Journal: Oncogene

Article Title: Suppression of heparan sulfation re-sensitizes YAP1-driven melanoma to MAPK pathway inhibitors

doi: 10.1038/s41388-022-02400-z

Figure Lengend Snippet: a Fold change of mRNA levels of the 22 genes from the YAP1 signature in YAP 5SA melanoma cells compared to controls (mean + SD of two biological replicates per cell line is presented). b Correlation of drug sensitivity of BRAF V600E mutant melanoma cell lines ( n = 30) and expression of known sensitivity and resistance marker genes (marked by asterisks) as well as the YAP1/TAZ score ( p values from unpaired, two-tailed t -tests; BRAF V600E i, BRAF V600E inhibitor; MEKi, MEK inhibitor). c Correlation of sensitivity and resistance genes and the YAP1/TAZ score across the TCGA melanoma cohort (SKCM; p values from unpaired, two-tailed t -tests). d Comparison of YAP1/TAZ score of tumors of the TCGA melanoma cohort with low versus high MITF/AXL ratio ( n = 236, respectively; p value from unpaired, two-tailed t -test). e Correlation of sensitivity and resistance genes and the YAP1 score across a melanoma patient cohort ( n = 19) treated with BRAF and/or MEK inhibitors (asterisks indicate patient tumors treated with a combination of a BRAF and MEK inhibitor; DP, disease progressive). f Immunoblotting of indicated antibodies using protein lysates from melanoma cell lines with acquired MAPKi resistance. g YAP1 activity was determined in melanoma cell lines with acquired MAPKi resistance and engineered YAP 5SA cell lines using a luciferase reporter assay (mean + SD of at least six replicates are presented). h YAP1, TAZ, and simultaneous YAP1/TAZ CRISPR/Cas9-mediated knockout cell lines were generated and protein levels determined using immunoblotting with indicated antibodies (left). Cell lines were tested for sensitivity toward 2 µM vemurafenib compared to DMSO in eGFP competition assays mixed with SKMEL28R non-targeting controls, respectively (right). The eGFP ratio was determined at different time points as indicated using flow cytometry (mean ± SD of two replicates is presented; * p < 0.05, ** p < 0.01, *** p < 0.001. f – h These experiments have been performed at least two times independently, one representative experiment is shown; competition assay in h was performed once with two replicates).

Article Snippet: The following antibodies were used: YAP1 (#ab52771; abcam, Cambridge, United Kingdom; 1:1000 or #sc-101199; Santa Cruz; 1:1000), pMEK (#9154; CST, Danvers, MA, United States; 1:1000), MEK (#4694; CST; 1:1000), pEGFR (#44-788G; Thermo Fisher; 1:1000), EGFR (#2239; CST; 1:1000), pERBB3 (#4791; CST; 1:1000), ERBB3 (#12708; CST; 1:1000), pEPHA2 (#3970; CST; 1:1000), EPHA2 (#6997; CST; 1:1000), pAXL (#5724; CST; 1:1000), AXL (#8661; CST; 1:1000), HSP90 (#610418; BD Biosciences, Franklin Lakes, NJ, United States; 1:5000), GAPDH (#sc-32233; Santa Cruz; 1:2000), β-ACTIN (#A3854, Sigma-Aldrich; 1:1000), SOX10 (#ab155279; abcam; 1:000), MITF (#97800; CST; 1:1000), TAZ (#8418; CST; 1:1000), EXT1 (#sc-515144; Santa Cruz; 1:1000).

Techniques: Mutagenesis, Expressing, Marker, Two Tailed Test, Comparison, Western Blot, Activity Assay, Luciferase, Reporter Assay, CRISPR, Knock-Out, Generated, Flow Cytometry, Competitive Binding Assay

Journal: Oncogene

Article Title: Suppression of heparan sulfation re-sensitizes YAP1-driven melanoma to MAPK pathway inhibitors

doi: 10.1038/s41388-022-02400-z

Figure Lengend Snippet: Graph illustrating the mechanism by which suppression of SLC35B2 re-sensitizes YAP1-driven melanoma cells to MAPK pathway inhibitors. YAP1-driven RTK activation through upregulation of RTKs and/or ligands mediates Vemurafenib resistance, the latter relying on heparan sulfate proteoglycans (HSPGs) for efficient RTK activation. In this context, heparan sulfation becomes critical for sufficient HSPG expression and loss of SLC35B2 abrogates HSPG synthesis by decreasing the transport of PAPS from cytosol into the Golgi apparatus. As a consequence, activity of multiple RTKs including ERBB3, AXL, and in part also EGFR is diminished, which is associated with re-sensitization toward Vemurafenib. Unfractionated heparin (UFH) and low molecular weight heparin (LMWH) can interfere with the interaction of HS, RTKs, and their ligands and thereby inhibit ERBB3, AXL, and (in the presence of Vemurafenib) EGFR activity, which re-sensitizes resistant melanoma cells toward MAPKis. The pan-ERBB inhibitor Afatinib inhibits the ERBB family of RTKs and shows MAPKi re-sensitization in MITF low /AXL high melanoma cells that are characterized by high YAP1/TAZ activity.

Article Snippet: The following antibodies were used: YAP1 (#ab52771; abcam, Cambridge, United Kingdom; 1:1000 or #sc-101199; Santa Cruz; 1:1000), pMEK (#9154; CST, Danvers, MA, United States; 1:1000), MEK (#4694; CST; 1:1000), pEGFR (#44-788G; Thermo Fisher; 1:1000), EGFR (#2239; CST; 1:1000), pERBB3 (#4791; CST; 1:1000), ERBB3 (#12708; CST; 1:1000), pEPHA2 (#3970; CST; 1:1000), EPHA2 (#6997; CST; 1:1000), pAXL (#5724; CST; 1:1000), AXL (#8661; CST; 1:1000), HSP90 (#610418; BD Biosciences, Franklin Lakes, NJ, United States; 1:5000), GAPDH (#sc-32233; Santa Cruz; 1:2000), β-ACTIN (#A3854, Sigma-Aldrich; 1:1000), SOX10 (#ab155279; abcam; 1:000), MITF (#97800; CST; 1:1000), TAZ (#8418; CST; 1:1000), EXT1 (#sc-515144; Santa Cruz; 1:1000).

Techniques: Activation Assay, Expressing, Activity Assay, Molecular Weight

Journal: Cellular and Molecular Life Sciences

Article Title: Cdk5 regulates IP3R1-mediated Ca 2+ dynamics and Ca 2+ -mediated cell proliferation

doi: 10.1007/s00018-022-04515-8

Figure Lengend Snippet: Cdk5 −/− MEFs exhibit increased Nrf2 level, and scavenging ROS with mito-tempo or GSH prevents the increase in ROS and Nrf2 level in these cells. A Cdk5 −/− MEFs show upregulated expression of Nrf2 and its downstream targets, Prx1 and Prx2. Lysates of wt and Cdk5 −/− MEFs were analyzed by SDS-PAGE and immunoblotting for Cdk5, Nrf2, Prx1 and Prx2. Actin blot was used to assess protein loading. B Wt and Cdk5 −/− MEFs treated with an ROS scavenger, mito-tempo (10 µM) or GSH (10 µM), and then stained with 5 µM DCFDA for 30 min were examined for cytoplasmic ROS level by live-cell imaging using an Olympus I ×71 fluorescence microscope at 160 × magnification. Scale bar = 100 µm. C MEFs treated with mito-tempo or GSH were also analyzed by SDS-PAGE and immunoblotting for Nrf2. The graph (lower panel) shows the ratios of levels of Nrf2 vs actin calculated following densitometric analysis of blots using NIH Image J 1.61

Article Snippet: Mito-tempo and antibodies against Cdk5 (C-8), tubulin (D-10), IP3R1 (E-8), p21 CIP1 (L-17), Prx1 (N-19) and actin (I-19) were from Santa Cruz Biotech (Manassas, VA, USA).

