Journal: Journal of Cellular and Molecular Medicine

Article Title: Occludin facilitates tumour angiogenesis in bladder cancer by regulating IL8/STAT3 through STAT4

doi: 10.1111/jcmm.17257

Figure Lengend Snippet: Occludin (OCLN) promotes tumour angiogenesis in vitro and in vivo. (a and b) The knockdown efficiency was confirmed by performing (A) Western blotting and (B) RT‐qPCR assays using 5637 and T24 cells. (C), Tube formation by EA.hy926 cells incubated with CM derived from 5637 and T24 OCLN‐silenced bladder cancer (BLCA) cells was assessed using staining with Calcein AM and imaging with a fluorescence microscope. Scale bar = 100 µm. (d and e) The OCLN plasmid was transfected into 5637 and T24 cells; the efficiency of overexpression was analysed using (D) Western blotting and (E) RT‐qPCR. (F) Tube formation by EA.hy926 cells incubated with CM derived from 5637 and T24 OCLN overexpressing cells was assessed using staining with Calcein AM and imaging with a fluorescence microscope. Scale bar = 100 µm. (G) The segment lengths were analysed, and the meshes were quantified using ImageJ software. (H) Matrigel plugs containing 5637 and T24 OCLN stable knockdown cells were removed from BALB/c nude mice. (I) CD31 staining in the indicated cells embedded in Matrigel plugs after growth in BALB/c nude mice. Scale bar = 100 µm. (J) The density of microvessels in Matrigel plugs from BALB/c nude mice injected with the indicated cells. (K) IHC staining showing CD31 levels in clinical patients with high‐/low‐grade BLCA. Scale bar = 100 µm. The results are shown as the mean ± SD. *.01 < p < .05; **.001 < p < .01

Article Snippet: BLCA cell lines, namely T24 (ATCC HTB‐4), 5637 (ATCC HTB‐9) were derived from laboratory retained cells.

Techniques: In Vitro, In Vivo, Knockdown, Western Blot, Quantitative RT-PCR, Incubation, Derivative Assay, Staining, Imaging, Fluorescence, Microscopy, Plasmid Preparation, Transfection, Over Expression, Software, Injection, Immunohistochemistry

Journal: Journal of Cellular and Molecular Medicine

Article Title: Occludin facilitates tumour angiogenesis in bladder cancer by regulating IL8/STAT3 through STAT4

doi: 10.1111/jcmm.17257

Figure Lengend Snippet: Occludin (OCLN) mediates bladder cancer (BLCA) angiogenesis by regulating IL8 expression. (A) Venn diagram showing the differentially expressed genes (DEGs) in the two T24 OCLN knockdown groups compared with the control groups (fold change ≥1, FDR < 0.1, p < .05). (B) Heatmap of the RNA sequencing analysis showing the relative levels of proangiogenic factors. Columns represent probe sets, and rows represent samples receiving the indicated treatments. (C) The relative mRNA levels of proangiogenic factors were detected in control and OCLN shRNA transfected 5637 and T24 cells. (D) The relative IL8 levels in control and OCLN shRNA‐transfected 5637 and T24 cells were measured using an ELISA (pg/ml). (E) The relative IL8 mRNA levels were detected in 5637 and T24 cells following transfection with the vector or OCLN plasmid. (F) Tube formation by EA.hy926 cells cultured with CM derived from 5637 and T24 OCLN‐silenced BLCA cells. IL8 was added, and the cells were stained with Calcein AM and then imaged with a fluorescence microscope. Scale bar = 100 µm. (G) The segment lengths were analysed, and the meshes were quantified using ImageJ software. The results are shown as the mean ± SD. *.01 < p < .05; **.001 < p < .01; *** p < .001

Article Snippet: BLCA cell lines, namely T24 (ATCC HTB‐4), 5637 (ATCC HTB‐9) were derived from laboratory retained cells.

