Journal: Nature cell biology

Article Title: Non-canonical function of IRE1α determines mitochondria-associated endoplasmic reticulum composition to control calcium transfer and bioenergetics

doi: 10.1038/s41556-019-0329-y

Figure Lengend Snippet: a, IRE1α KO cells reconstituted with IRE1α–HA were processed to obtain purified MAM fractions followed by western blot analysis of indicated proteins (n = 3 independent experiments). H, homogenate; C, cytosol; Cr, crude mitochondria; M, MAMs; P, pure mitochondria; Cyt c, cytochrome c; CNX, calnexin. b, Liver extracts were processed to obtain subcellular fractions enriched for MAMs and analysed by western blot (n = 9 independent experiments). c,d, IRE1α KO cells reconstituted with IRE1α–HA or mock control were simultaneously imaged for calcium signals in the cytosol (Fura2; c) and mitochondria with Rhod2 (d). Left, the Fura2 ratio (c) and mean Rhod2 intensity (d) of normalized data before and after ATP is added; arrow, 100 μM ATP. Right, the data for the maximum peak are shown (total cells analysed: mock, n = 116 cells; IRE1α–HA, n = 138 cells). e,f, Similar experiments for Fura2 (e) and Rhod2 (f) were performed in CRISPR control and IRE1α KO cells (total cells analysed: control, n = 129 cells; IRE1α KO, n = 117 cells). WT, wild type. g, Indicated cell lines were processed for western blot analyses to monitor the levels of indicated proteins (n = 4 independent experiments). h, IRE1α null and control cells were imaged for calcium levels in mitochondria by transiently expressing CEPIA2mt mitochondrial calcium probe (left) after addition of 50 μM M3M3FBS (arrow), (Mito red; Mitochondrila Ds-Cherry control). Scale bars, 10 μm. Right, maximum CEPIA2mt intensity for every cell analysed (mock, n = 14 cells; IRE1α–HA, n = 14 cells). i, Maximum peaks from Fura2/Rhod2 measurements from samples described in c and d were calculated using nonlinear regression analyses to determine the correlation constant (K) and s.e.m. (mock, K = 0.199 ± 0.009; IRE1α–HA, K = 0.231 ± 0.01). j, Cells were imaged for calcium levels in the ER after loading with Mag-Fluo4 in permeabilized cells followed by stimulation with InsP3R (n = 5 independent experiments; left). Middle, percentage activity for InsP3R for each condition normalized to maximum release (ionomycin). Right, the first derivative was calculated. Data in c–f,h–j are mean ± s.e.m. Statistical differences were detected using two-tailed unpaired Student’s t-tests except for j; right, which was one-tailed. Source data for statistical analyses are provided in Supplementary Table 6.

Article Snippet: Alternatively, we generated CRISPR cells using a double nickase that was targeted to IRE1α or scrambled as a control (sc-429758-NIC and sc-437281; Santa Cruz).

Techniques: Purification, Western Blot, CRISPR, Expressing, Activity Assay, Two Tailed Test, One-tailed Test

Journal: Nature cell biology

Article Title: Non-canonical function of IRE1α determines mitochondria-associated endoplasmic reticulum composition to control calcium transfer and bioenergetics

doi: 10.1038/s41556-019-0329-y

Figure Lengend Snippet: a, Ern1 and Ern1ΔK liver samples were processed for metabolomics studies (n = 4 animals per group). The heat map for the metabolites indicates significantly different metabolite levels in each experimental animal. b, Pathway analysis and statistical significance (two-tailed Student’s t-test) for the metabolites shown in a. c, The affected pathways and main hits from a are indicated. Altered metabolites and their associated pathways are indicated using the same colour code (coloured dots, size stands for P value as in b)) in a–c. d, Whisker and dot plots of the indicated metabolites of the TCA, indicating median and quartiles derived from samples in a (n = 4 animals per group) levels represent the log2 of the normalized area in a.u. e, Schematic of the TCA cycle. Metabolites with increased or decreased levels in Ern1 and Ern1ΔK samples are indicated by arrows. f, Glucose tolerance test in Ern1 control and Ern1ΔK mice (left). Right, data represent the area under the curve (AUC) for the whole glucose tolerance test (n = 4 animals per group). g, Proposed model: IRE1α expressed at MAMs docks the InsP3Rs at the mitochondrial–ER contact sites—possibly through a physical interaction, which may enhance InsP3R channel activity. The presence of IRE1α at MAMs favours calcium transfer into the mitochondria and bursts in ATP production. IRE1α deficiency leads to a metabolic stress condition that is characterized by the constitutive activation of AMPK, enhanced compensatory autophagy and altered mitochondrial morphology. Data are mean ± s.e.m. Statistical differences were detected with one-tailed (d) or two-tailed Student’s t-tests (f) or two-way ANOVA (f). Source data for statistical analyses are provided in Supplementary Table 6.

Article Snippet: Alternatively, we generated CRISPR cells using a double nickase that was targeted to IRE1α or scrambled as a control (sc-429758-NIC and sc-437281; Santa Cruz).