Techniques: Expressing, SDS Page, Western Blot, Staining, Live Cell Imaging, Fluorescence, Microscopy

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Ginsenoside Rb1 Prevents Oxidative Stress-Induced Apoptosis and Mitochondrial Dysfunction in Muscle Stem Cells via NF- κ B Pathway

doi: 10.1155/2022/9159101

Figure Lengend Snippet: Ginsenoside Rb1 attenuated the decrease of mitochondrial content by promoting mitochondrial biogenesis. (a, b) Representative images of MitoTracker staining of C2C12 myoblasts in different groups. (c) Relative mRNA expression levels of mitochondrial DNA (normalized by nuclear DNA, n = 3 in each group). (d, e) Immunoblot detection of Tom20. (f, g) Immunoblot analyses of the genes related to mitochondrial biogenesis (Nrf1, PGC-1 α , TFEB). (h) Representative TEM images of C2C12 myoblasts from different groups. (i) The percentage of abnormal mitochondria in different groups. Data show mean ± SEM ( ∗ P < 0.05: control vs. other groups; & P < 0.05: H 2 O 2 vs. Rb1 groups).

Article Snippet: The C2C12 mouse myoblasts were cultured in confocal dishes and stained with JC-1 (JC-1 Mitochondrial Membrane Potential Assay Kit, Yeason, China), MitoTracker (MitoTracker Red CMXRos, Beyotime, Beijing, China), MitoSOX (MitoSOX Red Mitochondrial Superoxide Indicator, Yeason, Shanghai, China), or DCFH-DA (ROS Assay Kit, Beyotime, Beijing, China) according to the manufacturer's protocol.

Techniques: Staining, Expressing, Western Blot, Control

Journal: Oncogene

Article Title: Mitf is the key molecular switch between mouse or human melanoma initiating cells and their differentiated progeny.

doi: 10.1038/onc.2010.598

Figure Lengend Snippet: Figure 1 Tumorigenic properties and stem cell features increase in Mitf depleted mouse melanoma cells. (a) B16-F10 cells were grown in stem cell medium (SCM); the picture shows the melanospheres that arise after 96 h. Bar ¼ 80 mM. (b) 0.5 106 cells grown in normal condition (normal condition medium (NCM)) or in melanospheres (SCM) were injected subcutaneously, and tumor volume was monitored for 12 days (mean volume in mm3±s.e.m.). Picture of representative tumors at 12 days is shown (n ¼ 8 mice, *Po0.05). (c) Mitf, Oct4, Nanog and tyrosinase (Tyro) mRNA from NCM and SCM was measured by quantitative PCR; results are expressed as the mean of three independent experiments (fold induction±s.d.). Results were normalized to Gapdh. (d) Western blot analysis of Mitf, Oct4, Nanog and tyrosinase protein expression in cells grown in normal condition (NCM) or in melanosphere (SCM). One representative experiment is shown. (e) B16-F10 cells were transfected with siRNA against Mitf (siMitf) or with the scrambled sequence (siScr). Mitf expression was evaluated by immunofluorescence with anti-Mitf antibody (2–4). Nuclei were counterstained with DAPI (1–3). Bar ¼ 20 mM. (f). Relative mRNA levels expression of Mitf, Oct4, Nanog and tyrosinase were assayed by quantitative PCR. Results were normalized using Gapdh (fold induction±s.d.). (g) Western blot analysis of Mitf, Oct4, Nanog and tyrosinase protein expression in cells transfected with siRNA against Mitf (siMitf) or with the scrambled sequence (siScr). ERK2 was used as loading control. (h) Tumorigenicity of cells transfected with siScr or siMitf, was assessed by subcutaneous injection as in (b). Tumor volume was measured after 15 days of injection; results are expressed as the mean of volume in mm3±s.e.m. Pictures of representative tumors are displayed (n ¼ 8 mice, *Po0.05).

Article Snippet: Cells were transfected with siRNA against Mitf (Larribere et al., 2005), p27 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or a scrambled sequence using Lipofectamine RNAiMax (Invitrogen) according manufacturer protocol.

Techniques: Injection, Real-time Polymerase Chain Reaction, Western Blot, Expressing, Transfection, Sequencing, Control

Journal: Oncogene

Article Title: Mitf is the key molecular switch between mouse or human melanoma initiating cells and their differentiated progeny.

doi: 10.1038/onc.2010.598

Figure Lengend Snippet: Figure 2 Mitf depletion increases the number of melanoma initiating cells. (a) B16-F10 cells were labeled with 10 mM CFSE according to the manufacturer’s protocol and transfected with siScr (black line) or siMitf (filled in red). After 48 h, cells were analyzed by flow cytometry for CFSE intensity. Analysis of cells fixed immediately after the CFSE staining, (gray line) define the T0 CFSE intensity. Number of divisions and cell percentages for each division were determined according to CFSE intensity (M1–M6). (b) After CFSE labeling and siRNA transfection, 0.5 106 cells were injected into the mice. Four days later, mice were killed, tumors were dissociated with collagenase/dispase mix, and then analyzed by flow cytometry for CFSE intensity; siScr (black line) or siMitf (filled in red). The gray line shows the T0 CFSE intensity. Number of divisions and cell percentages for each division were determined according to CFSE intensity (M1–M9). (c) Western blot analysis of the B16 melanoma cells transfected with siScr or siMitf. (d) Graphic is displayed as the mean±s.d. of the number of initiating cells determined according to the method described in Materials and methods (data from Supplementary Table 1) (siScr- (white) or siMitf- (black) treated cells). (e) Extrapolation on day 16, graphic is displayed as the mean±s.d. of the number of cells that underwent six or more divisions for siScr- (white) or siMitf- (black) treated cells (data from Supplementary Table 2), *Po0.05.

Article Snippet: Cells were transfected with siRNA against Mitf (Larribere et al., 2005), p27 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or a scrambled sequence using Lipofectamine RNAiMax (Invitrogen) according manufacturer protocol.

Techniques: Labeling, Transfection, Cytometry, Staining, Injection, Western Blot

Journal: Oncogene

Article Title: Mitf is the key molecular switch between mouse or human melanoma initiating cells and their differentiated progeny.

doi: 10.1038/onc.2010.598

Figure Lengend Snippet: Figure 3 The p27kip1 upregulation is required for the induction of the MIC phenotype. (a) B16-F10 cells were transfected with siRNA to Mitf, p27 and/or appropriated scrambled siRNA. Lysates were probed for p27, Oct4 and Erk2 as loading control. (b) The same transfected cells (0.25 106 cells in 200 ml of phosphate-buffered saline) were injected subcutaneously. Tumor volume was measured 10 days after injection; results are expressed as the mean of volume in mm3±s.e.m. (c) Western blots analysis of B16-F10 infected with control or p27 expressing adenovirus. Membrane was probed for p27, Oct4, Nanog and Erk2 as loading control. (d) The same infected cells (0.25 106 cells in 200 ml of phosphate-buffered saline) were injected subcutaneously. Tumor volume was measured 15 days after injection; results are expressed as the mean of volume in mm3±s.e.m. Results are the mean±s.d. of three independent experiments; *Po0.05.

Article Snippet: Cells were transfected with siRNA against Mitf (Larribere et al., 2005), p27 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or a scrambled sequence using Lipofectamine RNAiMax (Invitrogen) according manufacturer protocol.