Techniques: Expressing, Knockdown, Control, RNA Sequencing, shRNA, Transfection, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Cell Culture, Derivative Assay, Staining, Fluorescence, Microscopy, Software

Journal: Journal of Cellular and Molecular Medicine

Article Title: Occludin facilitates tumour angiogenesis in bladder cancer by regulating IL8/STAT3 through STAT4

doi: 10.1111/jcmm.17257

Figure Lengend Snippet: IL8/ STAT3 is involved in the process of Occludin (OCLN)‐mediated angiogenesis in bladder cancer (BLCA). (A) p‐STAT3 and STAT3 protein levels were detected in OCLN knockdown 5637 and T24 cells. (B) 5637 and T24 cells were transfected with the OCLN plasmid and treated with or without the STAT3 inhibitor Stattic, and tube formation by EA.hy926 cells incubated with CM derived from the indicated cells was assessed by performing staining with Calcein AM and imagining using a fluorescence microscope. Scale bar = 100 µm. (C) The segment lengths in these images were analysed, and the meshes were quantified using ImageJ software. D, p‐STAT3 and STAT3 protein levels were detected in OCLN knockdown 5637 and T24 cells after IL8 supplementation. (E) p‐STAT3 and STAT3 protein levels were detected in OCLN‐overexpressing 5637 and T24 cells after blocking IL8. (F) 5637 and T24 cells were transfected with the OCLN plasmid or cultured with the IL8‐neutralizing antibody; tube formation by EA.hy926 cells incubated with CM derived from the indicated cells was assessed using staining with Calcein AM and imaging with a fluorescence microscope. Scale bar = 100 µm. G, The segment lengths in these images were analysed, and the meshes were quantified using ImageJ software. The results are shown as the mean ± SD. *.01< p < .05; **.001< p < .01

Article Snippet: BLCA cell lines, namely T24 (ATCC HTB‐4), 5637 (ATCC HTB‐9) were derived from laboratory retained cells.

Techniques: Knockdown, Transfection, Plasmid Preparation, Incubation, Derivative Assay, Staining, Fluorescence, Microscopy, Software, Blocking Assay, Cell Culture, Imaging

Journal: Molecular and cellular endocrinology

Article Title: SDHB and SDHD silenced pheochromocytoma spheroids respond differently to tumour microenvironment and their aggressiveness is inhibited by impairing stroma metabolism.

doi: 10.1016/j.mce.2022.111594

Figure Lengend Snippet: Fig. 1. Characterization of stable SDHB and SDHD silenced cell lines. (A) Representative blots of the expression of mitochondrial SDHA, B and D subunits. (B) Densitometric analysis of western blot bands, performed by Bio-Rad imaging and analysis software (Quantity One), showed significant differences in the SDHB and SDHD subunit expression levels in SDHB (light grey) and in SDHD silenced cells (dark grey) respectively, compared to Wt (black). Bars are the means of three independent preparations ± SD, ***p < 0.001. (C) Representative traces of SDH enzymatic activity measured in cell homogenates. The silenced SDHB and SDHD cells (dotted and continuous lines, respectively) showed a similar decrease of the SDH activity, significantly different compared with Wt (dashed line). (D) Histogram represents the SDH activity expressed as the percentages. SDHB and SDHD silenced cells (light and dark grey, respectively) showed a significantly decreased of SDH activity compared to Wt (black). Bars are the means of three independent experiments (each of them conducted in duplicate samples) ± SD, ***p < 0.01. (E) The bar graph represents the means of intracellular succinate/fumarate ratio ± SD, measured by GC/MS, in three independent experiments with two replicates. SDHB silenced cells (light grey) showed a significant increase of the metabolites ratio compared with both Wt (black) and to SDHD silenced cells (dark grey), *p < 0.05, **p < 0.01. (F) Representative immunoblot of HIF1α expression in Wt, SDHB and SDHD silenced cells. (G) Optical density analysis of western blot bands. Actin im munoblots was used as loading control. For all the analyses One-way ANOVA post-test Bonferroni was used.

Article Snippet: Bound antibodies were detected using ECL reagents (Immobilon) and analysed with a Bio-Rad ChemiDoc Imaging System (Quantity One) for dedicated chemiluminescent image acquisition.

Techniques: Expressing, Western Blot, Imaging, Software, Activity Assay, Gas Chromatography-Mass Spectrometry, Control

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Adiponectin triggers breast cancer cell death via fatty acid metabolic reprogramming

