Journal: bioRxiv

Article Title: Distinct tumor architectures for metastatic colonization of the brain

doi: 10.1101/2023.01.27.525190

Figure Lengend Snippet: (A) Schematic of the workflow of isolating, culturing, and comparing the transcriptome of brain metastatic (BrM) derivatives to the parental breast cancer cell lines they were isolated from. (B) GSEA showing that, in comparison to corresponding parental cell lines, the ECM organization pathway is enriched in the brain metastatic derivatives of HCC1954 and MMTV-ErbB2 HER2BC cells and not in MDA231 TNBC cells. Color shades indicate BH-adjusted P values of normalized enrichment scores. (C) Z-scored expression of the matrisome genes TNC , COL4A1 , IL24 , and SEMA7A that are differentially upregulated in HCC1954 and MMTV-Erbb2 HER2BC brain metastatic derivatives (BrM) in reference to their corresponding parental (Par) cell lines. (D) Forest plots showing the hazard ratio of the expression of matrisome genes COL4A1 , CST6 , SEMA7A , TNC , IL24 , and S100A7A for relapse-free survival of HER2BC breast cancer patients. Gene names in red indicate core matrisome genes. P values from top to bottom are as follows: 0.0012, 0.0135, 0.0894, 0.0641, 0.1649, 0.0889. (E) Schematic of the MetMap workflow using barcoded cancer cell line pools for high-throughput metastatic potential profiling (adapted from Ref. ). Relative metastatic potential was quantified by deep sequencing of barcode abundance from tissue. Comparing the transcriptome of in vivo brain metastases to that of in vitro cell culture per multiplexed cell line pool yielded the log2 fold change (log2FC) of gene expression shown in (F). (F) Relative in vivo expression, visualized by the log2FC values shown in a descending order, of the top ECM component genes that are differentially upregulated in the brain metastasis samples composed predominantly of HER2BC cells (pink) than of TNBC cells (blue) (see for statistical analysis of the association between relative in vivo expression and percent of HER2BC cells across multiplexed brain metastasis samples). (G) GSEA showing that ECM-related pathways are enriched in multiplexed brain metastasis samples composed predominantly of HER2BC cells (denoted in pink in (F)) than of TNBC cells (in blue in (F)). Top four positively enriched gene sets are Gene Ontology (GO) terms of 1, collagen containing extracellular matrix (GO:0062023), 2, extracellular matrix (GO:0031012), 3, extracellular structure organization (GO:0043062), and 4, extracellular matrix structural constituent (GO:0005201).

Article Snippet: TaqMan human COL4A1 (Hs00266237_m1) , Thermo Fisher , Cat #: 4453320.

Techniques: Isolation, Comparison, Expressing, High Throughput Screening Assay, Sequencing, In Vivo, In Vitro, Cell Culture, Gene Expression

Journal: bioRxiv

Article Title: Distinct tumor architectures for metastatic colonization of the brain

doi: 10.1101/2023.01.27.525190

Figure Lengend Snippet: (A-B) Effect of the suppression of COL4A1 and TNC expression on oncosphere formation (A) by shRNA in HCC1954-BrM cells and (B) by CRISPRi (2 sgRNAS per target gene) in MMTV-Erbb2-BrM cells, measured by the size of colonies after 5 days of growth in vitro . n = 100–175 colonies/group. Mean ± SEM. (C-D) Effect of the suppression of COL4A1 and TNC expression on brain colonization (C) by shRNA in HCC1954-BrM cells (n = 5 mice/group, 4 weeks after intracardiac inoculation into athymic mice, 2 shRNAs per target gene) and (D) by CRISPRi in MMTV-Erbb2-BrM cells (n = 5–9 mice/group, 3 weeks post-intracardiac inoculation into FVB/NJ mice, 2 sgRNAs per target gene), both quantified by whole-body BLI. (E-G) (E, G) Quantification and (F) representative IF staining of brain metastatic colonies formed by HCC1954-BrM cells expressing either a control vector or shRNAs that deplete COL4A1 or TNC expression. n = 3 mice/group. Mean ± SEM. Unpaired t test. (G) The percent of vascular coopting cells and (E) the number of colonies were determined 1 and 3 weeks post-intracardiac inoculation, respectively. (H) Schematic illustrates distinctive modes of colonization and stromal interface, various cancer cell-intrinsic mediators of colonization, and the induction of distinct DAM responses during the initiation of brain colonization in TNBC and HER2BC. Copyright © 2023 Memorial Sloan Kettering.

Article Snippet: TaqMan human COL4A1 (Hs00266237_m1) , Thermo Fisher , Cat #: 4453320.