Techniques: Transfection, Control, Saline, Injection, Western Blot, Infection, Expressing, Membrane

Journal: Oncogene

Article Title: Mitf is the key molecular switch between mouse or human melanoma initiating cells and their differentiated progeny.

doi: 10.1038/onc.2010.598

Figure Lengend Snippet: Figure 4 A Mitf-negative population spontaneously exists in mouse melanoma cells and is required for tumor formation. (a) B16-F10 cells grown in Dulbecco’s modified Eagle’s medium 7% fetal calf serum were analyzed for Mitf content by flow cytometry. Mitf intensity was plotted on abscissa, and forward scatter (FSC) on ordinate. (b) Mitf-low (gray) and Mitf-high (white) cells were sorted on a FACSaria, as described in Materials and methods. RNAs, prepared from these cells, were used to evaluate, by quantitative PCR, the expression of Mitf, Oct4, Nanog and tyrosinase. Gapdh was used to normalize the values. (c) CFSE-labeled B16-F10 cells were grown for 72 h and sorted on a FACSAria for high-CFSE content (red, right part) and low-CFSE content (discontinuous black line, left part) (middle panel). Cells were then stained for Mitf (Mitf intensity) and analyzed by flow cytometry on a FACScan cytometer. One representative histogram of Mitf content for each condition is displayed (upper panel). Subcutaneous injection (20 103 cells) of high- and low-CFSE content cells was performed in C57BL/6J mice (n ¼ 6 per condition). Thirty days later, mice were killed, and tumor volume was measured. Results are expressed as the mean of tumor volume in mm3 (lower panel), **Po0.01.

Article Snippet: Cells were transfected with siRNA against Mitf (Larribere et al., 2005), p27 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or a scrambled sequence using Lipofectamine RNAiMax (Invitrogen) according manufacturer protocol.

Techniques: Cytometry, Real-time Polymerase Chain Reaction, Expressing, Labeling, Staining, Injection

Journal: Oncogene

Article Title: Mitf is the key molecular switch between mouse or human melanoma initiating cells and their differentiated progeny.

doi: 10.1038/onc.2010.598

Figure Lengend Snippet: Figure 5 Mitf regulates MIC transition in human melanoma cell lines. (a) Human cell lines 501-mel, MeWo and Sk Mel-28 were grown in stem cell medium (SCM); melanosphere (SCM) and adherent normal cultured cells (normal condition medium (NCM)). Expression of Mitf, Oct4 and p27 was analyzed by western blot. GAPDH was used as loading control. (b) The same cell lines were transfected for 48 h with siMitf or the scrambled sequence. Expression of Mitf, Oct4 and p27 was analyzed by western blot. GAPDH was used as loading control. (c) MeWo cells or (d) Sk Mel-28 cells, were transfected with siScr or siMitf for 48 h and injected subcutaneously (2 106 cells) into nude mice. Tumor volume was measured during 25 days; results are expressed as the mean of volume in mm3±s.e.m. (n ¼ 8 mice, *Po0.05). (e) MeWo cells infected with control or p27 expressing adenovirus were injected subcutaneously. Tumor volume was measured 25 days after injection; results are expressed as the mean of volume in mm3±s.e.m. Results are the mean±s.d. of three independent experiments; *Po0.05.

Article Snippet: Cells were transfected with siRNA against Mitf (Larribere et al., 2005), p27 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or a scrambled sequence using Lipofectamine RNAiMax (Invitrogen) according manufacturer protocol.

Techniques: Cell Culture, Expressing, Western Blot, Control, Transfection, Sequencing, Injection, Infection

Journal: Oncogene

Article Title: Mitf is the key molecular switch between mouse or human melanoma initiating cells and their differentiated progeny.

doi: 10.1038/onc.2010.598

Figure Lengend Snippet: Figure 6 A Mitf-negative population spontaneously exists in human melanoma cell lines and is required for human melanoma tumor formation in nude mice. (a) MeWo (upper panel) or SKmel-28 (lower panel) cells grown in Dulbecco’s modified Eagle’s medium 7% fetal calf serum were analyzed for Mitf content by flow cytometry. Mitf intensity was plotted on abscissa, and forward scatter (FSC) on ordinate. (b) (middle panel) CFSE-labeled SKmel-28 cells were grown for 72 h and sorted using FACSAria for high-CFSE content, (red, right part) and low-CFSE content (discontinuous black line, left part). Cells were then stained for Mitf (Mitf intensity) and analyzed by flow cytometry on a FACScan cytometer. One representative histogram of Mitf content for each condition is displayed (upper panel). In all 70 103 high-CFSE and low-CFSE cells were subcutaneously injected in nude mice (n ¼ 7). Sixty days later, mice were killed and tumor volume was measured. Results are expressed as the mean in mm3 of the tumor volume (lower panel), **Po0.01.

Article Snippet: Cells were transfected with siRNA against Mitf (Larribere et al., 2005), p27 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or a scrambled sequence using Lipofectamine RNAiMax (Invitrogen) according manufacturer protocol.

Techniques: Cytometry, Labeling, Staining, Injection

Journal: Oncogene

Article Title: Mitf is the key molecular switch between mouse or human melanoma initiating cells and their differentiated progeny.

doi: 10.1038/onc.2010.598

Figure Lengend Snippet: Figure 7 Mitf regulates MIC transition in freshly isolated human melanoma cells. (a) Freshly isolated human melanoma cells from skin (#1) or lymph node metastasis (#2) were transfected for 48 h with siMitf or the scrambled sequence. Expression of Mitf, Oct4 and p27 was analyzed by western blot. ERK2 was used as loading control. (b) In all 2 106 cells from patient #1 or #2 transfected with siScr or siMitf for 48 h were injected subcutaneously, and tumor volume was monitored for 25 days (mean volume in mm3±s.e.m., n ¼ 8 mice, *Po0.05). (c) Freshly isolated human melanoma cells were labeled with CFSE. After 72 h, cells were sorted using FACSAria for high or low CFSE content (as in Figure 6). In all 70 103 high-CFSE and low-CFSE cells were subcutaneously injected in right and left side of nude mice, respectively. One representative picture of mouse injected with CFSE-sorted melanoma cells is shown. (d) Sixty days later, mice were killed and tumors were counted.

Article Snippet: Cells were transfected with siRNA against Mitf (Larribere et al., 2005), p27 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or a scrambled sequence using Lipofectamine RNAiMax (Invitrogen) according manufacturer protocol.

Techniques: Isolation, Transfection, Sequencing, Expressing, Western Blot, Control, Injection, Labeling

Journal: The Journal of investigative dermatology

Article Title: Reduced WIF-1 expression stimulates skin hyperpigmentation in patients with melasma.

doi: 10.1038/jid.2012.270

Figure Lengend Snippet: Figure 3. The effects of Wnt inhibitory factor-1 (WIF-1) knockdown in keratinocytes on hyperpigmentation. (a) Real-time PCR and western blot analysis for the transfection of WIF-1 siRNA (siWIF-1) and control siRNA (siControl) in keratinocytes. Data represent means±SD of three and five independent experiments, respectively. *Po0.001 (b–d) Western blot analysis of tyrosinase (b) and Wnt signaling factors in total cell lysates (c) and in nuclear fractions (d) from melanocyte/keratinocyte co-cultures in which the keratinocytes had been transfected with siWIF-1 or siControl. Data represent means±SD of five independent experiments. *Po0.005 and Po0.05 for tyrosinase and Wnt signaling factors, respectively. (e) FACS analysis and immunofluorescence (X–Y two-dimensional (2D) image) study using anti-TYRP-1 (red) and anti-K14 antibodies (green) with or without WIF-1 knockdown. The cells (white arrow), which contained various amount of TYRP-1-positive particles around/over the nuclei (blue), indicate positive cells. Data represent means±SD of three independent experiments. *Po0.05. FL (binding ability to fluorochromes) 1: FITC; FL2: phycoerythrin. GSK-3b, glycogen synthase kinase-3b; JNK, c-Jun N-terminal kinase; K14, cytokeratin 14; MITF, microphthalmia-associated transcription factor; NFATc2, nuclear factor of activated T cells, cytoplasmic, calcineurin-dependent 2; p, phospho; PKC, protein kinase C; siRNA, small interfering RNA; t, total; TYRP-1, tyrosinase-related protein-1. Bar¼ 50mm.