doi: 10.1186/s13046-021-02223-y

Figure Lengend Snippet: Roles of SIRT-1 in fatty acid metabolic reprogramming by globular adiponectin in breast cancer cells. A-B MCF-7 ( A ) and MDA-MB-231 ( B ) cells were treated with gAcrp as indicated. SIRT-1 expression was determined by western blot analysis. C MCF-7 cells were pretreated with EX527 (5 μM) for 2 h, followed by incubation with gAcrp (1 μg/mL) for further 8 h. The protein levels of SREBP-1 and FASN were examined by western blot analysis. D MCF-7 cells were transfected with a siRNA targeting SIRT-1 or a scramble control siRNA for 36 h, followed by treatment with gAcrp (1 μg/mL) for 8 h. (Upper panel) The gene silencing efficiency was monitored by western blot analysis. (Lower panel) The expression levels of FASN and SREBP-1 in SIRT-1 knockdown cells were examined after gAcrp treatment. E MCF-7 cells were pretreated with EX527 for 2 h, followed by further incubation with gAcrp (1 μg/mL) for 12 h. mRNA levels of FASN, ACC-1, ACLY, and FADS2 were measured by RT-qPCR. F-H MCF-7 cells were transfected with SIRT-1 siRNA (25 nM) for 36 h, followed by treatment with 1 μg/mL of gAcrp for 24 h ( F ), 48 h ( G ), or 6 h ( H ). F Cellular neutral lipid content was determined by Bodipy 493/503 uptake assay. G Cells were labeled with Alexa fluor 488-conjugated CT-B and lipid raft microdomains were observed under a confocal microscope. H Lipid droplets were stained with Nile red and autophagosomes were labeled with an Alexa fluor 488-conjugated anti-LC3 antibody. The overlapping between lipid droplets (red) and autophagosomes (green) were observed under a confocal microscope. The Mander’s overlap coefficient was used to test the colocalization of lipid droplets and autophagosomes. Scale bar: 20 μm. I-M MCF-7 cells were transfected with SIRT-1 siRNA (25 nM) ( I ) or pretreated with EX527 for 2 h ( J - M ), followed by further treatment with 1 μg/mL of gAcrp for 48 h. I and J Cell viability was measured by MTS assay. K - L The apoptosis level was determined using caspase-3/7 activity ( K ) and TUNEL assay ( L ) as indicated in the methods. M Expression levels of Bax and Bcl2 were examined by western blot analysis. * denotes p < 0.05 compared to control; # denotes p < 0.05 compared to cells treated with gAcrp alone; n=3 except where specifically indicated in Figures

Article Snippet: Antibodies against p-AMPKα (#2531), AMPKα (#2532), non-phospho (active) β-catenin (#8814), FASN (#3180), p-Akt (# 9018), Akt (#9272), LC3 (#2775), p-mTOR (#2971), mTOR (# 2972), Bax (#5023), Bcl2 (#3498), Acetylated lysine (#9441), p62 (#5114), and Beclin-1 (#3738) were purchased from Cell Signaling Technology Inc.; antibodies for LRP-6 (#sc-25317), SIRT-1 (#sc-15404), and ubiquitin (#sc-8017) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA); antibodies for Atg5 (#PA1-46178) and β-actin (#MA5-15739) were purchased from Thermo Scientific (Waltham, MA, USA); antibodies for SREBP-1 (#ab191857) and p-LRP6 (#GTX62033) were purchased from Abcam (Cambridge, MA, USA) and GeneTex Inc. (Irvine, CA, USA), respectively.

Techniques: Expressing, Western Blot, Incubation, Transfection, Control, Knockdown, Quantitative RT-PCR, Labeling, Microscopy, Staining, MTS Assay, Activity Assay, TUNEL Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Adiponectin triggers breast cancer cell death via fatty acid metabolic reprogramming

doi: 10.1186/s13046-021-02223-y

Figure Lengend Snippet: Role of SIRT-1 in adiponectin modulation of in vivo breast tumor lipid metabolism and growth. MDA-MB-231-luc orthotopic breast tumor s were generated in BALB/c nude mice, followed by treatment with gAcrp alone or gAcrp in combination with EX527 for 28 days. A and B Luminescent images of tumors ( A ) and tumor growth rate were monitored by luminescent in vivo imaging during treatment ( B ). C and D Tumor tissues were harvested after 4 weeks of treatment. Isolated tumors were captured at the end of experiment ( C ) and tumor weight was recorded ( D ). E Tissue section was prepared, and cleavage of caspase-3 was examined by immunohistochemistry (IHC). The percentage of cleaved caspase-3 positive tumor cells was determined by Image J software. F The expression levels of Bax and Bcl2 were measured by western blot analysis. The representative images from 3 mice each group were shown along with blot quantification for all collected tumor tissues. G The expression levels of FASN and SREBP-1 were analyzed by immunoblotting analysis. H SREBP-1 was detected in tumor tissues by IHC. The proportion of nuclear SREBP-1 positive cells were presented in bar diagram. Scale bar: 100 μm. I The mRNA levels of SREBP-1, FASN, ACC-1, FADS2, and ACLY in tumor tissues were measured by RT-qPCR. J The protein levels of p-mTOR, mTOR, β-catenin, and LC3I/II were determined by western blot analysis. K-L Single cells were isolated from tumor tissues by incubating with collagenase solution. K Tumor cells were incubated with Bodipy 493/503 for 15 min at 37 o C, followed by flow cytometry analysis. L The free fatty acid level was measured in tumor cells and normalized to tumor cell number