Techniques: Two Tailed Test, Whisker Assay, Derivative Assay, Activity Assay, Activation Assay, One-tailed Test

Journal: Nature cell biology

Article Title: Non-canonical function of IRE1α determines mitochondria-associated endoplasmic reticulum composition to control calcium transfer and bioenergetics

doi: 10.1038/s41556-019-0329-y

Figure Lengend Snippet: a, IRE1α KO cells that were reconstituted with either IRE1α–HA or an empty vector (mock) were imaged for TMRM signals before and after addition of 1 μM FCCP (carbonyl cyanide-p-trifluoromethoxyphenylhydrazone) (left). Scale bar, 20 μm. Right, mean TMRM intensity normalized to IRE1α–HA cells (n = 6 independent experiments). b, CRISPR control and IRE1α KO cells were analysed as described in a (n = 4 independent experiments). c,d, Percentage of ATP of the indicated cells using a luminescence assay (n = 18 biologically independent samples). e,f, ATP levels were measured in the indicated cell lines using the AT01 mitochondrial (yellow fluorescent protein (YFP)/cyan fluorescent protein (CFP)) FRET probe FRET labeling stands for 440 nm excitation emmited in YFP channel. White numbers indicate regions of interest (left). Right, quantification of YFP/CFP ratio excited at 440 nm (mock, n = 52 cells; IRE1α–HA, n = 58 cells; control, n = 145 cells; IRE1α KO, n = 151 cells). Scale bars, 10 μm and 2 μm. g,h, The indicated cell lines were analysed for oxygen consumption rate (OCR). O, 1 μM oligomycin, F, 0.5 μM FCCP; A/R = 1 μM antimycin/rotenone (n = 4 independent experiments). i, pAMPK was analysed in the indicated cells using western blots (left) and normalized to total AMPK levels (right; n = 6 independent experiments). j, Determination of LC3-II levels in the indicated cell lines using western blots (left), followed by quantification normalizing to actin (right; n = 6 independent experiments). k, TEM-derived morphological parameters of mitochondria were obtained from indicated cells. Scale bar, 4 μm (left). Right, the data represent the area in μm2 and circularity (mock, n = 52 cells; IRE1α–HA, n = 58 cells). l, Cells were stained for ERp72 and TOM20 by indirect immunofluorescence (left) followed by colocalization quantification (right; Mander’s index: mock, n = 33 cells; IRE1α–HA, n = 40 cells; Pearson’s index: mock, n = 68 cells; IRE1α–HA, n = 78 cells). Scale bar, 20 μm and 5 μm. m, The indicated cells were imaged using TEM to visualize MAMs (pointed with red arrows) (left) using two quantification methods (right; mock, n = 38 contacts; IRE1α–HA, n = 30 contacts). Scale bars, 500 nm. Data in a–m are mean ± s.e.m. Statistical differences detected with one-tailed (k) or two-tailed unpaired Student’s t-tests. A Wilcoxon signed-rank test was applied in a–d and paired Student’s t-tests were applied in h,i,m (right panel). Source data for statistical analyses are provided in Supplementary table 6.

Article Snippet: Alternatively, we generated CRISPR cells using a double nickase that was targeted to IRE1α or scrambled as a control (sc-429758-NIC and sc-437281; Santa Cruz).

Techniques: Plasmid Preparation, CRISPR, Luminescence Assay, Labeling, Western Blot, Derivative Assay, Staining, Immunofluorescence, One-tailed Test, Two Tailed Test

Journal: Nature cell biology

Article Title: Non-canonical function of IRE1α determines mitochondria-associated endoplasmic reticulum composition to control calcium transfer and bioenergetics

doi: 10.1038/s41556-019-0329-y

Figure Lengend Snippet: a, Indicated cells were processed to obtain subcellular fractions and analysed using western blots. Right, quantification of the MAM fractions for the indicated proteins (InsP3R1, n = 4; InsP3R1, n = 8 independent experiments). b,c, Cells described in a were stained with a PLA (red) and DAPI (Blue) using anti-InsP3R1 (b) or anti-InsP3R3 (c) antibodies paired with anti-VDAC1 antibodies. Scale bars, 20 μm (left). Right, quantification of the number of positive PLA dots per cell (n = 3 independent experiments). d, Cells were imaged using TEM (left) to calculate ER to mitochondrial width (right; n = 3 independent experiments; mock, n = 46 contacts; IRE1α–HA, n = 30 contacts). Scale bars, 200 nm. e, CRISPR control and IRE1α KO cells were were imaged using TEM (left) to calculate ER to mitochondrial width (right; n = 3 independent experiments; CRISPR control, n = 27 contacts; CRISPR IRE1α KO, n = 47 contacts). Scale bars, 200 nm. f, The same cells as described in a were transiently transfected with SPLICSL to visualize MAMs with a width of 40–50 nm (left). Nuclei were stained with DAPI. Scale bar, 25 μm. Right, quantification of SPLICSL signal as dots per cell (n = 5 independent experiments; total cells analysed: mock, n = 41 cells; IRE1α–HA, n = 38 cells). g, Schematic representation and representative TEM images of indicated cells transiently expressing either a AKAP1 (34–63)-linker 9x-mRFP (9xL) construct or a control linker construct. Scale bar, 500 nm (top). MAM width was determined by TEM (bottom; mock control linker, n = 14 contacts; IRE1α–HA control linker, n = 12 contacts; mock 9xL, n = 15 contacts). h, Cells described in g were stained with PLA (green) and DAPI (blue) to measure the close proximity between InsP3R1 and VDAC1 proteins (left) in mRFP positive cells. Right, the number of dot counts per cell was quantified (n = 4 independent experiments) Scale bars, 20 μm. Data in a–h are mean ± s.e.m. Statistical differences were detected using one-way ANOVA and Tukey post-tests for multiple comparison (g,h), two-tailed Student’s t-tests (b–f) or Wilcoxon signed-rank test (a). Source data for statistical analyses are provided in Supplementary Table 6.

Article Snippet: Alternatively, we generated CRISPR cells using a double nickase that was targeted to IRE1α or scrambled as a control (sc-429758-NIC and sc-437281; Santa Cruz).