Article Snippet: The membranes were incubated with antibodies to b-catenin, phospho-b-catenin, GSK3b, phospho-GSK3b, JNK, phospho-JNK, PKC-d/y, phospho-PKC-d/ y, WIF-1, and NFATc2 (rabbit polyclonal; Cell Signaling Technology, Beverly, MA), phospho-NFATc2, MITF (rabbit polyclonal; Santa Cruz Biotechnology, Santa Cruz, CA), tyrosinase (goat polyclonal; Santa Cruz Biotechnology), and Wnt-1 (rabbit polyclonal; Epitomics, Burlingame, CA).

Techniques: Knockdown, Real-time Polymerase Chain Reaction, Western Blot, Transfection, Control, Immunofluorescence, Binding Assay, Small Interfering RNA

Journal: The Journal of investigative dermatology

Article Title: Reduced WIF-1 expression stimulates skin hyperpigmentation in patients with melasma.

doi: 10.1038/jid.2012.270

Figure Lengend Snippet: Figure 4. Effects of Wnt inhibitory factor-1 (WIF-1) knockdown in fibroblasts on tyrosinase expression and melanosome transfer. (a) Real-time PCR and western blot analysis for the transfection of WIF-1 siRNA (siWIF-1) and control siRNA (siControl) in fibroblasts. Data represent means±SD of three and five independent experiments, respectively. *Po0.005. (b, c) Western blot analysis of (b) tyrosinase and (c) Wnt signaling factors in keratinocyte/melanocyte co- cultures that were cultured three-dimensionally with fibroblasts transfected with or without WIF-1 knockdown. Data represent means±SD of five independent experiments.(*Po0.005 and Po0.05 for tyrosinase and Wnt signaling factors, respectively. (d) FACS analysis using anti-TYRP-1 and anti-K14 antibodies with or without WIF-1 knockdown. Data represent means±SD of three independent experiments. *Po0.001. FL (binding ability to fluorochromes) 1: FITC; FL2: phycoerythrin. GSK-3b, glycogen synthase kinase-3b; JNK, c-Jun N-terminal kinase; K14, cytokeratin 14; MITF, microphthalmia-associated transcription factor; NFATc2, nuclear factor of activated T cells, cytoplasmic, calcineurin-dependent 2; p, phospho; PKC, protein kinase C; siRNA, small interfering RNA; t, total; TYRP-1, tyrosinase-related protein-1.

Article Snippet: The membranes were incubated with antibodies to b-catenin, phospho-b-catenin, GSK3b, phospho-GSK3b, JNK, phospho-JNK, PKC-d/y, phospho-PKC-d/ y, WIF-1, and NFATc2 (rabbit polyclonal; Cell Signaling Technology, Beverly, MA), phospho-NFATc2, MITF (rabbit polyclonal; Santa Cruz Biotechnology, Santa Cruz, CA), tyrosinase (goat polyclonal; Santa Cruz Biotechnology), and Wnt-1 (rabbit polyclonal; Epitomics, Burlingame, CA).

Techniques: Knockdown, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Transfection, Control, Cell Culture, Binding Assay, Small Interfering RNA

Journal: The Journal of investigative dermatology

Article Title: Reduced WIF-1 expression stimulates skin hyperpigmentation in patients with melasma.

doi: 10.1038/jid.2012.270

Figure Lengend Snippet: Figure 5. The effect of Wnt inhibitory factor-1 (WIF-1) overexpression on melanogenesis and melanosome transfer. (a) Western blot analysis of WIF-1 expression following treatment of keratinocytes (KCs), melanocytes (MCs), or keratinocyte/melanocyte co-cultures (Co-culture) with recombinant human WIF-1 (rhWIF-1). Data represent means±SD of five independent experiments. *Po0.05. (b, d) Western blot analysis of (b) tyrosinase and (c) Wnt signaling factors in total cell lysates from co-cultures and/or in (d) nuclear fractions from monocultures of melanocytes simultaneously treated with or without rhWIF-1. Data represent means±SD of three independent experiments. *Po0.001 and Po0.05 for tyrosinase and Wnt signaling factors, respectively. (e) FACS analysis using anti-TYRP-1 and anti-K14 antibodies in keratinocyte/melanocyte co-cultures treated with or without rhWIF-1. Data represent means±SD of three independent experiments. *Po0.005. FL (binding ability to fluorochromes) 1: FITC; FL2: phycoerythrin. GSK-3b, glycogen synthase kinase-3b; JNK, c-Jun N-terminal kinase; K14, cytokeratin 14; MITF, microphthalmia-associated transcription factor; NFATc2, nuclear factor of activated T cells, cytoplasmic, calcineurin-dependent 2; p, phospho; PKC, protein kinase C; siRNA, small interfering RNA; t, total; TYRP-1, tyrosinase-related protein-1.

Article Snippet: The membranes were incubated with antibodies to b-catenin, phospho-b-catenin, GSK3b, phospho-GSK3b, JNK, phospho-JNK, PKC-d/y, phospho-PKC-d/ y, WIF-1, and NFATc2 (rabbit polyclonal; Cell Signaling Technology, Beverly, MA), phospho-NFATc2, MITF (rabbit polyclonal; Santa Cruz Biotechnology, Santa Cruz, CA), tyrosinase (goat polyclonal; Santa Cruz Biotechnology), and Wnt-1 (rabbit polyclonal; Epitomics, Burlingame, CA).

Techniques: Over Expression, Western Blot, Expressing, Co-Culture Assay, Recombinant, Binding Assay, Small Interfering RNA

Journal: The Journal of investigative dermatology

Article Title: Reduced WIF-1 expression stimulates skin hyperpigmentation in patients with melasma.

doi: 10.1038/jid.2012.270

Figure Lengend Snippet: Figure 6. Expression of Wnts in the skin of melasma patients. (a) Real-time PCR analysis of Wnt-1 and Wnt-5A mRNA expression in hyperpigmented (L) and in normally pigmented (N) skin specimens from 13 patients with melasma. *Po0.05. (b) Western blot analysis of tyrosinase and Wnt signaling factors in cultured melanocytes treated with recombinant human Wnt-1 (rhWnt-1). Data represent means±SD of three independent experiments. *Po0.05. GSK-3b, glycogen synthase kinase-3b; JNK, c-Jun N-terminal kinase; MITF, microphthalmia-associated transcription factor; NFATc2, nuclear factor of activated T cells, cytoplasmic, calcineurin-dependent 2; p, phospho; PKC, protein kinase C; t, total.

Article Snippet: The membranes were incubated with antibodies to b-catenin, phospho-b-catenin, GSK3b, phospho-GSK3b, JNK, phospho-JNK, PKC-d/y, phospho-PKC-d/ y, WIF-1, and NFATc2 (rabbit polyclonal; Cell Signaling Technology, Beverly, MA), phospho-NFATc2, MITF (rabbit polyclonal; Santa Cruz Biotechnology, Santa Cruz, CA), tyrosinase (goat polyclonal; Santa Cruz Biotechnology), and Wnt-1 (rabbit polyclonal; Epitomics, Burlingame, CA).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Cell Culture, Recombinant

Journal: Theranostics

Article Title: TRPM2 protects against cisplatin-induced acute kidney injury and mitochondrial dysfunction via modulating autophagy.