Article Snippet: Antibodies against p-AMPKα (#2531), AMPKα (#2532), non-phospho (active) β-catenin (#8814), FASN (#3180), p-Akt (# 9018), Akt (#9272), LC3 (#2775), p-mTOR (#2971), mTOR (# 2972), Bax (#5023), Bcl2 (#3498), Acetylated lysine (#9441), p62 (#5114), and Beclin-1 (#3738) were purchased from Cell Signaling Technology Inc.; antibodies for LRP-6 (#sc-25317), SIRT-1 (#sc-15404), and ubiquitin (#sc-8017) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA); antibodies for Atg5 (#PA1-46178) and β-actin (#MA5-15739) were purchased from Thermo Scientific (Waltham, MA, USA); antibodies for SREBP-1 (#ab191857) and p-LRP6 (#GTX62033) were purchased from Abcam (Cambridge, MA, USA) and GeneTex Inc. (Irvine, CA, USA), respectively.

Techniques: In Vivo, Generated, In Vivo Imaging, Isolation, Immunohistochemistry, Software, Expressing, Western Blot, Quantitative RT-PCR, Incubation, Flow Cytometry

Journal: Cell reports

Article Title: β-Amyloid Clustering around ASC Fibrils Boosts Its Toxicity in Microglia

doi: 10.1016/j.celrep.2020.02.025

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Thereafter, cells were blocked using 5% normal goat serum (Vector Laboratories) in PTX for 20 min and primary antibodies were added for another 30 min. To check for ASC speck formation, the rabbit anti-ASC (1:250; clone AL177, AdipoGen) or mouse-specific rabbit anti-ASC/TMS1 (1:250, D2W8U, Cell Signaling Technology®) and rat anti-CD11b (1:250; Serotec by Bio-Rad) were used.

Techniques: Control, Recombinant, Magnetic Beads, Enzyme-linked Immunosorbent Assay, Bicinchoninic Acid Protein Assay, Quantitation Assay, Gel Extraction, Purification, Isolation, Software, Transmission Assay, Microscopy, Spectrophotometry, Fluorescence, Imaging

Journal: eLife

Article Title: MDGAs are fast-diffusing molecules that delay excitatory synapse development by altering neuroligin behavior

doi: 10.7554/eLife.75233

Figure Lengend Snippet:

Article Snippet: Commercial assay, kit , ClarityWestern blot ECL , Bio-Rad#170–5061 , , .

Techniques: Sequencing, Control, CRISPR, Biomarker Discovery, Reverse Transcription, Expressing, Recombinant, Electroporation, Plasmid Preparation, Blocking Assay, Transmission Assay, RNA Extraction, Lysis, Transfection, Cloning, cDNA Synthesis, Software, Microscopy, Imaging, Western Blot, Quantitation Assay

Journal: Molecular cell

Article Title: The Dystonia Gene THAP1 Controls DNA Double Strand Break Repair Choice

doi: 10.1016/j.molcel.2021.03.034

Figure Lengend Snippet: (A) Top: Venn diagram depicting differentially expressed genes (nascent RNA-seq, log2 fold-change >2 and FDR <0.05) in Thap1−/− versus WT and Thap1−/−Brca1Δ11 versus Brca1Δ11 MEFs in relation to THAP1-bound genes (ChIP-seq). The number of genes that were shown to be bound by THAP1 and were either downregulated or upregulated in THAP1-deficient MEFs are shown in blue and red, respectively.

Article Snippet: ChIP-seq was performed as described previously ( Shinoda et al., 2019 ) with a rabbit polyclonal antibody against THAP1 (Proteintech, 12584–1-AP).

Techniques: RNA Sequencing, ChIP-sequencing

Journal: Molecular cell

Article Title: The Dystonia Gene THAP1 Controls DNA Double Strand Break Repair Choice

doi: 10.1016/j.molcel.2021.03.034

Figure Lengend Snippet: (A) Western blot analysis of doxycycline-dependent expression of exogenous SHLD1 (left) and THAP1 (right) proteins in WT MEFs 24 to 96 hours after induction with doxycycline (Dox) as detected by anti-Flag antibody.