Techniques: Western Blot, Staining, CRISPR, Transfection, Expressing, Construct, Two Tailed Test

Journal: Nature cell biology

Article Title: Non-canonical function of IRE1α determines mitochondria-associated endoplasmic reticulum composition to control calcium transfer and bioenergetics

doi: 10.1038/s41556-019-0329-y

Figure Lengend Snippet: a, Schematic of IRE1α structure and the mutants analysed (left) (TM; transmembrane domain). Right, the indicated cell lines were treated with 0.1 μg ml−1 tunicamycin for 4 h and then Xbp1 mRNA splicing was evaluated using PCR analysis. The agarose gel image was sliced to eliminate irrelevant lanes. Xbp1u, unspliced Xbp1s, spliced (n = 2 independent experiments). b, Calcium levels in the cytosol after ATP stimulation were analysed in IRE1α KO cells reconstituted with the indicated constructs. Arrow, 100 μM ATP (left; Fura2; n = 4 independent experiments; total cells analysed: mock, n = 131 cells; IRE1α–HA, n = 149 cells; IRE1α-P830L–HA, n = 120 cells; IRE1α-ΔC–HA, n = 97 cells). The maximum peak for the normalized Fura2 ratio was measured (middle). The same cells were imaged simultaneously with Rhod2 to measure mitochondrial calcium uptake. Arrow, 100 μM ATP (right two panels). c–e, HEK293T cells were transiently transfected with the indicated constructs and immunoprecipitation (IP) was performed using anti-HA antibodies. Western blot (WB) analysis was performed for the indicated proteins in immunoprecipitations and total input (c, n = 3 independent experiments; d,e are representative of two independent experiments). f, The indicated MEF cell lines were processed for immunoprecipitation using anti-HA antibodies. Western blot analysis was performed for the indicated proteins in immunoprecipitations and total input. g, Cells described in f were stained for PLA (red) and DAPI (blue) using anti-InsP3R1 antibodies paired with anti-HA antibodies and analysed by confocal microscopy. Scale bar, 20 μm (left). Right, the number of dots per cell were quantified (n = 3 independent experiments). h, Schematic of InsP3R1 domains used to generate recombinant proteins and perform in vitro pull-down assays (left; the asterisk indicates that residues 167–169 and 267 are relevant for channel function). Right, in vitro pull-down assay for purified GST-fused domains of InsP3R1 with recombinant IRE1α cytosolic portion (IRE1α-ΔN) followed by western blot analysis (D1, domain 1; D2, domain 2; D3, domain 3; n = 3 independent experiments). Data in b and g are mean ± s.e.m. Statistical differences were detected using two-tailed unpaired Student’s t-test (g) or ANOVA with Tukey multiple comparison test (b). Source data for statistical analyses are provided in Supplementary Table 6.

Article Snippet: Alternatively, we generated CRISPR cells using a double nickase that was targeted to IRE1α or scrambled as a control (sc-429758-NIC and sc-437281; Santa Cruz).

Techniques: Agarose Gel Electrophoresis, Construct, Transfection, Immunoprecipitation, Western Blot, Staining, Confocal Microscopy, Recombinant, In Vitro, Pull Down Assay, Purification, Two Tailed Test

Journal: Nature cell biology

Article Title: Non-canonical function of IRE1α determines mitochondria-associated endoplasmic reticulum composition to control calcium transfer and bioenergetics

doi: 10.1038/s41556-019-0329-y

Figure Lengend Snippet: a, Strategy to generate CRa particles using the synergistic activator mediators and sgRNA complex. b, IRE1α KO cells were generated that stably express either a CRa that targets the InsP3R1 promoter or a control vector. Representative western blot analysis of the indicated proteins was performed to confirm InsP3R1 upregulation (n = 10 independent experiments). c, The cells described in a were stained with a PLA (red) DAPI (blue) using anti- InsP3R1 and anti-VDAC1 antibodies, and were analysed by confocal microscopy. Scale bar, 20 μm (left). Right, the number of dots per cell were quantified (n = 6 independent experiments). d, CRa-InsP3R1 or CRacontrol cells were imaged with Rhod2 to measure mitochondrial calcium uptake. Arrow, stimulation using 50 μM M3M3FBS (left). Right, the maximum peak for normalized Rhod2 was calculated (total cells analysed: CRa-InsP3R1, n = 46 cells; CRa-control, n = 42 cells). e, CRa-InsP3R1 or CRa-control cells were imaged for mitochondrial membrane potential after TMRM staining (left). Arrow, stimulation with 1 μM FCCP; AU, arbitrary units. Right, normalized TMRM intensity (n = 4 independent experiments). f, pAMPK and total AMPK levels were determined in CRa-InsP3R1 or CRa-control cells using western blot (left). Right, quantification of the pAMPK/AMPK ratio (n = 7 independent experiments). g, The indicated cells were lysed and ATP levels were determined using a luminescence assay (n = 13 biologically independent experiments). h, IRE1α KO cells were generated that stably express either a CRa that targets the InsP3R3 promoter or a control vector. Representative western blot analysis of the indicated proteins was performed to confirm InsP3R3 upregulation (n = 5 independent experiments). i, The indicated cells were imaged with Rhod2. Arrow, stimulation with 50 μM M3M3FBS (left). Right, the maximum peak for the normalized Rhod2 was calculated (total cells analysed: CRa-InsP3R1, n = 132 cells; CRa-control, n = 112 cells). j, ATP levels were determined in the indicated cells using a luminescence assay (n = 25 biologically independent experiments). Data in b–j are mean ± s.e.m. Statistical differences were detected with unpaired Student’s t-tests (c,d,g,i,j) or Wilcoxon signed-rank test (b,e,f,h). Source data for statistical analyses are provided in Supplementary Table 6.

Article Snippet: Alternatively, we generated CRISPR cells using a double nickase that was targeted to IRE1α or scrambled as a control (sc-429758-NIC and sc-437281; Santa Cruz).