doi: 10.7150/thno.84655

Figure Lengend Snippet: Figure 4. Mitochondrial ROS scavenger Mito-TEMPO protects mice from cisplatin-induced kidney injury and mitochondrial damage. (A) Schematic diagram of the experimental design. Briefly, Trpm2-/- and WT mice were injected daily with either Mito-TEMPO (Mito-T, 7 mg/kg) or vehicle one week before administration of cisplatin (CP, 18 mg/kg) until being sacrificed at 3 days. (B) Kidney function assessed by the serum creatinine level (n = 8). (C, D) Representative PAS staining images and quantification of tubular injury score (n = 8). Scale bars, 100 μm. (E, F) Representative images of TUNEL staining and quantification of apoptotic cells (n = 8). Scale bars, 100 μm. (G, H) Representative confocal images of Kim-1 staining and quantification of Kim-1 positive cells (n = 8). Scale bars, 100 μm. (I) Immunoblotting analysis and quantification of BAX and cleaved and total caspase-3 (CASP3) in mice. (J) Representative TEM images showing mitochondrial morphology in the renal tubules. Scale bars, 1 μm. (K, L) Representative confocal images of DHE staining and quantification of DHE positive cells (n = 8). Scale bars, 100 μm. (M, N) Representative immunohistochemistry images of 8-OHdG staining and quantification of 8-OHdG positive cells (n = 8). Scale bars, 100 μm. Data are presented as mean±SEM. Statistical analysis was performed using one-way ANOVA with Tukey post-hoc test. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Clotrimazole (CLT, 10 μM, Target Mol, T0506), 2-APB (20 μM, Sigma-Aldrich, D9754), ADPR (100 μM, MedChemExpress, HY-100973A), chloroquine (CQ, 10 μM, MedChemExpress, HY-17589A), Mito-TEMPO (200 nM, MedChemExpress, HY-112879), BAPTA-AM (5 μM, MedChemExpress, HY-100545), 3-methyladenine (3-MA, 5 mM, Selleck, S2767), VIII (5 μM, Beyotime, SF2784) and rapamycin (50 nM, MedChemExpress, HY-10219) were added to cell culture medium 1 h before incubation with cisplatin.

Techniques: Injection, Staining, TUNEL Assay, Western Blot, Immunohistochemistry

Journal: Theranostics

Article Title: TRPM2 protects against cisplatin-induced acute kidney injury and mitochondrial dysfunction via modulating autophagy.

doi: 10.7150/thno.84655

Figure Lengend Snippet: Figure 8. TRPM2 protects against cisplatin-induced cell apoptosis and mitochondrial dysfunction in vitro by the regulation of autophagy via suppressing the AKT-mTOR signaling pathway. Trpm2-/- MEFs or mRTECs were pre-incubated with AKT inhibitor VIII (5 μM) or mTOR inhibitor rapamycin (Rapa, 50 nM) for 1 h before cisplatin (CP) intervention. (A) Immunoblotting analysis and quantification of LC3B II, p-AKT and p-p70S6K in Trpm2-/- and WT mRTECs following treatment with 5 μM CP for 24 h. (B) Representative confocal images of LC3 puncta in Trpm2-/- and WT MEFs expressing mRFP-GFP-LC3 following treatment with 20 μM CP for 24 h. Scale bars, 20 μm. (C) Quantification of autophagosomes (yellow dots, RFP+GFP+) and autolysosomes (red dots, RFP+GFP-) (n = 4). (D, E) Representative confocal images and quantification of mitolysosomes evaluated by immunofluorescence double-labeled TOM20 and LAMP2 in mRTECs (n = 4). Scale bars, 20 μm. Then, Trpm2-/- mRTECs and MEFs were pre-incubated with rapamycin (50 nM), Mito-TEMPO (Mito-T, 200 nM) or Rapamycin (50 nM) plus 3-MA (5 mM) for 1 h followed by intervention of 5 and 20 μM CP for 24 h, respectively. (F) CP-induced apoptosis in mRTECs determined by flow cytometry (n = 3). (G) Level of mitochondrial ROS in mRTECs determined by the mean fluorescent intensity (MFI) of Mito-SOX (n = 3). (H) Mitochondrial membrane potential of mRTECs indicated by the ratio of red to green fluorescence intensity of JC-1 (n = 3). (I) Mitochondrial morphology of MEFs detected by the fluorescence of MitoTracker Red. Scale bars, 20 μm. Data are presented as mean±SEM. Statistical analysis was performed using one-way ANOVA with Tukey post-hoc test. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Clotrimazole (CLT, 10 μM, Target Mol, T0506), 2-APB (20 μM, Sigma-Aldrich, D9754), ADPR (100 μM, MedChemExpress, HY-100973A), chloroquine (CQ, 10 μM, MedChemExpress, HY-17589A), Mito-TEMPO (200 nM, MedChemExpress, HY-112879), BAPTA-AM (5 μM, MedChemExpress, HY-100545), 3-methyladenine (3-MA, 5 mM, Selleck, S2767), VIII (5 μM, Beyotime, SF2784) and rapamycin (50 nM, MedChemExpress, HY-10219) were added to cell culture medium 1 h before incubation with cisplatin.

Techniques: In Vitro, Incubation, Western Blot, Expressing, Immunofluorescence, Labeling, Flow Cytometry, Membrane, Fluorescence

Journal: FEBS letters

Article Title: MRPS27 is a pentatricopeptide repeat domain protein required for the translation of mitochondrially encoded proteins.

doi: 10.1016/j.febslet.2012.07.043

Figure Lengend Snippet: Fig. 5. MRPS27 affects mitochondrial protein synthesis. (A) MRPS27 knockdown lowers mitochondrial translation in cells. 143B cells were transfected with MRPS27 siRNAs, NT siRNAs, pMRPS27-TAP and pEYFP-TAP and protein synthesis was measured by pulse incorporation of 35S-labelled methionine and cysteine. Equal amounts of cell lysate protein were separated by SDS–PAGE and visualised by autoradiography. The gels were stained with Coomassie to confirm equal loading (Supplementary Fig. 5) (B) Protein isolated from mitochondria was analysed by immunoblotting after knockdown and overexpression of MRPS27. The blot was re- probed with antibodies against other mitochondria encoded (COXII) and nuclear encoded (COXIV) cytochrome c oxidase subunits, and porin was used as a loading control. (C) BN-PAGE of mitochondrial proteins from cells after knockdown and overexpression of MRPS27. (D) Ribosomal stability is not affected by decreasing MRPS27 levels in cells. Immunoblot of mitochondria isolated from 143B cells transfected with MRPS27 and NT siRNAs and blotted for markers of the small ribosomal subunit (MRPS15) or the large ribosomal subunit (MRPL11). (E) Cytochrome c oxidase activity normalised to citrate synthase for MRPS27 knock- down and overexpression. ⁄p <0.05.

Article Snippet: Specific proteins were detected using mouse monoclonal antibodies: COXI, COXII and COXIV (Mito Sciences, diluted 1:1000), and polyclonal rabbit antibodies: MRPL11 (Cell Signaling Technologies, diluted 1:1000), MRPS27 (Proteintech, diluted 1:500), MRPS15 (Abcam, diluted 1:1000) in 5% skim milk powder in phosphate-buffered saline (PBS).

Techniques: Knockdown, Transfection, SDS Page, Autoradiography, Staining, Isolation, Western Blot, Over Expression, Control, Activity Assay

Journal: bioRxiv

Article Title: An unconventional mechanism of IL-1β secretion that requires Type I IFN in lupus monocytes

doi: 10.1101/2023.08.03.551696

Figure Lengend Snippet: a, Confocal images (left) and quantification (right) of classical Mo isolated from JDM and SLE patients. Each dot represents the average count from at least ten different micrscopy fields from a study participant sample that was processed, stained and analyzed independently. Scale bar: 20 µm. b, Percentage of IL-1β − MxA + Mo and IL-1β + MxA + Mo in SLE patients without (blue, n=8) or with (red, n=6) circulating Mito + RBCs. c, Confocal images of classical Mo isolated from SLE patients showing evidence of erythrophagocytosis. GPA: Glycophorin A. Scale bar: 20 µm. d, Two-tailed Pearson’s correlation between SLEDAI and the percentage of IL-1β + ISGs + Mo (n=20). Dashed lines represent 95% confidence intervals. Each dot represents a study participant sample that was processed, stained and analyzed independently.

Article Snippet: To generate Mito + RBCs, MG132 (5 mM; Santa Cruz Biotech) was added to the culture during phase 3.