Article Snippet: ChIP-seq was performed as described previously ( Shinoda et al., 2019 ) with a rabbit polyclonal antibody against THAP1 (Proteintech, 12584–1-AP).

Techniques: Western Blot, Expressing

Journal: Molecular cell

Article Title: The Dystonia Gene THAP1 Controls DNA Double Strand Break Repair Choice

doi: 10.1016/j.molcel.2021.03.034

Figure Lengend Snippet: (A-B) Quantification of RPA2 (A) and RAD51 (B) foci in individual EdU-positive (S-phase) nuclei of WT, Brca1Δ11, Trp53bp1−/−Brca1Δ11 and two individual clones of Thap1−/− Brca1Δ11 MEFs. Cells were irradiated with 10 Gy and analyzed 4 h post-IR. Statistical significance was determined by Welch’s t-test.

Article Snippet: ChIP-seq was performed as described previously ( Shinoda et al., 2019 ) with a rabbit polyclonal antibody against THAP1 (Proteintech, 12584–1-AP).

Techniques: Clone Assay, Irradiation

Journal: Molecular cell

Article Title: The Dystonia Gene THAP1 Controls DNA Double Strand Break Repair Choice

doi: 10.1016/j.molcel.2021.03.034

Figure Lengend Snippet: (A) Representative flow cytometry plots of IgM-to-IgA class switch recombination (CSR) in WT, Trp53bp1−/−, Shld1−/−, Shld3−/− and two individual clones of Thap1−/− (#4 and #12) CH12-F3 cells 24 hours after cytokine stimulation (IL-4, CD40L and TGFβ). Unstimulated WT cells are shown as a negative control. Quantification of IgM-to-IgA CSR is shown on the right and represents mean ± s.d., n=3.

Article Snippet: ChIP-seq was performed as described previously ( Shinoda et al., 2019 ) with a rabbit polyclonal antibody against THAP1 (Proteintech, 12584–1-AP).

Techniques: Flow Cytometry, Clone Assay, Negative Control

Journal: Molecular cell

Article Title: The Dystonia Gene THAP1 Controls DNA Double Strand Break Repair Choice

doi: 10.1016/j.molcel.2021.03.034

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: ChIP-seq was performed as described previously ( Shinoda et al., 2019 ) with a rabbit polyclonal antibody against THAP1 (Proteintech, 12584–1-AP).

Techniques: Purification, Blocking Assay, Virus, Bacteria, Expressing, CRISPR, Knock-Out, Recombinant, Transfection, Cloning, PCR Cloning, Protease Inhibitor, Ligation, Library Quantification, Selection, Flow Cytometry, Cell Viability Assay, cDNA Synthesis, SYBR Green Assay, Cell Culture, Mutagenesis, Illumina Sequencing, Software, Microscopy, Imaging, Irradiation

Journal: Frontiers in Immunology

Article Title: Metallothionein 3-Zinc Axis Suppresses Caspase-11 Inflammasome Activation and Impairs Antibacterial Immunity

doi: 10.3389/fimmu.2021.755961

Figure Lengend Snippet: The MT3-Zn 2+ axis suppresses TRIF signaling resulting in decreased IRF3 phosphorylation. When MT3 is absent, TRIF-IRF3-STAT1 signaling and non-canonical inflammasome activation are exaggerated. A lack of MT3 augments immunity to gram-negative bacteria, an effect, that is further enhanced by the combined absence of MT3 and caspase-11 in vivo . Thus, while MT3 curtails caspase-11 activation, the two molecules act together in compromising antibacterial immunity.

Article Snippet: MT3 , Applied Biosystems , Hs00359394_g1.

Techniques: Phospho-proteomics, Activation Assay, Bacteria, In Vivo

Journal: Frontiers in Immunology

Article Title: Metallothionein 3-Zinc Axis Suppresses Caspase-11 Inflammasome Activation and Impairs Antibacterial Immunity

doi: 10.3389/fimmu.2021.755961

Figure Lengend Snippet: See also Supplementary Table S1 and Files S1 , S2 |Protein interaction network of Mus musculus MT3 to determine functionally enriched GO BP categories using the STRING database.

Article Snippet: MT3 , Applied Biosystems , Hs00359394_g1.