Techniques: Expressing, shRNA, In Vitro, Staining, Control, Plasmid Preparation

Journal: bioRxiv

Article Title: Distinct tumor architectures for metastatic colonization of the brain

doi: 10.1101/2023.01.27.525190

Figure Lengend Snippet:

Article Snippet: TaqMan human COL4A1 (Hs00266237_m1) , Thermo Fisher , Cat #: 4453320.

Techniques: Blocking Assay, Virus, Derivative Assay, Recombinant, Modification, Saline, Electron Microscopy, cDNA Synthesis, Sample Prep, Microarray, Gene Expression, Synthesized, Plasmid Preparation, Software, Magnetic Beads

Journal: bioRxiv

Article Title: Distinct tumor architectures for metastatic colonization of the brain

doi: 10.1101/2023.01.27.525190

Figure Lengend Snippet:

Article Snippet: TaqMan human COL4A1 (Hs00266237_m1) , Thermo Fisher , Cat #: 4453320.

Techniques: shRNA, Plasmid Preparation, Selection, Marker, Retroviral

Journal: bioRxiv

Article Title: Distinct tumor architectures for metastatic colonization of the brain

doi: 10.1101/2023.01.27.525190

Figure Lengend Snippet:

Article Snippet: TaqMan human COL4A1 (Hs00266237_m1) , Thermo Fisher , Cat #: 4453320.

Techniques: Sequencing

Journal: Molecular Cell

Article Title: HuD Is a Neural Translation Enhancer Acting on mTORC1-Responsive Genes and Counteracted by the Y3 Small Non-coding RNA

doi: 10.1016/j.molcel.2018.06.032

Figure Lengend Snippet: HuD Increases Global and Target-Specific Translation (A) Top enriched Gene Ontology terms among HuD mRNA targets are related to RNA processes, including splicing, transport, stability, and translation (highlighted in bold). (B) Metaprofile of HuD binding sites along protein coding transcripts, showing binding enrichment in 3′UTRs. (C) Right panel: representative sucrose gradient profiles in control and HuD overexpressing NSC-34 cells. Left panel: calculation of the global translation efficiency upon HuD silencing and overexpression. (D) Right: schematic representation of Click-iT AHA assay to quantify de novo protein synthesis in NSC-34 cells. Left: detection of de novo protein synthesis upon HuD silencing and overexpression. Puromycin, a translation inhibitor, was used as negative control. (E) Transcriptome-wide translation efficiency changes upon HuD overexpression in NSC-34 cells. Scatterplot displaying for each gene the average expression signal (CPM) against the log2 change in translation efficiency (delta TE) upon HuD overexpression. Genes with increased or decreased TE are highlighted. (F) Enrichment analysis of HuD RNA targets among genes with increased or decreased TE upon HuD overexpression, compared to enrichments associated with genes changing at either the polysomal or the total RNA level. Fisher’s test ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. (G) Enrichment of mTOR responsive mRNAs among HuD targets, as listed in multiple literature sources. (H) Western blot analysis of HuD targets (Eef1a1, Eif4a1, Eif4a2, Pabpc1) and negative control (Eif4a3) in HEK293 cells transiently transfected with HuD. Tubulin was used as reference. Experiments were performed at least in triplicate. In (C), (D), and (H), data are represented as mean ± SEM; t test ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. See also Figure S2 .

Article Snippet: The following antibodies were used: mouse anti HuD (sc-28299, Santa Cruz), rabbit anti HA (A190-1081, Bethyl Laboratories), rabbit anti eIF4A1 (ab312-17, Abcam), rabbit anti-eIF4A2 (31218, Abcam), rabbit anti-eIF4A3 (homemade, generously provided from Prof. Macchi’s lab), rabbit anti eEF1A1 (SAB2108050, Sigma), mouse anti Tubulin (sc-53140, Santa Cruz) and rabbit Anti-PABPC1 (SAB2101708, Sigma), rabbit anti Rpl26 (Ab59567, Abcam), rabbit anti S6 (2217, Cell Signaling Technology).

Techniques: Binding Assay, Control, Over Expression, Negative Control, Expressing, Western Blot, Transfection

Journal: Molecular Cell

Article Title: HuD Is a Neural Translation Enhancer Acting on mTORC1-Responsive Genes and Counteracted by the Y3 Small Non-coding RNA

doi: 10.1016/j.molcel.2018.06.032

Figure Lengend Snippet: Y3 Counteracts HuD-Induced Neurogenesis (A) Differentiating ESCs cultures assayed for Y3 and HuD expression levels by Northern blot and western blot, respectively. Cultures were immunostained for stage-specific markers: Oct4 (ESCs; red), Nestin (NPCs; red), and beta3-tubulin (early neurons; red); the scale bar corresponds to 75 μm. Relative quantification of Y3 and HuD levels are shown (right). (B) Differentiated NSC-34 cells (control or silenced for Y3) immunostained with anti-tubulin antibody (yellow) to detect neurites (left panel); GFP (green) identified transfected cells subjected to high content analysis; the scale bar corresponds to 100 μm. Multiple parameters were analyzed using Operetta HCS device (right panel). (C) Differentiation assay in control Y3 silenced cells, Y3 silenced cells transfected with wild-type HuD or with mutant HuD. A schematic representation of HuD constructs used in the experiment is provided. (D) PC12 cells were co-transfected with HA-tagged HuD and mock or Y3 WT or Y3 “deleted” vectors. Co-transfected cells were immunostained with anti-HA antibody, and the neurites were stained for tubulin. In (A)–(D), data are represented as mean ± SEM t test ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001.