Techniques: Isolation, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: An unconventional mechanism of IL-1β secretion that requires Type I IFN in lupus monocytes

doi: 10.1101/2023.08.03.551696

Figure Lengend Snippet: a, Phagocytosis of CFSE-labeled RBCs in the presence or absence of IgG opsonizing antibodies. b, Quantification of phagocytosis of CFSE-labeled Mito - or Mito + RBCs by blood Mo. (n=3). c, Immunoblot analysis of whole cell lysate (WCL) and supernatants (Sup) from blood Mo that were treated as described. One representative of two experiments. d, Levels of IP-10 and IL-1β in the supernatants of bone marrow (BM) Mo cultured with media, Mito - or Mito + opsonized RBCs. (n=3). e, Phenotype of BLaER1 cells before (Day 1) and after (Day 6) differentiation into BLaER1 Mo. f, Quantification of phagocytosis of CFSE-labeled Mito - or Mito + RBCs by BLaER1 Mo. (n=3). g, Immunoblot analysis of WCL and Sup from BLaER1 Mo that were treated as described. One representative of two. h, Relative mtDNA abundance in Mito + RBCs generated with vehicle, EtBr (ρ 0 ) or CAM. (n=3). i, Quantification of phagocytosis of CFSE-labeled Mito + RBCs RBCs generated with vehicle or EtBr (ρ 0 ). (n=3). j, Western blot analysis of Mito + RBCs generated with vehicle, EtBr (ρ 0 ) or CAM. One representative of two.

Article Snippet: To generate Mito + RBCs, MG132 (5 mM; Santa Cruz Biotech) was added to the culture during phase 3.

Techniques: Labeling, Western Blot, Cell Culture, Generated

Journal: bioRxiv

Article Title: An unconventional mechanism of IL-1β secretion that requires Type I IFN in lupus monocytes

doi: 10.1101/2023.08.03.551696

Figure Lengend Snippet: a, Heat map of differentially expressed genes in blood Mo that phagocytized either Mito - or Mito + RBCs. Data are normalized to Mito - RBCs sample . b, Levels of IP-10 and IL-1β in the supernatants of blood Mo cultured with media, Mito - or Mito + RBCs. (n=6). c, Heat map of differentially expressed genes in BLaER1 Mo that phagocytized Mito - or Mito + RBCs. Data are normalized to Mito - RBCs sample . d, Levels of IP-10 and IL-1β in the supernatants of BLaER1 Mo cultured with media, Mito - or Mito + RBCs. (n=4). e, Experiment scheme (left) and normalized cytokine levels (right) in the supernatants of BLaER1 Mo activated with Mito + RBCs generated in the presence of vehicle, ethidium bromide (EtBr; ρ 0 ) or chloramphenicol (CAM). (n=4).

Article Snippet: To generate Mito + RBCs, MG132 (5 mM; Santa Cruz Biotech) was added to the culture during phase 3.

Techniques: Cell Culture, Generated

Journal: bioRxiv

Article Title: An unconventional mechanism of IL-1β secretion that requires Type I IFN in lupus monocytes

doi: 10.1101/2023.08.03.551696

Figure Lengend Snippet: a, Normalized IP-10 levels in the supernatants of Mito + RBCs-activated wild type, cGAS KOor STING1 KO BLaER1 Mo. (n=3). b, Normalized IL-1β levels in the supernatants of Mito + RBCs-activated wild type or Caspase-1 KO BLaER1 Mo. (n=3). c, Experimental scheme (left), dot/Western blot analysis (middle) and quantification (left; n=3) of Mito + RBCs (1 - 3) or BLaER1 Mo (2 − 4) labeled with bromodeoxyuridine (BrdU) and then immunoprecipitated (IP) with cGAS or NLRP3 antibodies. As a loading control, cGAS or NLRP3 immunoblot or double-stranded DNA (dsDNA) dot blot (DB) was performed. A.U.: Arbitrary Units.

Article Snippet: To generate Mito + RBCs, MG132 (5 mM; Santa Cruz Biotech) was added to the culture during phase 3.

Techniques: Western Blot, Labeling, Immunoprecipitation, Control, Dot Blot

Journal: bioRxiv

Article Title: An unconventional mechanism of IL-1β secretion that requires Type I IFN in lupus monocytes

doi: 10.1101/2023.08.03.551696

Figure Lengend Snippet: a, Western blot analysis confirming CRISPR/Cas9-mediated KO of cGAS and STING1. b, Normalized IP-10 levels in the supernatants of Mito + RBC-activated BLaER1 Mo in the presence of the cGAS inhibitor RU.521 or the STING inhibitor H151. (n=3). c, Western blot analysis of phosphorylated TBK1 and IRF3 in BLaER1 Mo cultured with media or Mito + RBCs. d, Western blot analysis confirming CRISPR/Cas9-mediated KO of AIM2. e, Normalized IL-1β levels in the supernatants of Mito + RBCs-activated wild type or AIM2 KO BLaER1 Mo. (n=3). f, Flow cytometry analysis showing the internalization of unlabeled (control) or AlexaFluor 488 (AF488)-labeld IgG upon electroporation. g, Percentages of cell death measured by 7AAD incorporation. h, Western blot analysis confirming Trim-Away knockdown of AIM2 (left). Normalized IL-1β levels in the supernatants of activated BLaER1 Mo upon Trim-Away knockdown of AIM2 (right). (n=3). i, Western blot analysis confirming CRISPR/Cas9-mediated KO of Caspase-1. j, Normalized IL-1β levels in the supernatants of Mito + RBCs-activated BLaER1 Mo in the presence of the NLRP3 inhibitor MCC950 or the caspase-1 inhibitor Ac-YVAD-cmk. (n=4). k, Phagocytosis of CFSE-labeled RBCs in wild type, cGAS KO, STING1 KO or caspase-1 KO BLaER1 Mo. (n=3).

Article Snippet: To generate Mito + RBCs, MG132 (5 mM; Santa Cruz Biotech) was added to the culture during phase 3.

Techniques: Western Blot, CRISPR, Cell Culture, Flow Cytometry, Control, Electroporation, Knockdown, Labeling

Journal: bioRxiv

Article Title: An unconventional mechanism of IL-1β secretion that requires Type I IFN in lupus monocytes

doi: 10.1101/2023.08.03.551696

Figure Lengend Snippet: a, Normalized cytokine levels in the supernatants of blood Mo activated with Mito + RBCs generated in the presence of vehicle, EtBr (ρ 0 ) or CAM. b, Normalized cytokine levels in the supernatants of Mito + RBC-activated blood Mo in the presence of RU.521, H151, MCC950 or Ac-YVAD-cmk. (n=3). c, Normalized cytokine levels in the supernatants of BM Mo activated with Mito + RBCs generated in the presence of vehicle, EtBr (ρ 0 ) or CAM. d, Normalized cytokine levels in the supernatants of Mito + RBCs-activated BM Mo in the presence of RU.521, H151, MCC950 or Ac-YVAD-cmk. (n=3).

Article Snippet: To generate Mito + RBCs, MG132 (5 mM; Santa Cruz Biotech) was added to the culture during phase 3.

Techniques: Generated

Journal: bioRxiv

Article Title: An unconventional mechanism of IL-1β secretion that requires Type I IFN in lupus monocytes

doi: 10.1101/2023.08.03.551696

Figure Lengend Snippet: a, Western blot analysis of cytosolic fraction obtained from media, Mito + RBCs- or dsDNA/Lyovec-activated BLaER1 Mo. WCL: whole cell lysate. Quantification of lactate dehydrogenase (LDH) release (b) or propidium iodide (PI) internalization (c) in BLaER1 Mo activated Mito + RBCs or dsDNA/Lyovec over time. Data are normalized to media-treated cells. (n=3). d, Western blot analysis of ASC oligomers (pyroptosome) formation in BLaER1 Mo activated Mito + RBCs or dsDNA/Lyovec for 18 h. Triton X-100 soluble (TX100 Sol ) and insoluble (TX100 Ins ) fractions were immunoblotted with ASC antibody. One representative of two experiments. e, Normalized IL-1β levels in the supernatants of Mito + RBC- or dsDNA/Lyovec-activated BLaER1 Mo in the presence of the lysosmal acidification inhibitor Bafilomycin A1 (BAf A1). (n=3). f, Normalized IL-1β levels in the supernatants of Mito + RBC- or dsDNA/Lyovec-activated BLaER1 Mo in the presence of extracellular K + . (n=3).