Techniques: Transduction, Cell Differentiation

Journal: Frontiers in Immunology

Article Title: Metallothionein 3-Zinc Axis Suppresses Caspase-11 Inflammasome Activation and Impairs Antibacterial Immunity

doi: 10.3389/fimmu.2021.755961

Figure Lengend Snippet: See also Supplementary Figure S1 MT3 suppresses caspase-11 inflammasome activation in BMDMϕ. qRT-PCR analysis of Mt3 expression in WT BMDMϕ stimulated with (A) iLPS (2 μg/ml) or vehicle control, 3-5 independent experiments and (B) exLPS (10 μg/ml) for 48h, 3 independent experiments, two-tailed t-test. (C) Western Blots of pro- and active-caspase-11, pro-caspase-1, pro-IL1β and β-actin in cell lysates and active-caspase-1 and active-IL-1β in supernatants of WT and Mt3 -/- BMDMϕ stimulated with iLPS (10 μg/ml) or vehicle for 48h. Bar graphs are densitometric analysis of targets normalized to β-actin, 3-4 independent experiments, one-way ANOVA, data are mean ± SEM. (D) Western Blots of pro- and active-caspase-11 and β-actin in lysate + supernatant samples from WT and Mt3 -/- BMDMϕ stimulated with iLPS (2 μg/ml) or vehicle for 48h. Bar graphs are densitometric analysis of targets normalized to β-actin. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: MT3 , Applied Biosystems , Hs00359394_g1.

Techniques: Activation Assay, Quantitative RT-PCR, Expressing, Control, Two Tailed Test, Western Blot

Journal: Frontiers in Immunology

Article Title: Metallothionein 3-Zinc Axis Suppresses Caspase-11 Inflammasome Activation and Impairs Antibacterial Immunity

doi: 10.3389/fimmu.2021.755961

Figure Lengend Snippet: See also Supplementary Figure S2 . MT3 curtails CASPASE-4 and caspase-11 signaling and antibacterial immunity in hMϕ and in vivo . (A) MT3 and MT2A expression analyzed by qRT-PCR in hMϕ transfected with scramble siRNA or MT3 siRNA for 24h, 3 independent experiments, two-tailed t-test. (B) Scramble siRNA or MT3 siRNA treated hMϕ stimulated with iLPS (10 μg/ml) or vehicle for 48h. Immunoblots of pro-CASPASE-4 and active-CASPASE-4 in cell extracts, 3 independent experiments, one-way ANOVA. (C) Active-IL-1β measured by ELISA in supernatants of hMϕ treated as above, 3 independent experiments, one-way ANOVA. (D) E . coli growth inhibition in hMϕ transfected with MT3 siRNA and infected with 25 E . coli (K12): 1 hMϕ for 24h compared to scramble siRNA treated hMϕ, 3 independent experiments, two-tailed t-test. (E) E . coli growth inhibition in WT and Mt3 -/- BMDMϕ infected with 25 E . coli (K12):1 hMϕ for 24h, 4 independent experiments, two-tailed t-test. (F) WT and Mt3 -/- mice infected i.p. with 1X10 9 E . coli for 6h, log CFUs of E . coli in blood, kidney and peritoneal lavage samples, n = 12-15 per group, two-tailed t-test. (G) Western blots of inflammasome mediators in kidney homogenates of WT and Mt3 -/- mice infected as above, n = 6 per group, two-tailed t-test. (H) WT and Mt3 -/- mice infected i.p. with 1 X10 9 E . coli for 1h and IL-1β measured in peritoneal lavage and serum by ELISA. n = 3 per group, two-tailed t-test. (I) WT and Mt3 -/- mice primed i.p. with poly(I:C) (10 mg/kg) for 6h and challenged with LPS (2 mg/kg) i.p. After 18h, IL-1β was measured in peritoneal lavage and serum by ELISA, n = 3/group, two-tailed t-test. (J) Bacterial growth in spleen, lung and kidney of WT and Mt3 -/- mice infected i.n. with K. pneumoniae (4 X10 4 CFUs/mouse) for 48h, n = 8-12 per group, two-tailed t-test, data are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: MT3 , Applied Biosystems , Hs00359394_g1.