Article Snippet: The following antibodies were used: mouse anti HuD (sc-28299, Santa Cruz), rabbit anti HA (A190-1081, Bethyl Laboratories), rabbit anti eIF4A1 (ab312-17, Abcam), rabbit anti-eIF4A2 (31218, Abcam), rabbit anti-eIF4A3 (homemade, generously provided from Prof. Macchi’s lab), rabbit anti eEF1A1 (SAB2108050, Sigma), mouse anti Tubulin (sc-53140, Santa Cruz) and rabbit Anti-PABPC1 (SAB2101708, Sigma), rabbit anti Rpl26 (Ab59567, Abcam), rabbit anti S6 (2217, Cell Signaling Technology).

Techniques: Expressing, Northern Blot, Western Blot, Quantitative Proteomics, Control, Transfection, High Content Screening, Differentiation Assay, Mutagenesis, Construct, Staining

Journal: Molecular Cell

Article Title: HuD Is a Neural Translation Enhancer Acting on mTORC1-Responsive Genes and Counteracted by the Y3 Small Non-coding RNA

doi: 10.1016/j.molcel.2018.06.032

Figure Lengend Snippet:

Article Snippet: The following antibodies were used: mouse anti HuD (sc-28299, Santa Cruz), rabbit anti HA (A190-1081, Bethyl Laboratories), rabbit anti eIF4A1 (ab312-17, Abcam), rabbit anti-eIF4A2 (31218, Abcam), rabbit anti-eIF4A3 (homemade, generously provided from Prof. Macchi’s lab), rabbit anti eEF1A1 (SAB2108050, Sigma), mouse anti Tubulin (sc-53140, Santa Cruz) and rabbit Anti-PABPC1 (SAB2101708, Sigma), rabbit anti Rpl26 (Ab59567, Abcam), rabbit anti S6 (2217, Cell Signaling Technology).

Techniques: Virus, Recombinant, Magnetic Beads, Western Blot, Luciferase, Amplified Luminescent Proximity Homogenous Assay, Imaging, Sequencing, CRISPR, Knock-Out, Control, Northern Blot, Mutagenesis, Binding Assay, Activity Assay, Plasmid Preparation, Software, Microscopy, High Content Screening

Journal: JCI Insight

Article Title: ZNFX1 promotes AMPK-mediated autophagy against Mycobacterium tuberculosis by stabilizing Prkaa2 mRNA

doi: 10.1172/jci.insight.171850

Figure Lengend Snippet: ( A ) Heatmap of expression data of several ZFPs identified using quantitative real-time PCR (qPCR). ( B ) Quantification of mRNA coding for ZNFX1 in different macrophages following H37Rv infection (MOI = 2), using qPCR. hpi, hours postinfection; RQ, relative quantification. ( C ) Western blot analysis of ZNFX1 expression in H37Rv-infected BMDMs. ( D ) Immunohistochemistry detection and statistical analysis of ZNFX1 expression in the lung tissues and LNs of patients with CI or TB ( n = 4). A 2-way ANOVA with Holm-Šídák post hoc test ( B ) or an unpaired t test ( D ) was used for statistical analysis. Data are presented as mean ± SD and are representative of at least 3 experiments with similar observations. *** P < 0.001; **** P < 0.0001.

Article Snippet: The obtained cellular fractions of protein were analyzed using Western blotting, with Na,K-ATPase (catalog 3010), Lamin A/C (catalog 4777) (both from CST), and GAPDH (catalog 10494-1-AP) (Proteintech) as the reference proteins of membrane, nucleus fractions, and cytosol, respectively. co-IP assays were performed using Protein A/G magnetic beads (MCE) with 2 μg of antibodies against ZNFX1 (customized by Dia-an Biosciences), AMPKα2 (catalog 18167-1-AP), and STK11/LKB1 (catalog 10746-1-AP) (both from Proteintech) and rabbit IgG isotype control (catalog I5006) (Merck Millipore), according to the following protocol.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Infection, Quantitative Proteomics, Western Blot, Immunohistochemistry