Article Snippet: To generate Mito + RBCs, MG132 (5 mM; Santa Cruz Biotech) was added to the culture during phase 3.

Techniques: Western Blot

Journal: bioRxiv

Article Title: An unconventional mechanism of IL-1β secretion that requires Type I IFN in lupus monocytes

doi: 10.1101/2023.08.03.551696

Figure Lengend Snippet: a, Experiment scheme (left) and normalized cytokine levels (right) in the supernatants of BLaER1 Mo, generated in the presence of vehicle, EtBr (ρ 0 ) or CAM and activated with Mito + RBCs. (n=4). b, Relative levels of mtDNA-encoded genes in cGAS (left) or NLRP3 (right) immunocomplexes isolated from Mito + RBC-activated wild type or MAVS KO BLaER1 Mo. (n=3). c, Experiment scheme (left) and normalized cytokine levels (right) in the supernatants of BLaER1 Mo cultured with Mito + RBCs generated in the presence of Actinomycin D (Act D). (n=4). d, Normalized IL-1β levels in the supernatants of Mito + RBCs- or Poly I:C/Lyovec-activated wild type, MAVS KO or RIG- I/MDA5 KO BLaER1 Mo. (n=3).

Article Snippet: To generate Mito + RBCs, MG132 (5 mM; Santa Cruz Biotech) was added to the culture during phase 3.

Techniques: Generated, Isolation, Cell Culture

Journal: bioRxiv

Article Title: An unconventional mechanism of IL-1β secretion that requires Type I IFN in lupus monocytes

doi: 10.1101/2023.08.03.551696

Figure Lengend Snippet: a, Western blot analysis confirming CRISPR/Cas9-mediated KO of mitochondrial DNA polymerase gamma (POLG; Polγ). b, Normalized IL-1β levels in the supernatants of Mito + RBC-activated wild type or POLG KO BLaER1 Mo. (n=3). c, DB analysis of cGAS or NLRP3 immunocomplexes isolated from Mito + RBCs-activated BLaER1 Mo. As a loading control, dsDNA dot blot (DB) was performed. d, 8-OHdG quantification by ELISA in cGAS or NLRP3 immunocomplexes isolated from Mito + RBC-activated BLaER1 Mo. (n=3). e, Experimental scheme (left) and DB analysis (right) of IP cGAS or NLRP3 upon incubation with biotinylated intact (Int) or fragmented (Fgt) mtDNA. As a loading control, cGAS or NLRP3 immunoblot was performed. f, SDD-AGE of MAVS oligomerization in BLaER1 Mo in response to Mito + RBCs (generated with or without Act D) or to transfection with Poly I:C. One representative of three. g, Western blot analysis confirming CRISPR/Cas9-mediated KO of MAVS. h, Relative caspase-1 activity in the lysate of Mito + RBC-activated wild type, MAVS or RIG-I/MDA5 KOBLaER1 Mo. (n=3). i, Normalized IL-1β levels in the supernatants of Poly I:C/Lyovec-activated BLaER1 Mo in the presence of MCC950 or Ac-YVAD-cmk or that were generated in the presence of EtBr. (n=3). j, Relative levels of mtDNA-encoded genes in NLRP3 immunocomplexes isolated from Poly I:C/Lyovec-activated wild type or MAVS KO BLaER1 Mo. (n=3). k, Relative caspase-1 activity in the lysate of Poly I:C/Lyovec-activated wild type, MAVS or RIG-I/MDA5 KO BLaER1 Mo. (n=3).

Article Snippet: To generate Mito + RBCs, MG132 (5 mM; Santa Cruz Biotech) was added to the culture during phase 3.

Techniques: Western Blot, CRISPR, Isolation, Control, Dot Blot, Enzyme-linked Immunosorbent Assay, Incubation, Generated, Transfection, Activity Assay

Journal: bioRxiv

Article Title: An unconventional mechanism of IL-1β secretion that requires Type I IFN in lupus monocytes

doi: 10.1101/2023.08.03.551696

Figure Lengend Snippet: a, Western blot analysis confirming CRISPR/Cas9-mediated KO of RIG-I or MDA5. b, Normalized IL-1β levels in the supernatants of Mito + RBCs or Poly I:C/Lyovec-activated wild type, RIG-I KO or MDA5 KO BLaER1 Mo. (n=3). c, Western blot analysis confirming CRISPR/Cas9-mediated KO of DHX33. d, Normalized IL-1β levels in the supernatants of Mito + RBCs or Poly I:C/Lyovec-activated wild type or DHX33 KO BLaER1 Mo. (n=3). e, Western blot analysis confirming CRISPR/Cas9-mediated KO of both RIG-I and MDA5. Relative levels of mtDNA-encoded genes in NLRP3 immunocomplexes isolated from Mito + RBCs (f) or Poly I:C/Lyovec (g) activated wild type or RIG-I/MDA5 KO BLaER1 Mo. (n=3).

Article Snippet: To generate Mito + RBCs, MG132 (5 mM; Santa Cruz Biotech) was added to the culture during phase 3.

Techniques: Western Blot, CRISPR, Isolation

Journal: bioRxiv

Article Title: An unconventional mechanism of IL-1β secretion that requires Type I IFN in lupus monocytes

doi: 10.1101/2023.08.03.551696

Figure Lengend Snippet: a, Western blot analysis of gasdermin-D N-terminal fragment (GSDMD-NT) in total cell lysate isolated from Mito + RBCs, Poly I:C/Lyovec and Nigericin-activated BLaER1 Mo. b , Quantification of lactate dehydrogenase (LDH) release or propidium iodide (PI) internalization in BLaER1 Mo activated Mito + RBCs, Poly I:C/Lyovec or Nigericin over time. Data are normalized to media-treated cells. (n=3). c, Western blot analysis confirming CRISPR/Cas9-mediated KO of GSDMD (left). Normalized IL-1β levels in the supernatants from Mito + RBCs, Poly I:C/Lyovec and Nigericin-activated wild type or GSDMD KO BLaER1 Mo (right). (n=3). Confocal images of classical Mo isolated from SLE patients and stained for IL-1β and ISG15 (d) or IFIT1 (e). Scale bar: 15 µm.

Article Snippet: To generate Mito + RBCs, MG132 (5 mM; Santa Cruz Biotech) was added to the culture during phase 3.

Techniques: Western Blot, Isolation, CRISPR, Staining

Journal: bioRxiv

Article Title: An unconventional mechanism of IL-1β secretion that requires Type I IFN in lupus monocytes

doi: 10.1101/2023.08.03.551696

Figure Lengend Snippet: a, Experimental scheme (left) and Western blot analysis (right) of the microsomal pellet (100k P) and postmicrosomal supernatant (100k S) isolated from Poly I:C/Lyovec-activated BLaER1 Mo. b , Western blot analysis of different fractions collected after sucrose fractionation of microsomal membranes isolated from Poly I:C/Lyovec-activated BLaER1 Mo. ER: endoplasmic reticulum; ERGIC: ER-Golgi intermediate compartment. c, Western blot analysis of the 100k P and 100k S fractions isolated from Poly I:C/Lyovec-activated wild type or MxA KOBLaER1 Mo. d, Normalized IL-1β levels in the supernatants from Mito + RBCs, Poly I:C/Lyovec and dsDNA/Lyovec-activated wild type or MxA KO BLaER1 Mo. (n=3).

Article Snippet: To generate Mito + RBCs, MG132 (5 mM; Santa Cruz Biotech) was added to the culture during phase 3.