Techniques: In Vivo, Expressing, Quantitative RT-PCR, Transfection, Two Tailed Test, Western Blot, Enzyme-linked Immunosorbent Assay, Inhibition, Infection

Journal: Frontiers in Immunology

Article Title: Metallothionein 3-Zinc Axis Suppresses Caspase-11 Inflammasome Activation and Impairs Antibacterial Immunity

doi: 10.3389/fimmu.2021.755961

Figure Lengend Snippet: See also Supplementary Figure S3 Caspase-11 synergizes with MT3 in impairing bacterial clearance. WT, C asp-11 -/- , Mt3 -/- and Casp-11 -/- Mt3 -/- mice were infected i.p. with E . coli (1 X10 9 CFUs/mouse) for 6h. (A) Bacterial CFUs measured in kidney, blood and peritoneal lavage, n = 3-6 per group, one-way ANOVA. (B) Western blots of pro-GSDMD, active-GSDMD (p31), pro-caspase-1, active-caspase-1, pro-IL1β and active-IL-1β in kidney homogenates, n = 3-6 per group, one-way ANOVA, data are mean ± SEM. (C) WT and Mt3 -/- mice treated i.p. with MCC950 (1 mg/mouse) or PBS and infected i.p. with E . coli (1 X10 9 CFUs/mouse) for 6h. IL1β was measured by ELISA in peritoneal lavage, n = 6 per group, one-way ANOVA, data are mean ± SEM. Bacterial CFUs in whole blood and kidney, n = 4 per group, one-way ANOVA, data are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: MT3 , Applied Biosystems , Hs00359394_g1.

Techniques: Infection, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Metallothionein 3-Zinc Axis Suppresses Caspase-11 Inflammasome Activation and Impairs Antibacterial Immunity

doi: 10.3389/fimmu.2021.755961

Figure Lengend Snippet: See also Supplementary Figure S4 Myeloid-MT3 suppresses non-canonical inflammasome activation and blunts gram-negative bacterial clearance in vivo . (A) Generation of Mt3 fl/fl mice by inserting loxp sites flanking exon 3 of the Mt3 gene using the CRISPR-Cas9 gene targeting approach. Mt3 fl/fl mice crossed with Lys2Cre mice to obtain Lys2Cre Mt3 fl/fl mice. (B) Efficacy of myeloid Mt3 deletion assessed by genotyping peritoneal Mϕ (PMϕ) and BMDMϕ from Lys2Cre , Mt3 fl/fl and Lys2Cre Mt3 fl/fl mice. Gel electrophoresis analysis demonstrating efficient deletion of the Mt3 gene from BMDMϕ and PMϕ of Lys2Cre Mt3 fl/fl mice. (C) Western blots of pro-caspase-11, active-caspase-11, pro-GSDMD, active-GSDMD (p31), pro-caspase-1, active-caspase-1, pro-IL1β and active-IL-1β in whole kidney homogenates of mice infected as above, n = 3-5 per group, two-tailed t-test. (D) Bacterial CFUs in kidney and whole blood of Lys2Cre and Lys2Cre Mt3 fl/fl mice infected i.p. with E . coli (1 X10 9 CFUs/mouse) for 6h, n = 3-5 per group, two-tailed t-test, data are mean ± SEM. **p < 0.01, ***p < 0.001.

Article Snippet: MT3 , Applied Biosystems , Hs00359394_g1.

Techniques: Activation Assay, In Vivo, CRISPR, Nucleic Acid Electrophoresis, Western Blot, Infection, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Metallothionein 3-Zinc Axis Suppresses Caspase-11 Inflammasome Activation and Impairs Antibacterial Immunity

doi: 10.3389/fimmu.2021.755961

Figure Lengend Snippet: See also Supplementary Figure S5 MT3 thwarts TRIF-IRF3-STAT1 signaling to suppress non-canonical inflammasome activation. (A) Functional enrichment analysis of differentially expressed genes using RNA-seq data from resting WT and Mt3 -/- BMDMϕ (NCBI SRA: PRJNA533616) FDR, false detection rates. (B, C) Heat map (left) and table (right) show differentially expressed IFN-related genes in resting Mt3 -/- BMDMϕ compared to resting WT BMDMϕ obtained from RNA-seq analysis. (D) Western blots of pIRF3, pSTAT1, STAT1, GBP2 and GBP5 in vehicle or iLPS (10 μg/ml)-treated WT and Mt3 -/- BMDMϕ lysates, 3-4 independent experiments, one-way ANOVA. (E) Western blots of TRIF in lysates from WT and Mt3 -/- BMDMϕ stimulated as above, 3 independent experiments, one-way ANOVA. (F) Scramble and Ticam1 siRNA treated WT and Mt3 -/- BMDMϕ treated with iLPS (10 μg/ml) or vehicle for 48h. Immunoblots of TRIF (2 independent experiments), pro-caspase-11, and active-caspase-11 in lysates and active-IL-1β in supernatants, 3 independent experiments, one-way ANOVA, data are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001; NS, not significant.