Techniques: Generated, Stable Transfection, Plasmid Preparation, Western Blot, Staining, Confocal Microscopy, Luminescence Assay

Journal: Nature cell biology

Article Title: Non-canonical function of IRE1α determines mitochondria-associated endoplasmic reticulum composition to control calcium transfer and bioenergetics

doi: 10.1038/s41556-019-0329-y

Figure Lengend Snippet: a, Schematic of Ern1 structure (the gene encoding IRE1α) (TM; transmembrane domain) and the strategy to delete the kinase domain (Ern1ΔK; left). Middle, livers from Ern1 control and Ern1ΔK mice were processed for western blot analysis to measure the levels of indicated proteins (n = 3 independent experiments). Right, mice were intraperitoneally injected with 1 mg kg−1 of tunicamycin or vehicle for 6 h. Xbp1s mRNA splicing was evaluated by RT–PCR analysis of cDNA obtained from total liver extracts. b–f, Ern1 and Ern1ΔK livers were processed for TEM analysis (b) to determine morphological parameters including mitochondrial area (arrows indicate MAMs, scale bar, 500 nm) (c), MAM width (d), mitochondrial circularity (e) and MAM length (f; n = 4 animals per group). g, Ern1 and Ern1ΔK MAM fractions were processed for quantitative mass spectrometry analysis (see Methods). The volcano plot shows all of the detected proteins (grey) and those that are known to be present at MAMs (red dots; n = 3 animals per group). h, A summary of statistically significant hits observed in the proteomic screening of MAMs. i,j, Ern1 and Ern1ΔK liver samples were processed to obtain subcellular fractions, and were analysed by western blot for the indicated proteins (i). Quantification of protein expression was performed for the indicated proteins by normalizing to calnexin (CNX; j; Ern1, n = 9 animals; Ern1ΔK, n = 7 animals). k, The protein content (in mg) from liver MAM fractions (left) or pure mitochondria (right) was quantified and normalized by total liver extract (in g) to obtain the percentage of MAM proteins in the liver (Ern1, n = 6 animals; Ern1ΔK, n = 4 animals). l, Liver homogenates from Ern1 and Ern1ΔK were immunoprecipitated for IRE1α and analysed for the indicated proteins by western blot. Ab, antibody (representative of three independent experiments). Data in c–f,j and k are mean ± s.e.m. Statistical differences were detected using two-tailed unpaired Student’s t-tests (c,d). For j,k one-tailed Student’s t-tests were applied. Source data for statistical analyses are provided in Supplementary Table 6.

Article Snippet: Alternatively, we generated CRISPR cells using a double nickase that was targeted to IRE1α or scrambled as a control (sc-429758-NIC and sc-437281; Santa Cruz).

Techniques: Western Blot, Injection, Reverse Transcription Polymerase Chain Reaction, Mass Spectrometry, Expressing, Immunoprecipitation, Two Tailed Test, One-tailed Test

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: CD44 mediates shear stress mechanotransduction in an in vitro blood-brain barrier model through small GTPases RhoA and Rac1

doi: 10.1096/fj.202100822RR

Figure Lengend Snippet: Expression of CD44 regulates barrier formation. (A) Western blot of CD44 and beta-actin in CRISPR-modified cells. The ratio of intensities of CD44 and beta-actin signals normalized to scrambled levels. *Indicates p < .05 compared to scrambled and upregulated conditions. (B) Permeability coefficients of the channels seeded with transfected cells after 4 days of culture * denotes p < .05 compared to all conditions. (C) TEER measurements taken over the course of 4 days for different conditions, * indicates p < .05 compared to Day 1 timepoint for all conditions. (D,E) Fluorescent images of channels stained with DAPI (blue), anti-ZO-1 (red) (isolated in ii), and anti-adducin-γ (isolated in iii) for two conditions: scrambled control cells in collagen/HA hydrogels exposed to flow (D) and knockout cells in collagen/HA hydrogels exposed to flow (E). Scale: 50 μm. (F) Relative intensity (RQ) of RhoA activation in channels measured with ELISA. (G) Relative intensity (RQ) of Rac1 activation in channels measured with ELISA. *Indicates p < .05

Article Snippet: Commercially available CRISPR plasmids (Santa Cruz) were used to alter the expression of CD44 within the hCMEC/D3 cells prior to introduction into the 3D BBB model. To knockout CD44 in the cells, two plasmids encoding a D10A mutated Cas9 nuclease and a CD44-specific 20 nucleotide guide RNA were transfected into cells (Santa Cruz, sc-400209-NIC).

Techniques: Expressing, Western Blot, CRISPR, Modification, Permeability, Transfection, Staining, Isolation, Control, Knock-Out, Activation Assay, Enzyme-linked Immunosorbent Assay

Journal: Advances in Clinical and Experimental Medicine

Article Title: mGluR5 promotes the progression of multiple myeloma in vitro via Ras–MAPK signaling pathway

doi: 10.17219/acem/130445

Figure Lengend Snippet: Fig. 1. mGluR5 had higher expression in multiple myeloma (MM) cell lines. A. Quantitative real-time polymerase chain reaction (qRT-PCR) was applied to evaluate the mGluR5 expression in MM cell lines and MM1S, OPM-2, U266, NCI-H929, and RPMI-8226, and normal cell line CD143; B,D. MM1S cell line was treated with dimethyl sulfoxide (DMSO) as a control group and 3,5-dihydroxyphenylglycine (DHPG) as an agonist group. The qRT-PCR was used to measure mGluR5 RNA expression in each group in MM1S cell line. Western blot was used to measure mGluR5 protein expression in each group in MM1S cell line; C,E. OPM2 cell line was treated with DMSO as a control group, 2-methyl-6-(phenylethynyl)-pyridine (MPEP) or 3-[(2-methyl-1,3- thiazol-4-yl)ethynyl]pyridine (MTEP) as antagonist groups. The qRT-PCR was used to measure mGluR5 RNA expression in each group in the MM1S cell line. Western blot was used for mGluR5 protein expression in each group in MM1S cell line. Each assay was performed thrice independently. Kruskal–Wallis test (K–W) with Dunn’s post hoc test and Kolmogorov–Smirnov test (K–S) were applied in the statistical analysis (Fig. 1A. human umbilical vein endothelial cells (HUVECs) compared to RPMI82226, p = 0.0197; HUVECs compared to U266, p = 0.0324; HUVECs compared to OPM2, p = 0.0029; HUVECs compared to MM1S, p = 0.0452; HUVECs compared to NCI-H929, p = 0.0298; K–W, Dunn’s post hoc. Fig. 1B. p = 0.0237, K–S; Fig. 1C. DMSO compared to MPEP, p = 0.0376; DMSO compared to MTEP, p = 0.0425; K–W, Dunn’s post hoc; Fig. 1D. p = 0.0198, K–S; Fig. 1E. DMSO compared to MPEP1. p = 0.0487; DMSO compared to MTEP2, p = 0.0341; K–W, Dunn’s post hoc)