Techniques: Western Blot, Isolation, Fractionation

Journal: bioRxiv

Article Title: An unconventional mechanism of IL-1β secretion that requires Type I IFN in lupus monocytes

doi: 10.1101/2023.08.03.551696

Figure Lengend Snippet: a, Immunoblot analysis of mIL-1β present within liposomes (pellet), or supernatants (sup) after ultracentrifugation of liposomes that were treated with buffer or recombinant human MxA for 0 (control), 30 and 120 min. Densitometry quantification of Western blot band density for mIL-1β release from liposomes is also shown. (n=3). b, Quantification of LDH enzymatic activity release from Lp loaded with LDH and treated with buffer or MxA for indicated times. (n=3). c, Normalized IL-1β levels in the supernatants from Mito + RBCs-activated BLaER1 Mo with the indicated genotype. (n=3). Immunoblot analysis of the total cell lysate is also shown. d, Western blot analysis confirming Trim-Away knockdown of MxA (left). Normalized IL-1β levels in the supernatants of activated BLaER1 Mo upon Trim-Away knockdown of MxA (right). (n=3). e, Western blot analysis of whole cell lysate (WCL) and supernatants (Sup) isolated from BLaER1 Mo overexpressing mIL-1β that were treated with or without recombinant Type I IFN (IFNα2β). One representative of two.

Article Snippet: To generate Mito + RBCs, MG132 (5 mM; Santa Cruz Biotech) was added to the culture during phase 3.

Techniques: Western Blot, Liposomes, Recombinant, Control, Activity Assay, Knockdown, Isolation

Journal: bioRxiv

Article Title: An unconventional mechanism of IL-1β secretion that requires Type I IFN in lupus monocytes

doi: 10.1101/2023.08.03.551696

Figure Lengend Snippet: (1) In SLE patients with active disease, internalization of Mito + RBCs induces Mo to co-express ISGs and mIL-1β. ISG expression depends on cGAS activation by Mito + RBC-derived mtDNA (2), while mIL-1β production requires NLRP3 activation. Upstream of NLRP3, Mito + RBC-derived mt dsRNA activates the RLRs/MAVS pathway, which induces the release of Mo-derived mtDNA fragments that bind NLRP3 (3). Importantly, mIL-1β triggers the oligomerization of MxA on the surface or the TGN (4), which favors the entry of this cytokine into a TGN-mediated unconventional secretory (vesicular?) pathway (5). (6) Cytosolic dsRNA of microbial origin (i.e. Poly I:C/Lyovec) could also activate these pathways via MAVS/IRF3.

Article Snippet: To generate Mito + RBCs, MG132 (5 mM; Santa Cruz Biotech) was added to the culture during phase 3.

Techniques: Expressing, Activation Assay, Derivative Assay

Journal: Shock (Augusta, Ga.)

Article Title: Adenosine 5’-monophosphate protects from hypoxia by lowering mitochondrial metabolism and oxygen demand

doi: 10.1097/SHK.0000000000001440

Figure Lengend Snippet: A-B, SH-SY5Y cells expressing the mitochondrial Ca2+ sensor mito-CAR-GECO1 were treated with the indicated concentrations of AMP or ATP and changes in mitochondrial Ca2+ levels were recorded by fluorescence imaging. C-F, SH-SY5Y cells (C-D) or mouse hippocampal neurons (E-F) were labeled with the cytosolic Ca2+ indicator Fluo-4 and changes in cytosolic Ca2+ levels after addition of ATP or AMP were analyzed by fluorescence microscopy. Fluorescence traces of one representative experiment (mean + SD) are shown in A (n=5–9 cells), C (n=20–25 cells) and E (n=8–14 cells). Averaged mitochondrial Ca2+ changes measured 15 s after addition of AMP and cytosolic Ca2+ changes measured 10 s after ATP or 50 s after AMP treatment are shown in B, D, and F. (B: mean + SD; n=10–50 cells from 3–8 separate experiments; D: mean + SD, n=70–117 cells from 4–6 separate experiments; F: mean + SD, n=15–30 cells from 3 separate experiments; *p<0.05 vs. control, Kruskal-Wallis test).

Article Snippet: Mitochondrial Ca 2+ uptake was assessed in SH-SY5Y cells expressing the mitochondrial Ca 2+ biosensor mito-CAR-GECO1 (plasmid 46022, Addgene, Cambridge, MA) ( 18 ).

Techniques: Expressing, Fluorescence, Imaging, Labeling, Microscopy

Journal: Shock (Augusta, Ga.)

Article Title: Adenosine 5’-monophosphate protects from hypoxia by lowering mitochondrial metabolism and oxygen demand

doi: 10.1097/SHK.0000000000001440

Figure Lengend Snippet: A, SH-SY5Y cells were stained with JC-1 and the change in mitochondrial membrane potential (Δψm) after addition of adenosine (ADO; 1 mM), AMP (20 mM), CCCP (10 μM), or vehicle control was assessed by fluorescence microscopy. B, Δψm was assessed in JC-1-stained SH-SY5Y cells 1.5 min after addition of CCCP (10 μM), AMP (20 mM), or adenosine. C, SH-SY5Y cells expressing the mitochondrial Ca2+ indicator mito-CAR-GECO1 were treated with CCCP (10 μM), AMP (10 mM), the A2a receptor agonist CGS21680 (100 nM), the unspecific P2 receptor antagonist suramin (100 μM), the ATP release blocker CBX (10 μM), or cell culture medium (control). The change in mitochondrial Ca2+ 1 min after treatment was analyzed by fluorescence imaging. D-F, SH-SY5Y cells expressing the mitochondrial Ca2+ sensor mito-CAR-GECO1 were stimulated with ATP (10 μM), UTP (10 μM), AMP (10 mM), AMP+ATP, AMP+UTP, or vehicle control and analyzed by fluorescence microscopy. Data in A and B are means ± SD of 2 separate experiments (n≥6 analyzed cells per experiment). Data in C and F show averaged results of the change in fluorescence 10 s after treatment (mean + SD, n=24–44 cells from n≥4 different experiments). D-E, Calcium traces of one representative experiment (mean + SD, n≥3 cells); *p<0.05 vs. control (Kruskal-Wallis and Dunn’s test).

Article Snippet: Mitochondrial Ca 2+ uptake was assessed in SH-SY5Y cells expressing the mitochondrial Ca 2+ biosensor mito-CAR-GECO1 (plasmid 46022, Addgene, Cambridge, MA) ( 18 ).

Techniques: Staining, Fluorescence, Microscopy, Expressing, Cell Culture, Imaging

Journal: Shock (Augusta, Ga.)

Article Title: Adenosine 5’-monophosphate protects from hypoxia by lowering mitochondrial metabolism and oxygen demand

doi: 10.1097/SHK.0000000000001440

Figure Lengend Snippet: A-B, SH-SY5Y cells were treated with the indicated concentrations of AMP for 10 min (A) or with 10 mM AMP for the indicated times (B) and concentrations of intracellular adenine compounds were determined by HPLC. Data are shown as mean ± SEM (A: n=4, B: n=3). C, SH-SY5Y cells were treated with AMP (10 mM) for the indicated times and mTORC1, P70S6K, and AMPK activity were measured using phosphospecific antibodies and immunoblotting. MAPK p38 was used as a loading control. A representative immunoblot and averaged normalized results (mean + SD, n=3) are shown. D, SH-SY5Y cells expressing the mitochondrial Ca2+ indicator mito-CAR-GECO1 were treated with CCCP (10 μM), AMP (10 mM), the AMPK agonist AICAR (500 μM), or cell culture medium (control). Mitochondrial Ca2+ uptake was monitored by fluorescence imaging and the relative change in mitochondrial Ca2+ levels 1 min after the addition of drugs was determined and normalized to the control cells. Data are mean values + SD of single cells (n≥28) analyzed in 4 separate experiments; *p<0.05 vs. control (one-way ANOVA).

Article Snippet: Mitochondrial Ca 2+ uptake was assessed in SH-SY5Y cells expressing the mitochondrial Ca 2+ biosensor mito-CAR-GECO1 (plasmid 46022, Addgene, Cambridge, MA) ( 18 ).

Techniques: Activity Assay, Western Blot, Expressing, Cell Culture, Fluorescence, Imaging