Article Snippet: MT3 , Applied Biosystems , Hs00359394_g1.

Techniques: Activation Assay, Functional Assay, RNA Sequencing, Western Blot

Journal: Frontiers in Immunology

Article Title: Metallothionein 3-Zinc Axis Suppresses Caspase-11 Inflammasome Activation and Impairs Antibacterial Immunity

doi: 10.3389/fimmu.2021.755961

Figure Lengend Snippet: See also Supplementary Figures S6 , 7 MT3-Zn 2+ axis drives negative regulation of the non-canonical inflammasome. (A) SEC-ICP-MS of WT and Mt3 -/- BMDMϕ exposed to vehicle or iLPS (10 ug/ml) for the indicated time points, chromatograms depict Zn 2+ distribution in cell lysates across various molecular masses, arrow indicates Zn 2+ associated with the MT-peak (18-21 min.) on the chromatogram, Y axis is off-set to allow easy comparison under the same scale. (B) Bar graphs of total Zn 2+ and MT-Zn 2+ in WT and Mt3 -/- BMDMϕ post iLPS (10 μg/ml) or vehicle exposure. Two-way t-test against respective BMDMϕ controls at each time point, 3 independent experiments, data are mean ± SD. (C) WT BMDMϕ treated with iLPS (10 μg/ml) or vehicle for 24h in Zn 2+ sufficient or Zn 2+ deficient Opti-MEM media, immunoblots of pIRF3, pro-caspase-11, active-caspase-11 and pro-IL-1β in lysates and active-IL-1β in media supernatants, one-way ANOVA, data are mean ± SEM. (D, E) Mt3 -/- BMDMϕ transfected with Pro-Ject™ or Pro-Ject™ complexed with apo-MT3, 4Zn 2+ MT3 or 6Zn 2+ MT3 and treated with iLPS (10 μg/ml) or vehicle for 24h in Zn 2+ deficient Opti-MEM media. (D) Chromatograms depict Zn 2+ distribution in cell lysates across various molecular masses, arrow indicates Zn 2+ signal associated with the MT-peak (18-21 min.) on the chromatogram, Y axis is off-set to allow easy comparison under the same scale. (E) Western blots of pIRF3, pro-caspase-11, active-caspase-11 and pro-IL1β in lysates and active-IL-1β in supernatants, 3 independent experiments, one-way ANOVA, data are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001; NS, not significant.

Article Snippet: MT3 , Applied Biosystems , Hs00359394_g1.

Techniques: Comparison, Western Blot, Transfection

Journal: Frontiers in Immunology

Article Title: Metallothionein 3-Zinc Axis Suppresses Caspase-11 Inflammasome Activation and Impairs Antibacterial Immunity

doi: 10.3389/fimmu.2021.755961

Figure Lengend Snippet: Reagents and resources.

Article Snippet: MT3 , Applied Biosystems , Hs00359394_g1.

Techniques: Expressing, Plasmid Preparation, Control, Extraction, Blocking Assay, Injection, Cell Culture, Enzyme-linked Immunosorbent Assay, Transgenic Assay, Filtration, Transfection, Reverse Transcription, Cytotoxicity Assay, Isolation, Software, Imaging, Real-time Polymerase Chain Reaction

Journal: Cell reports

Article Title: The lactate-NAD + axis activates cancer-associated fibroblasts by downregulating p62

doi: 10.1016/j.celrep.2022.110792

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Mouse anti-PAR Polymer , Cell Signaling Technology , Cat# 4947; RRID: AB_823547.

Techniques: Polymer, Virus, Recombinant, Transfection, Western Blot, Infection, Electroporation, Quantitation Assay, Control, Staining, Real-time Polymerase Chain Reaction, Plasmid Preparation, Software, Imaging, Spectrophotometry, Microscopy

Journal: Cell reports

Article Title: The lactate-NAD + axis activates cancer-associated fibroblasts by downregulating p62

doi: 10.1016/j.celrep.2022.110792

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Human DU145 , ATCC , Cat# HTB-81, RRID: CVCL_0105.

Techniques: Polymer, Virus, Recombinant, Transfection, Western Blot, Infection, Electroporation, Quantitation Assay, Control, Staining, Real-time Polymerase Chain Reaction, Plasmid Preparation, Software, Imaging, Spectrophotometry, Microscopy