Article Snippet: Therefore, mGluR5 siRNA plasmid, lentivirus plasmid and respective empty vectors (Santa Cruz Biotechnology, Santa Cruz, USA) were used to regulate the expression levels of mGluR5 in MM1S and OPM2 cells.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Control, RNA Expression, Western Blot

Journal: Advances in Clinical and Experimental Medicine

Article Title: mGluR5 promotes the progression of multiple myeloma in vitro via Ras–MAPK signaling pathway

doi: 10.17219/acem/130445

Figure Lengend Snippet: Fig. 2. Agonist-induced mGluR5 upregulation promoted cell viability in multiple myeloma (MM) cells. A,B. The MTT assay measured the cell viability situation when MM1S cell line was treated with dimethyl sulfoxide (DMSO) as a control group and 3,5-dihydroxyphenylglycine (DHPG) as an agonist group, and OPM2 cell line was treated with DMSO as control group, with 2-methyl-6-(phenylethynyl)-pyridine (MPEP) or 3-[(2-methyl-1,3- thiazol-4-yl) ethynyl]pyridine (MTEP) as antagonist groups. Each assay was performed thrice independently. Kruskal–Wallis test (K–W) with Dunn’s post hoc test and Kolmogorov–Smirnov test (K–S) were applied in the statistical analysis (Fig. 2A. p = 0.0032, K–S; Fig. 2B. DMSO compared to MPEP, p = 0.0356; DMSO compared to MTEP, p = 0.0225; K–W, Dunn’s post hoc)Fig. 1. mGluR5 had higher expression in multiple myeloma (MM) cell lines. A. Quantitative real-time polymerase chain reaction (qRT-PCR) was applied to evaluate the mGluR5 expression in MM cell lines and MM1S, OPM-2, U266, NCI-H929, and RPMI-8226, and normal cell line CD143; B,D. MM1S cell line was treated with dimethyl sulfoxide (DMSO) as a control group and 3,5-dihydroxyphenylglycine (DHPG) as an agonist group. The qRT-PCR was used to measure mGluR5 RNA expression in each group in MM1S cell line. Western blot was used to measure mGluR5 protein expression in each group in MM1S cell line; C,E. OPM2 cell line was treated with DMSO as a control group, 2-methyl-6-(phenylethynyl)-pyridine (MPEP) or 3-[(2-methyl-1,3- thiazol-4-yl)ethynyl]pyridine (MTEP) as antagonist groups. The qRT-PCR was used to measure mGluR5 RNA expression in each group in the MM1S cell line. Western blot was used for mGluR5 protein expression in each group in MM1S cell line. Each assay was performed thrice independently. Kruskal–Wallis test (K–W) with Dunn’s post hoc test and Kolmogorov–Smirnov test (K–S) were applied in the statistical analysis (Fig. 1A. human umbilical vein endothelial cells (HUVECs) compared to RPMI82226, p = 0.0197; HUVECs compared to U266, p = 0.0324; HUVECs compared to OPM2, p = 0.0029; HUVECs compared to MM1S, p = 0.0452; HUVECs compared to NCI-H929, p = 0.0298; K–W, Dunn’s post hoc. Fig. 1B. p = 0.0237, K–S; Fig. 1C. DMSO compared to MPEP, p = 0.0376; DMSO compared to MTEP, p = 0.0425; K–W, Dunn’s post hoc; Fig. 1D. p = 0.0198, K–S; Fig. 1E. DMSO compared to MPEP1. p = 0.0487; DMSO compared to MTEP2, p = 0.0341; K–W, Dunn’s post hoc)Fig. 2. Agonist-induced mGluR5 upregulation promoted cell viability in multiple myeloma (MM) cells. A,B. The MTT assay measured the cell viability situation when MM1S cell line was treated with dimethyl sulfoxide (DMSO) as a control group and 3,5-dihydroxyphenylglycine (DHPG) as an agonist group, and OPM2 cell line was treated with DMSO as control group, with 2-methyl-6-(phenylethynyl)-pyridine (MPEP) or 3-[(2-methyl-1,3- thiazol-4-yl)ethynyl]pyridine (MTEP) as antagonist groups. Each assay was performed thrice independently. Kruskal–Wallis test (K–W) with Dunn’s post hoc test and Kolmogorov–Smirnov test (K–S) were applied in the statistical analysis (Fig. 2A. p = 0.0032, K–S; Fig. 2B. DMSO compared to MPEP, p = 0.0356; DMSO compared to MTEP, p = 0.0225; K–W, Dunn’s post hoc)

Article Snippet: Therefore, mGluR5 siRNA plasmid, lentivirus plasmid and respective empty vectors (Santa Cruz Biotechnology, Santa Cruz, USA) were used to regulate the expression levels of mGluR5 in MM1S and OPM2 cells.