Journal: JCI Insight

Article Title: ZNFX1 promotes AMPK-mediated autophagy against Mycobacterium tuberculosis by stabilizing Prkaa2 mRNA

doi: 10.1172/jci.insight.171850

Figure Lengend Snippet: ( A ) Schematic diagram of the time points of assays during the in vivo evaluation of Znfx1 –/– -induced immune responses against M . tuberculosis infection. CK, cytokine. ( B ) In vivo M . tuberculosis load in the lung and spleen tissues of Znfx1 –/– mice at 1 and 4 weeks after H37Rv infection ( n = 5). ( C ) H&E staining of the lung and spleen tissues of Znfx1 –/– mice. The splenic MGCs were quantified ( n = 5, with 30 randomly selected fields of view for statistics). Yellow arrows indicate MGCs. ( D ) Measurement of NO production indicated as the concentration of NO 2 – in the lung and spleen tissues 1 week postinfection ( n = 5). ( E ) Luminex multiplex assays of cytokine expression in the peripheral blood of mice 1 week after H37Rv infection ( n = 5). ( F ) ELISA of cytokine expression in the lung and spleen of mice 1 week after H37Rv infection ( n = 5). A 2-way ANOVA with Holm-Šídák post hoc test ( B ) or an unpaired 2-tailed t test was used ( C – F ) was used for statistical analysis. Data are presented as mean ± SD and are representative of at least 3 experiments with similar observations. ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: The obtained cellular fractions of protein were analyzed using Western blotting, with Na,K-ATPase (catalog 3010), Lamin A/C (catalog 4777) (both from CST), and GAPDH (catalog 10494-1-AP) (Proteintech) as the reference proteins of membrane, nucleus fractions, and cytosol, respectively. co-IP assays were performed using Protein A/G magnetic beads (MCE) with 2 μg of antibodies against ZNFX1 (customized by Dia-an Biosciences), AMPKα2 (catalog 18167-1-AP), and STK11/LKB1 (catalog 10746-1-AP) (both from Proteintech) and rabbit IgG isotype control (catalog I5006) (Merck Millipore), according to the following protocol.

Techniques: In Vivo, Infection, Staining, Concentration Assay, Luminex, Multiplex Assay, Expressing, Enzyme-linked Immunosorbent Assay

Journal: JCI Insight

Article Title: ZNFX1 promotes AMPK-mediated autophagy against Mycobacterium tuberculosis by stabilizing Prkaa2 mRNA

doi: 10.1172/jci.insight.171850

Figure Lengend Snippet: ( A ) Flow cytometry analysis of red fluorescence–positive WT and Znfx1 –/– BMDMs infected with H37Rv carrying the red fluorescence protein (RFP) gene (i.e., H37Rv-RFP) at MOI = 10 at 2 hpi ( n = 3). ( B ) Flow cytometry analysis of green fluorescence–positive WT and Znfx1 –/– BMDMs incubated with FITC-conjugated latex beads ( n = 4). ( C ) CFU assays of intracellular M . tuberculosis levels in H37Rv-infected WT and Znfx1 –/– BMDMs at MOI = 5 ( n = 4). ( D ) Flow cytometry analysis of CD80, CD86, MHC-II, and CD206 expression on the surface of H37Rv-infected WT and Znfx1 –/– BMDMs at MOI = 2 at 24 hpi ( n = 3). ( E ) Western blot assay of the regulatory effects of ZNFX1 on activation of signaling pathways following M . tuberculosis infection at MOI = 5. p-, phosphorylated. ( F ) Western blot assay of the regulatory effects of ZNFX1 on activation of the autophagy-associated mTOR signaling pathways. An unpaired 2-tailed t test ( A and B ) or a 2-way ANOVA with Holm-Šídák post hoc test ( C – F ) was used for statistical analysis. Data are presented as mean ± SD and are representative of at least 3 experiments with similar observations. ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: The obtained cellular fractions of protein were analyzed using Western blotting, with Na,K-ATPase (catalog 3010), Lamin A/C (catalog 4777) (both from CST), and GAPDH (catalog 10494-1-AP) (Proteintech) as the reference proteins of membrane, nucleus fractions, and cytosol, respectively. co-IP assays were performed using Protein A/G magnetic beads (MCE) with 2 μg of antibodies against ZNFX1 (customized by Dia-an Biosciences), AMPKα2 (catalog 18167-1-AP), and STK11/LKB1 (catalog 10746-1-AP) (both from Proteintech) and rabbit IgG isotype control (catalog I5006) (Merck Millipore), according to the following protocol.

