean + SEM (n = 3). *p ≤.05 determined by two-way ANOVA followed by Bonferroni post hoc test. (e) SYK phosphorylation relative to total SYK assessed by AlphaLISA. Untreated iPSdMiG revealed an increased pSYK/tSYK ratio followed by knockout of CD33 (dark gray) or expression of CD33ΔE2 (light gray) compared to WT iPSdMiG (black). Data are shown as mean + SEM (n = 6). **p ≤.01, *p ≤.05 determined by one-way ANOVA followed by Bonferroni post hoc test. (f) Treatment of iPSdMiG with activating antibodies for TREM2 resulted in an increased pSYK/tSYK ratio in WT (black), CD33−/−(dark gray) and CD33ΔE2 iPSdMiG (light gray) compared to isotype antibody-treated controls. Further, pSYK/tSYK was sharply increased in CD33−/−iPSdMiG following anti-TREM2 treatment. Data are shown as mean + SEM (n = 3). ***p ≤.001, *p ≤.05 determined by two-way ANOVA followed by Bonferroni post hoc test

Article Snippet: Then, 24 hr after plating, the cells were stimulated for 5 min with anti-TREM2 (5 μg/ml, R&D Systems #AF1828), corresponding IgG isotype control (5 μg/ml Abcam #ab224187) or left untreated. pSYK levels were detected by the AlphaLISA SureFire Ultra p-SYK (Tyr525/526) Assay Kit (PerkinElmer) and normalized to the values of tSYK using the AlphaLISA SureFire Ultra Total SYK Assay Kit (PerkinElmer) according to the two-plate assay pro- tocol for adherent cells.

Techniques: Activation Assay, Protein-Protein interactions, Knock-Out, Derivative Assay, Western Blot, Phospho-proteomics, Expressing