Techniques: MTT Assay, Control, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, RNA Expression, Western Blot

Journal: Advances in Clinical and Experimental Medicine

Article Title: mGluR5 promotes the progression of multiple myeloma in vitro via Ras–MAPK signaling pathway

doi: 10.17219/acem/130445

Figure Lengend Snippet: Fig. 3. Agonist-induced mGluR5 upregulation inhibited apoptosis in multiple myeloma (MM) cells. A,B. Flow cytometry (FCM) apoptosis assays measured the cell apoptosis changes when MM1S cell line was treated with dimethyl sulfoxide (DMSO) as a control group and 3,5-dihydroxyphenylglycine (DHPG) as an agonist group, and OPM2 cell line was treated with DMSO as a control group, with 2-methyl-6-(phenylethynyl)-pyridine (MPEP) or 3-[(2-methyl-1,3- thiazol-4-yl)ethynyl]pyridine (MTEP) as antagonist groups. Each assay was performed thrice independently. Kruskal–Wallis test (K–W) with Dunn’s post hoc test and Kolmogorov–Smirnov test (K–S) were applied in the statistical analysis (Fig. 3A. p = 0.0269, K–S; Fig. 3B. DMSO compared to MPEP, p = 0.0274; DMSO compared to MPTP, p = 0.0225; K–W, Dunn’s post hoc)Fig. 3. Agonist-induced mGluR5 upregulation inhibited apoptosis in multiple myeloma (MM) cells. A,B. Flow cytometry (FCM) apoptosis assays measured the cell apoptosis changes when MM1S cell line was treated with dimethyl sulfoxide (DMSO) as a control group and 3,5-dihydroxyphenylglycine (DHPG) as an agonist group, and OPM2 cell line was treated with DMSO as a control group, with 2-methyl-6-(phenylethynyl)-pyridine (MPEP) or 3-[(2-methyl-1,3- thiazol-4-yl)ethynyl]pyridine (MTEP) as antagonist groups. Each assay was performed thrice independently. Kruskal–Wallis test (K–W) with Dunn’s post hoc test and Kolmogorov–Smirnov test (K–S) were applied in the statistical analysis (Fig. 3A. p = 0.0269, K–S; Fig. 3B. DMSO compared to MPEP, p = 0.0274; DMSO compared to MPTP, p = 0.0225; K–W, Dunn’s post hoc)

Article Snippet: Therefore, mGluR5 siRNA plasmid, lentivirus plasmid and respective empty vectors (Santa Cruz Biotechnology, Santa Cruz, USA) were used to regulate the expression levels of mGluR5 in MM1S and OPM2 cells.

Techniques: Flow Cytometry, Control

Journal: Advances in Clinical and Experimental Medicine

Article Title: mGluR5 promotes the progression of multiple myeloma in vitro via Ras–MAPK signaling pathway

doi: 10.17219/acem/130445

Figure Lengend Snippet: Fig. 4. Transfection-induced upregulation of mGluR5 promoted cell viability and inhibited cell death in multiple myeloma (MM) cells. MM1S cell line was transfected with si-NC or si-mGluR5 plasmids while OPM2 cell line was selected to knock down by transfection with oe-NC and oe-mGluR5 plasmids. A. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to evaluate the relative mGluR5 mRNA expression among all the groups in both cell lines; B. Cell viability was measured with MTT method; C,D. Flow cytometry (FCM) apoptosis assay was used to measure cell apoptosis. Each assay was performed thrice independently. Kruskal–Wallis test (K–W) with Dunn’s post hoc test and Kolmogorov–Smirnov test (K–S) were applied in the statistical analysis (Fig. 4A. oe-NC (MMIS) compared to oe-mGluR5 (MMIS), p = 0.0163; si-NC (OPM2) compared to si-mGluR5 (OPM2), p = 0.0319; K–W, Dunn’s post hoc; Fig. 4B. oe-NC (MMIS) compared to oe-mGluR5 (MMIS), p = 0.0094; si-NC (OPM2) compared to si-mGluR5 (OPM2), p = 0.0308; K–W, Dunn’s post hoc. Fig. 4C; oe-NC(MMIS) compared to oe-mGluR5 (MMIS), p = 0.0268; si-NC (OPM2) compared to si-mGluR5 (OPM2), p = 0.0325; K–W, Dunn’s post hoc)Fig. 4. Transfection-induced upregulation of mGluR5 promoted cell viability and inhibited cell death in multiple myeloma (MM) cells. MM1S cell line was transfected with si-NC or si-mGluR5 plasmids while OPM2 cell line was selected to knock down by transfection with oe-NC and oe-mGluR5 plasmids. A. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to evaluate the relative mGluR5 mRNA expression among all the groups in both cell lines; B. Cell viability was measured with MTT method; C,D. Flow cytometry (FCM) apoptosis assay was used to measure cell apoptosis. Each assay was performed thrice independently. Kruskal–Wallis test (K–W) with Dunn’s post hoc test and Kolmogorov–Smirnov test (K–S) were applied in the statistical analysis (Fig. 4A. oe-NC (MMIS) compared to oe-mGluR5 (MMIS), p = 0.0163; si-NC (OPM2) compared to si-mGluR5 (OPM2), p = 0.0319; K–W, Dunn’s post hoc; Fig. 4B. oe-NC (MMIS) compared to oe-mGluR5 (MMIS), p = 0.0094; si-NC (OPM2) compared to si-mGluR5 (OPM2), p = 0.0308; K–W, Dunn’s post hoc. Fig. 4C; oe-NC(MMIS) compared to oe-mGluR5 (MMIS), p = 0.0268; si-NC (OPM2) compared to si-mGluR5 (OPM2), p = 0.0325; K–W, Dunn’s post hoc)

Article Snippet: Therefore, mGluR5 siRNA plasmid, lentivirus plasmid and respective empty vectors (Santa Cruz Biotechnology, Santa Cruz, USA) were used to regulate the expression levels of mGluR5 in MM1S and OPM2 cells.