Techniques: Flow Cytometry, Fluorescence, Infection, Incubation, Expressing, Western Blot, Activation Assay, Protein-Protein interactions

Journal: JCI Insight

Article Title: ZNFX1 promotes AMPK-mediated autophagy against Mycobacterium tuberculosis by stabilizing Prkaa2 mRNA

doi: 10.1172/jci.insight.171850

Figure Lengend Snippet: ( A ) Western blot assay of the regulatory effects of ZNFX1 on AMPK activation. mpi, minutes postinfection. ( B ) Western blot assay of the regulatory effects of ZNFX1 on the levels of p62 and LC3-I/II conversion. ( C ) Immunofluorescence assays of LC3 puncta in Znfx1 –/– BMDMs treated with BafA1, followed by infection with H37Rv-RFP ( n = 3, with 10 randomly selected fields of view for statistics). ( D ) Double-staining immunofluorescence assays of LC3 in F4/80 + macrophages in the lung and spleen tissues of WT and Znfx1 –/– mice following H37Rv infection ( n = 5, with 5 randomly selected fields of view for statistics). “Pearson’s R value” refers to Pearson’s correlation coefficient. ( E ) CFU assays of M . tuberculosis load in WT and Znfx1 –/– BMDMs treated with rapamycin ( n = 5). A 2-way ANOVA with Holm-Šídák post hoc test ( A – D ) was used for statistical analysis. Data are presented as mean ± SD and are representative of at least 3 experiments with similar observations. * P < 0.05; *** P < 0.001; **** P < 0.0001.

Article Snippet: The obtained cellular fractions of protein were analyzed using Western blotting, with Na,K-ATPase (catalog 3010), Lamin A/C (catalog 4777) (both from CST), and GAPDH (catalog 10494-1-AP) (Proteintech) as the reference proteins of membrane, nucleus fractions, and cytosol, respectively. co-IP assays were performed using Protein A/G magnetic beads (MCE) with 2 μg of antibodies against ZNFX1 (customized by Dia-an Biosciences), AMPKα2 (catalog 18167-1-AP), and STK11/LKB1 (catalog 10746-1-AP) (both from Proteintech) and rabbit IgG isotype control (catalog I5006) (Merck Millipore), according to the following protocol.

Techniques: Western Blot, Activation Assay, Immunofluorescence, Infection, Double Immunofluorescence Staining

Journal: JCI Insight

Article Title: ZNFX1 promotes AMPK-mediated autophagy against Mycobacterium tuberculosis by stabilizing Prkaa2 mRNA

doi: 10.1172/jci.insight.171850

Figure Lengend Snippet: ( A ) High-throughput RNA sequencing analysis of H37Rv-infected (MOI = 2) WT and Znfx1 –/– BMDMs at 6 and 24 hpi identified Prkaa2 as the downstream target of ZNFX1. ( B and C ) Expression levels of Prkaa2 and its coded protein AMPKα2 following H37Rv (MOI = 2) in WT and Znfx1 –/– BMDMs, using qPCR ( B ) and Western blotting ( C ), respectively. ( D ) Double-staining immunofluorescence assays of p-AMPK in F4/80 + macrophages in the lung and spleen tissues of WT and Znfx1 –/– mice following H37Rv infection ( n = 5, with 5 randomly selected fields of view for statistics). “Pearson’s R value” refers to Pearson’s correlation coefficient. ( E ) Flow cytometry analysis of red fluorescence–positive WT and Znfx1 –/– BMDMs treated with EX229, followed by infection with H37Rv-RFP (MOI = 10, n = 4). ( F and G ) CFU assays of intracellular M . tuberculosis levels in H37Rv-infected (MOI = 5) WT and Znfx1 –/– BMDMs ( F ) and ZNFX1 -silenced hMDMs ( G ) following EX229 treatment ( n = 4). si, siRNA. ( H ) Flow cytometry analysis of CD80, CD86, and MHC-II expression on the surface of WT and Znfx1 –/– BMDMs treated with EX229 and infected with H37Rv (MOI = 2, n = 3). A 2-way ANOVA with Holm-Šídák post hoc test ( B and E – H ) or an unpaired 2-tailed t test ( D ) was used for statistical analysis. Data are presented as mean ± SD and are representative of at least 3 experiments with similar observations. ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: The obtained cellular fractions of protein were analyzed using Western blotting, with Na,K-ATPase (catalog 3010), Lamin A/C (catalog 4777) (both from CST), and GAPDH (catalog 10494-1-AP) (Proteintech) as the reference proteins of membrane, nucleus fractions, and cytosol, respectively. co-IP assays were performed using Protein A/G magnetic beads (MCE) with 2 μg of antibodies against ZNFX1 (customized by Dia-an Biosciences), AMPKα2 (catalog 18167-1-AP), and STK11/LKB1 (catalog 10746-1-AP) (both from Proteintech) and rabbit IgG isotype control (catalog I5006) (Merck Millipore), according to the following protocol.

Techniques: High Throughput Screening Assay, RNA Sequencing, Infection, Expressing, Western Blot, Double Immunofluorescence Staining, Flow Cytometry, Fluorescence

Journal: JCI Insight

Article Title: ZNFX1 promotes AMPK-mediated autophagy against Mycobacterium tuberculosis by stabilizing Prkaa2 mRNA

doi: 10.1172/jci.insight.171850

Figure Lengend Snippet: WT and Znfx1 –/– mice were treated with EX229 and infected with H37Rv. ( A ) CFU assays of M . tuberculosis load in the lung and spleen ( n = 5). ( B ) ELISA of cytokine expression in the lung and spleen of mice 1 week after H37Rv infection ( n = 5). ( C ) Assays of NO production in the lung and spleen 1 week postinfection ( n = 5). ( D and E ) H&E staining of the lung and spleen. The MGCs in the spleen were quantified. Yellow arrows indicate MGCs ( n = 5, with 30 randomly selected fields of view for statistics). A 2-way ANOVA with Holm-Šídák post hoc test ( A – C and E ) was used for statistical analysis. Data are presented as mean ± SD and are representative of at least 3 experiments with similar observations. * P < 0.05; *** P < 0.001; **** P < 0.0001.

Article Snippet: The obtained cellular fractions of protein were analyzed using Western blotting, with Na,K-ATPase (catalog 3010), Lamin A/C (catalog 4777) (both from CST), and GAPDH (catalog 10494-1-AP) (Proteintech) as the reference proteins of membrane, nucleus fractions, and cytosol, respectively. co-IP assays were performed using Protein A/G magnetic beads (MCE) with 2 μg of antibodies against ZNFX1 (customized by Dia-an Biosciences), AMPKα2 (catalog 18167-1-AP), and STK11/LKB1 (catalog 10746-1-AP) (both from Proteintech) and rabbit IgG isotype control (catalog I5006) (Merck Millipore), according to the following protocol.

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Expressing, Staining

Journal: JCI Insight

Article Title: ZNFX1 promotes AMPK-mediated autophagy against Mycobacterium tuberculosis by stabilizing Prkaa2 mRNA

doi: 10.1172/jci.insight.171850

Figure Lengend Snippet: ( A ) Western blotting of ZNFX1 in the cytosol, membrane, and nucleus fraction of BMDMs. ( B ) qPCR analysis of Prkaa1 and Prkaa2 in Znfx1 –/– BMDMs treated with DRB ( n = 3). ( C ) qPCR analysis of Prkaa2 in WT and Znfx1 –/– BMDMs infected with LV- Prkaa2 ( n = 3). LV, lentiviral. ( D ) RIP assay using anti-ZNFX1 antibody and qPCR analysis of the association between ZNFX1 protein and Prkaa1 and Prkaa2 mRNA in BMDMs ( n = 3). ( E ) RNA pulldown assay using biotinylated Prkaa2 and GFP transcripts and Western blotting of the association between Prkaa2 mRNA and ZNFX1 protein in BMDMs. ( F ) Schematic diagram of recombinant plasmids carrying full-length or various truncated forms of the Znfx1 gene accompanied by a FLAG tag. ( G ) RIP assay using anti-FLAG antibody and qPCR analysis of the association between full-length or truncated forms of ZNFX1 protein and Prkaa2 mRNA in HEK293T cells transfected with various Znfx1 expression plasmids ( n = 5). ( H ) qPCR analysis of Prkaa2 in HEK293T cells transfected with various Znfx1 expression plasmids and treated with DRB ( n = 3). ( I ) CFU assay of M . tuberculosis load in WT and Znfx1 –/– BMDMs transfected with the F2 truncated form of Znfx1 ( n = 4). ( J ) Schematic diagram of the molecular mechanism of ZNFX1 in the regulation of autophagy against M . tuberculosis infection. OE, overexpression. A 2-way ANOVA with Holm-Šídák post hoc test ( D and I ) or a 1-way ANOVA followed by multiple-comparison test ( G ) was used for statistical analysis. Data are presented as mean ± SD and are representative of at least 3 experiments with similar observations. *** P < 0.001; **** P < 0.0001.

Article Snippet: The obtained cellular fractions of protein were analyzed using Western blotting, with Na,K-ATPase (catalog 3010), Lamin A/C (catalog 4777) (both from CST), and GAPDH (catalog 10494-1-AP) (Proteintech) as the reference proteins of membrane, nucleus fractions, and cytosol, respectively. co-IP assays were performed using Protein A/G magnetic beads (MCE) with 2 μg of antibodies against ZNFX1 (customized by Dia-an Biosciences), AMPKα2 (catalog 18167-1-AP), and STK11/LKB1 (catalog 10746-1-AP) (both from Proteintech) and rabbit IgG isotype control (catalog I5006) (Merck Millipore), according to the following protocol.