Techniques: Transfection, Knockdown, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Flow Cytometry, Apoptosis Assay

Journal: Advances in Clinical and Experimental Medicine

Article Title: mGluR5 promotes the progression of multiple myeloma in vitro via Ras–MAPK signaling pathway

doi: 10.17219/acem/130445

Figure Lengend Snippet: Fig. 5. Transfection-induced upregulation activated the Ras–mitogen activated protein kinase (MAPK) signaling pathway in multiple myeloma (MM) cells. MM1S cell line was transfected with si-NC or si-mGluR5 plasmids, while the OPM2 cell line was selected to be knocked down by transfection with oe-NC and oe-mGluR5 plasmids. Western blot was used to examine the protein levels of mGluR5 (A) and RAS (B) as well as phosphorylation of Raf1 (C) among all the groups in both cell lines after transfection. Each assay was performed thrice independently. Kruskal–Wallis test (K–W) with Dunn’s post hoc test and Kolmogorov–Smirnov test (K–S) were applied in the statistical analysis (Fig. 5A. oe-NC (MMIS) compared to oe-mGluR5 (MMIS), p = 0.0039, si-NC (OPM2) compared to si-mGluR5 (OPM2), p = 0.0254; K–W, Dunn’s post hoc; Fig. 5B. oe-NC (MMIS) compared to oe-mGluR5 (MMIS), p = 0.0174, si-NC (OPM2) compared to si-mGluR5 (OPM2), p = 0.0232; K–W, Dunn’s post hoc; Fig. 5D. oe-NC (MMIS) compared to oe-mGluR5 (MMIS), p = 0.0094, si-NC (OPM2) compared to si-mGluR5 (OPM2), p = 0.0163; K–W, Dunn’s post hoc)Fig. 5. Transfection-induced upregulation activated the Ras–mitogen activated protein kinase (MAPK) signaling pathway in multiple myeloma (MM) cells. MM1S cell line was transfected with si-NC or si-mGluR5 plasmids, while the OPM2 cell line was selected to be knocked down by transfection with oe-NC and oe-mGluR5 plasmids. Western blot was used to examine the protein levels of mGluR5 (A) and RAS (B) as well as phosphorylation of Raf1 (C) among all the groups in both cell lines after transfection. Each assay was performed thrice independently. Kruskal–Wallis test (K–W) with Dunn’s post hoc test and Kolmogorov–Smirnov test (K–S) were applied in the statistical analysis (Fig. 5A. oe-NC (MMIS) compared to oe-mGluR5 (MMIS), p = 0.0039, si-NC (OPM2) compared to si-mGluR5 (OPM2), p = 0.0254; K–W, Dunn’s post hoc; Fig. 5B. oe-NC (MMIS) compared to oe-mGluR5 (MMIS), p = 0.0174, si-NC (OPM2) compared to si-mGluR5 (OPM2), p = 0.0232; K–W, Dunn’s post hoc; Fig. 5D. oe-NC (MMIS) compared to oe-mGluR5 (MMIS), p = 0.0094, si-NC (OPM2) compared to si-mGluR5 (OPM2), p = 0.0163; K–W, Dunn’s post hoc)

Article Snippet: Therefore, mGluR5 siRNA plasmid, lentivirus plasmid and respective empty vectors (Santa Cruz Biotechnology, Santa Cruz, USA) were used to regulate the expression levels of mGluR5 in MM1S and OPM2 cells.

Techniques: Transfection, Western Blot, Phospho-proteomics

Journal: bioRxiv

Article Title: Macropinocytosis mediates neurotropism of Cryptococcus neoformans in a human organoid model of the blood-brain barrier

doi: 10.1101/2025.09.23.678106

Figure Lengend Snippet: a Representative fluorescent image of BBB organoids formed from EphA2 knockout (KO) human brain endothelial cells. EphA2 KO cells express RFP (magenta) against DAPI (blue) counterstaining. b Western blot analysis (1:1000 dilution of rabbit anti-Human EphA2; Cell signaling technology Inc. D4A2) (1:10,000 dilution of Goat Anti-Rabbit IgG H&L HRPab6721; Abcam Inc., Cambridge, MA, USA) confirming loss of EphA2 protein expression in EphA2 KO organoids, in contrast to controls (EphA2+ and EphA2 over-expression (o/e)). c Internalization of Cn ( Cryptococcus neoformans ) by BBB organoids is dependent on EphA2 expression. Organoids (EphA2+ or EphA2 KO brain endothelial cells) were exposed to CFSE-stained Cn for 48 h, after which organoids were sectioned and analyzed for Cn invasion (Welch two-sample t-test: t = 4.28, df = 13.94, p < 0.0008)(R v.4.4.2). d Comparison of GFAP expression between BBB organoids formed with EphA2 KO versus EphA2+ brain endothelial cells. Organoids deficient in EphA2 show significantly higher levels of GFAP expression compared to EphA2+ organoids. Quantification was performed on GFAP-antibody probed organoid sections by applying a uniform threshold to images and measuring the percent area in the relevant channel above the threshold (Welch two-sample t-test: t = 2.42, df = 10.68, p = 0.0343)(R v.4.2.2). e Organoids exposed to Cd ( Cryptococcus deuterogattii ) show increased GFAP expression in stark contrast to organoids exposed to Cn after 48 h of exposure (Welch two-sample t-test comparing Cn & Cd treatments: t = 3.84, df = 9.38, p = 0.003, comparing Cd & Control treatments: t = 4.25, df = 10.57, p = 0.0015)(R v.4.4.2). For panels c – e, each data point represents the average value for an organoid across several images.

Article Snippet: Two sets of plasmids were used in the CRISPR/Cas9 method - the EphA2 CRISPR/Cas9 KO plasmids and the EphA2 HDR plasmids, both of which were ordered from Santa Cruz Biotechnology.