Techniques: Western Blot, Membrane, Infection, Recombinant, FLAG-tag, Transfection, Expressing, Colony-forming Unit Assay, Over Expression, Comparison

Journal: bioRxiv

Article Title: Distinct tumor architectures for metastatic colonization of the brain

doi: 10.1101/2023.01.27.525190

Figure Lengend Snippet: (A) Schematic of the workflow of isolating, culturing, and comparing the transcriptome of brain metastatic (BrM) derivatives to the parental breast cancer cell lines they were isolated from. (B) GSEA showing that, in comparison to corresponding parental cell lines, the ECM organization pathway is enriched in the brain metastatic derivatives of HCC1954 and MMTV-ErbB2 HER2BC cells and not in MDA231 TNBC cells. Color shades indicate BH-adjusted P values of normalized enrichment scores. (C) Z-scored expression of the matrisome genes TNC , COL4A1 , IL24 , and SEMA7A that are differentially upregulated in HCC1954 and MMTV-Erbb2 HER2BC brain metastatic derivatives (BrM) in reference to their corresponding parental (Par) cell lines. (D) Forest plots showing the hazard ratio of the expression of matrisome genes COL4A1 , CST6 , SEMA7A , TNC , IL24 , and S100A7A for relapse-free survival of HER2BC breast cancer patients. Gene names in red indicate core matrisome genes. P values from top to bottom are as follows: 0.0012, 0.0135, 0.0894, 0.0641, 0.1649, 0.0889. (E) Schematic of the MetMap workflow using barcoded cancer cell line pools for high-throughput metastatic potential profiling (adapted from Ref. ). Relative metastatic potential was quantified by deep sequencing of barcode abundance from tissue. Comparing the transcriptome of in vivo brain metastases to that of in vitro cell culture per multiplexed cell line pool yielded the log2 fold change (log2FC) of gene expression shown in (F). (F) Relative in vivo expression, visualized by the log2FC values shown in a descending order, of the top ECM component genes that are differentially upregulated in the brain metastasis samples composed predominantly of HER2BC cells (pink) than of TNBC cells (blue) (see for statistical analysis of the association between relative in vivo expression and percent of HER2BC cells across multiplexed brain metastasis samples). (G) GSEA showing that ECM-related pathways are enriched in multiplexed brain metastasis samples composed predominantly of HER2BC cells (denoted in pink in (F)) than of TNBC cells (in blue in (F)). Top four positively enriched gene sets are Gene Ontology (GO) terms of 1, collagen containing extracellular matrix (GO:0062023), 2, extracellular matrix (GO:0031012), 3, extracellular structure organization (GO:0043062), and 4, extracellular matrix structural constituent (GO:0005201).

Article Snippet: TaqMan mouse Col4a1 (Mm01210125_m1) , Thermo Fisher , Cat #: 4453320.

Techniques: Isolation, Comparison, Expressing, High Throughput Screening Assay, Sequencing, In Vivo, In Vitro, Cell Culture, Gene Expression

Journal: bioRxiv

Article Title: Distinct tumor architectures for metastatic colonization of the brain

doi: 10.1101/2023.01.27.525190

Figure Lengend Snippet: (A-B) Effect of the suppression of COL4A1 and TNC expression on oncosphere formation (A) by shRNA in HCC1954-BrM cells and (B) by CRISPRi (2 sgRNAS per target gene) in MMTV-Erbb2-BrM cells, measured by the size of colonies after 5 days of growth in vitro . n = 100–175 colonies/group. Mean ± SEM. (C-D) Effect of the suppression of COL4A1 and TNC expression on brain colonization (C) by shRNA in HCC1954-BrM cells (n = 5 mice/group, 4 weeks after intracardiac inoculation into athymic mice, 2 shRNAs per target gene) and (D) by CRISPRi in MMTV-Erbb2-BrM cells (n = 5–9 mice/group, 3 weeks post-intracardiac inoculation into FVB/NJ mice, 2 sgRNAs per target gene), both quantified by whole-body BLI. (E-G) (E, G) Quantification and (F) representative IF staining of brain metastatic colonies formed by HCC1954-BrM cells expressing either a control vector or shRNAs that deplete COL4A1 or TNC expression. n = 3 mice/group. Mean ± SEM. Unpaired t test. (G) The percent of vascular coopting cells and (E) the number of colonies were determined 1 and 3 weeks post-intracardiac inoculation, respectively. (H) Schematic illustrates distinctive modes of colonization and stromal interface, various cancer cell-intrinsic mediators of colonization, and the induction of distinct DAM responses during the initiation of brain colonization in TNBC and HER2BC. Copyright © 2023 Memorial Sloan Kettering.

Article Snippet: TaqMan mouse Col4a1 (Mm01210125_m1) , Thermo Fisher , Cat #: 4453320.

Techniques: Expressing, shRNA, In Vitro, Staining, Control, Plasmid Preparation

Journal: bioRxiv

Article Title: Distinct tumor architectures for metastatic colonization of the brain

doi: 10.1101/2023.01.27.525190

Figure Lengend Snippet:

Article Snippet: TaqMan mouse Col4a1 (Mm01210125_m1) , Thermo Fisher , Cat #: 4453320.