Techniques: Knock-Out, Western Blot, Expressing, Over Expression, Staining, Comparison, Control

Journal: bioRxiv

Article Title: Macropinocytosis mediates neurotropism of Cryptococcus neoformans in a human organoid model of the blood-brain barrier

doi: 10.1101/2025.09.23.678106

Figure Lengend Snippet: a Cn induced stimulation of GTP-bound Cdc42 is contingent on EphA2 expression and b CD44 engagement. EphA2+ and EphA2 KO human brain endothelial cells were treated with either a Cn or b cps1Δ Cn deletion mutant at an MOI of 10 for 30 min and then lysed. Untreated and treated cell lysates were analyzed for GTP bound Cdc42 (active Cdc42) using the Cytoskeleton G-LISA assay. Data are expressed as a mean ± SD of three independent experiments. Statistical analysis was done using a two tailed unpaired t-test (n = 8, t = 0.2156, df = 14, **** p < 0.0001)(GraphPad Prism 10 software). c, d Expression of Cdc42 protein in lysates was confirmed by western blot analysis of nitrocellulose membrane probed with primary antibody to amino acid 150-182 of Cdc42 protein. (1:800 dilution of mouse anti-human Cdc42; cytoskeleton Inc. ACD04) (1:10,000 dilution of Goat anti-Mouse IgG H&L HRP ab6789; Abcam Inc., Cambridge, MA, USA). e, f Cn recruits a CD44-EphA2 protein complex in brain endothelial cells, detected by Proximity Labeling Assays (PLA - DuoLink) of EphA2 and CD44 in e mouse primary brain endothelial cells and f human brain endothelial cells (EphA2+, iBMEC). The in-situ interaction was quantified as the area in an image above a universally applied threshold. Significantly higher fluorescence was observed in Cn (+) versus Cn (-) treatments in both mouse primary cells (t = 4.40, df = 43.13, p < 0.0001) and human cells (t = 3.63, df = 131.88, p = 0.0004). In mouse primary cells, there was significantly higher signal in the Cn (+) compared to the Cd (+) treatments (t = 4.08, df = 43.63, p < 0.0002)(R v.4.4.2). Mouse primary brain endothelial cells were exposed to Cd , Cn media or a no primary antibody control. g Representative fluorescent images of PLA in human brain endothelial cells (EphA2+, iBMECs). Top row: iBMECs exposed to Cn for 90 min. Middle row: control panel iBMECs not exposed to Cn . Bottom row: control treatment in which iBMECs were exposed to Cn but omitting primary antibodies (anti-CD44 and anti-EphA2) Left column: Nuclear stain. Middle column: Fluorescent puncta (PLA-red probe) are indicative of target proteins (EphA2 and CD44) in proximity (within 40 nm) to each other. Right column: merged images.

Article Snippet: Two sets of plasmids were used in the CRISPR/Cas9 method - the EphA2 CRISPR/Cas9 KO plasmids and the EphA2 HDR plasmids, both of which were ordered from Santa Cruz Biotechnology.

Techniques: Expressing, Mutagenesis, Two Tailed Test, Software, Western Blot, Membrane, Labeling, In Situ, Fluorescence, Control, Staining

Journal: bioRxiv

Article Title: Macropinocytosis mediates neurotropism of Cryptococcus neoformans in a human organoid model of the blood-brain barrier

doi: 10.1101/2025.09.23.678106

Figure Lengend Snippet: Alpha-Fold-Multimer structure prediction indicates reliable binding between EphA2 and CD44 . a Cartoon representation of the AlphaFold3-predicted model of the EphA2 structure. extracellular region in complex with CD44, highlighting two distinct putative binding interfaces: the ligand-binding domain (LBD) (ipTM = 0.13; pTM = 0.49) and the fibronectin domains (FN1-FN2) (ipTM = 0.11; pTM = 0.49). b-c Representative binding conformations of the LBD: CD44 and FN: CD44 complexes obtained from molecular dynamics simulations. Shown here are the major conformational clusters derived from all replica trajectories. EphA2 is depicted in green and CD44 in red cartoon representations. For clarity, only the principle interfacial hydrogen bonds are displayed.

Article Snippet: Two sets of plasmids were used in the CRISPR/Cas9 method - the EphA2 CRISPR/Cas9 KO plasmids and the EphA2 HDR plasmids, both of which were ordered from Santa Cruz Biotechnology.

Techniques: Binding Assay, Ligand Binding Assay, Derivative Assay

Journal: bioRxiv

Article Title: Macropinocytosis mediates neurotropism of Cryptococcus neoformans in a human organoid model of the blood-brain barrier

doi: 10.1101/2025.09.23.678106

Figure Lengend Snippet: Upon binding to CD44, Cn promotes the association of CD44 with the ligand binding domain (LBD) and interactions involving both FN1 and FN2 domains, suggesting that CD44 can also engage EphA2 via a membrane-proximal site and/or the fibronectin 2 domain (FN2) (black arrows). The CD44-EphA2 molecular interaction leads to a PKC-dependent phosphorylation of EphA2, and the downstream activation of GTP-bound Cdc42 (green arrows). Active Cdc42 stimulates membrane ruffling by remodeling the cytoskeleton via actin polymerization. The ensuing macropinocytosis of Cn is a result of macropinosomes large enough to internalize and transport Cn across the brain endothelium. Cd does not cross the brain endothelium (crossed-out black arrow), but stimulates the expression of GFAP+ astrocytes by as yet an unknown mechanism (dashed arrow). Figure created in BioRender.com.

Article Snippet: Two sets of plasmids were used in the CRISPR/Cas9 method - the EphA2 CRISPR/Cas9 KO plasmids and the EphA2 HDR plasmids, both of which were ordered from Santa Cruz Biotechnology.

Techniques: Binding Assay, Ligand Binding Assay, Membrane, Phospho-proteomics, Activation Assay, Expressing