Techniques: Blocking Assay, Virus, Derivative Assay, Recombinant, Modification, Saline, Electron Microscopy, cDNA Synthesis, Sample Prep, Microarray, Gene Expression, Synthesized, Plasmid Preparation, Software, Magnetic Beads

Journal: bioRxiv

Article Title: Distinct tumor architectures for metastatic colonization of the brain

doi: 10.1101/2023.01.27.525190

Figure Lengend Snippet:

Article Snippet: TaqMan mouse Col4a1 (Mm01210125_m1) , Thermo Fisher , Cat #: 4453320.

Techniques: shRNA, Plasmid Preparation, Selection, Marker, Retroviral

Journal: bioRxiv

Article Title: Distinct tumor architectures for metastatic colonization of the brain

doi: 10.1101/2023.01.27.525190

Figure Lengend Snippet:

Article Snippet: TaqMan mouse Col4a1 (Mm01210125_m1) , Thermo Fisher , Cat #: 4453320.

Techniques: Sequencing

Journal: Molecular cell

Article Title: MRI is a DNA Damage Response Adaptor during Classical Non-Homologous End Joining

doi: 10.1016/j.molcel.2018.06.018

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit anti-cytoskeletal actin , Bethyl Laboratories , Cat#A300-485A.

Techniques: Blocking Assay, Virus, Recombinant, Cell Isolation, Magnetic Beads, Mass Spectrometry, Sequencing, Magnetic Resonance Imaging, Plasmid Preparation, Software, High Content Screening, Flow Cytometry, Inverted Microscopy, Laser-Scanning Microscopy, Spectrophotometry, Irradiation

Journal: Science Advances

Article Title: Glutathione dynamics determine the therapeutic efficacy of mesenchymal stem cells for graft-versus-host disease via CREB1-NRF2 pathway

doi: 10.1126/sciadv.aba1334

Figure Lengend Snippet: ( A to C ) FR plots of hES-MSCs carrying CREB1 shRNA (sh CREB1 ) (A) or DN inhibitor ( CREB1- DN) (B) and FR plots of cells treated with 2 μM FSK for 12 hours (C). FR plots are presented as means ± SEM ( n = 6). *** P < 0.001 (two-way ANOVA). GI values are depicted below each FR plot. ( D to H ) CFU-F ( n = 6) (D); chemotaxis ( n = 7) (E) to PDGF-AA (10 ng/ml) in the absence or presence of STI571 (0.5 μg/ml), a PDGFR inhibitor; chemotaxis in the response to ( n = 7) (F) SDF1α (150 ng/ml) (left) or SDF1β (right) in the absence or presence of AMD3100 (10 μM), a CXCR4 antagonist; in vitro Matrigel tube formation ( n = 7) (G); and immune modulation ( n = 4) (H) assays in FSK (2 μM for 12 hours)–primed hES-MSCs. Representative results for each assay are presented in the left [×200 magnification; scale bars, 100 μm; (E and F)] or bottom panel [×40 magnification; scale bars, 200 μm; (G)] of quantitative data. For the Matrigel tube formation assay, CM was prepared from FSK-primed or NT cells. For negative and positive controls, saline and recombinant human vascular endothelial growth factor A (VEGF-A) (50 ng/ml) were used, respectively. The immune modulatory capacity (H) of MSCs were evaluated by suppression of T cell proliferation (CFSE − /CD3 + ) in human PBMCs stimulated by PHA. Data are presented as ratios relative to the value in the NT cells and are expressed as means ± SEM. Statistical analyses were performed using nonparametric Mann-Whitney U test (D), one-way ANOVA (E to G), or two-way ANOVA (H). ** P < 0.01 and *** P < 0.001 relative to NT group. ## P < 0.01 and ### P < 0.001.

Article Snippet: Imatinib mesylate (STI571) , Selleck Chemicals , S1026.

Techniques: shRNA, Chemotaxis Assay, In Vitro, Tube Formation Assay, Saline, Recombinant, MANN-WHITNEY

Journal: Science Advances

Article Title: Glutathione dynamics determine the therapeutic efficacy of mesenchymal stem cells for graft-versus-host disease via CREB1-NRF2 pathway

doi: 10.1126/sciadv.aba1334

Figure Lengend Snippet: Key resource table.

Article Snippet: Imatinib mesylate (STI571) , Selleck Chemicals , S1026.

Techniques: Virus, shRNA, Recombinant, Magnetic Beads, Lysis, Protease Inhibitor, Reporter Assay, Kinase Assay, Luminex, Screening Assay, Selection, Plasmid Preparation, Blocking Assay, Expressing, Software