Journal: Science (New York, N.Y.)

Article Title: Rotavirus induces intercellular calcium waves through ADP signaling

doi: 10.1126/science.abc3621

Figure Lengend Snippet: (A-B) Number of Ca2+ spikes in MA104-GCaMP cells in RV-infected (SA11cl3-mRuby3) at MOI 0.01 and neighboring (NB)+3 cell, treated with: (A) DMSO, 30μM 18β-glycyrrhetinic acid (18β-gly), or 50μM TAT-Gap19 (Gap19) and imaged ~8–24 hpi or (B) anti-VP7 M60 MAb, anti-NSP4 MAb 622, or rabbit anti-NSP4 antisera 120–147 (Rb Ab) and imaged ~8–24 hpi. (data combined from N=3 independent experiments) (C) Representative images of intercellular calcium waves in MA104-GCaMP cells infected with RV (SA11cl3-mRuby3) at MOI 0.1 and mock- or 10U/mL apyrase-treated (5 U/mL apyrase VI and 5U/mL apyrase VII) and imaged ~10 hpi with (D) representative Ca2+ traces from ~8–25 hpi. (E) Ca2+ spikes in RV-infected and NB cells. (F) Average magnitude of Ca2+ spikes/cell in RV-infected and NB cells, (data combined from N=3 independent experiments). (G) qPCR of purinergic receptor mRNA normalized to 18S mRNA and fold change relative to P2X3 mRNA transcript levels in MA104 cells, (data combined from N=3 independent experiments) (H-I) Ca2+ spikes in MA104-GCaMP cells RV (SA114F)-infected (H) or neighboring (NB) cells (I) and treated with DMSO, 10μM BPTU, 10μM AR-C 118925XX (ARC), 10μM Bx430, or 10μM 5-BDBD (data combined from N=3 independent experiments). (A-B, E-F, H-I) Kruskal-Wallis with Dunn’s multiple comparisons test used. Scale bar = 100 μm. Data represented as mean ± SD, (*p<0.05, ****p<0.0001).

Article Snippet: Chemicals BPTU, AR-C 118925XX, Bx430, 5-BDBD, MRS2179, MRS2279, MRS2500, 10-Panx, suramin, TAT-Gap19, PPADS (Pyridoxalphosphate-6-azophenyl-2’,4’-disulfonic acid), NOC-7, prostaglandin E2, ARL 67156, and ω-agatoxin were purchased from Tocris Bioscience.

Techniques: Infection

Journal: Science (New York, N.Y.)

Article Title: Rotavirus induces intercellular calcium waves through ADP signaling

doi: 10.1126/science.abc3621

Figure Lengend Snippet: (A) J3 jHIE-GCaMP6s monolayers mock- or RV (Ito)-infected produce intercellular calcium waves originating at a cell (arrowheads), imaged at ~4 hpi. (B) IF images of (J3)HIE-GCaMP6s monolayers mock- or RV-infected, fixed at 24 hpi, and immunostained for RV antigen (pink) and counterstained with DAPI (gray). (C) Ca2+ spikes per field-of-view (FOV), in (J3)HIEs mock- or RV-infected and treated with vehicle, 100 μM carbenoxolone (CBX), or 10 μM 10Panx at 8–22 hpi (data combined from N=3 independent experiments). (D) qPCR of purinergic receptor mRNA normalized to 18S mRNA and fold change relative to P2X3 mRNA transcript levels in (J3)HIE monolayers (data combined from N=3 independent experiments). (E) Representative Ca2+ traces/FOV of (J3)HIE-GCaMP6s monolayers either mock- or RV-infected and treated with DMSO, 100 U/mL apyrase, or 10 μM BPTU between 8.5–22 hpi. Mock- or RV-infected (J3)HIEs, treated with DMSO, apyrase, or purinergic receptor blockers. (F) Ca2+ spikes/FOV in (J3)HIE-GCaMP6s monolayers treated with DMSO, 100U/mL apyrase (50 U/mL apyrase VI and 50U/mL apyrase VII), 10 μM BPTU, 10 μM AR-C 118925XX, 10 μM Bx430, or 10 μM 5-BDBD and (G) average magnitude of Ca2+ spikes/FOV for 8.5–22 hpi, (data combined from N=3 independent experiments) (H) Ca2+ spikes/FOV of patient J2 jHIE-GCaMP6s monolayers mock- or RV-infected and treated with DMSO, 10 μM BPTU, 10 μM MRS2179, 10 μM MRS2279, or 10 μM MRS2500 and (I) average magnitude of Ca2+ spikes/FOV for 8–22 hpi, (data combined from N=3 independent experiments). (C,H,I) One-way ANOVA with Bonferroni’s and (F,G) Kruskal-Wallis with Dunn’s multiple corrections test used. Scale bar = 50 μm. Data represented as mean ± SD, (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001)

Article Snippet: Chemicals BPTU, AR-C 118925XX, Bx430, 5-BDBD, MRS2179, MRS2279, MRS2500, 10-Panx, suramin, TAT-Gap19, PPADS (Pyridoxalphosphate-6-azophenyl-2’,4’-disulfonic acid), NOC-7, prostaglandin E2, ARL 67156, and ω-agatoxin were purchased from Tocris Bioscience.

Techniques: Infection

Journal: Science (New York, N.Y.)

Article Title: Rotavirus induces intercellular calcium waves through ADP signaling

doi: 10.1126/science.abc3621

Figure Lengend Snippet: (A) Rotavirus yield from SA114F-infected MA104 cells treated with DMSO vehicle, 10U/mL apyrase, 10μM BPTU, 10μM AR-C 118925XX, 20μM Bx430, 20μM 5-BDBD, 300μM suramin, or 10μM PPADS by fluorescent focus assay (data combined from N=3 independent experiments). (B) Plaque assay yield of RV (SA114F)-infected MA104-GCaMP (Par) or MA104-GCaMP P2Y1 knockout (KO) cells (data combined from N=3 independent experiments). (C-E) qPCR of mRNA transcripts normalized to 18S mRNA transcripts and fold change relative to the Mock-DMSO transcript levels in (J3)HIE monolayers mock- or RV (Ito)-infected and treated with DMSO vehicle, 100U/mL apyrase, or 10 μM BPTU (data combined from N=3 independent experiments). (F-G) Serotonin secretion from RV (Ito)-infected jHIE (F) monolayers and (G) transwells treated with DMSO, 100U/mL apyrase, 10μM BPTU, or 300nM ω-agatoxin (data combined from N=3 independent experiments). (H) Enteroid swelling assay: 3D jHIE-GCaMP6s enteroids mock- or RV (Ito)-infected and treated with DMSO or 10μM BPTU. Cross-sectional area of the internal lumen (pink outline) used for percent increase between basal and max swelling (left panels). Scale bar = 100μm. (n ≥ 68 HIEs per condition, data combined from N=3 independent experiments) (I-K) C57Bl/6J mouse pups with diarrhea infected with Rhesus RV and vehicle- or BPTU-treated and the (J) mean diarrhea score. (data combined from 4 cages of each condition, 26–30 pups total per condition, mean ± SEM) (K) Summary model of RV-induced ICWs mediated by extracellular ADP. (A-B, F-G) One-way ANOVA with Bonferroni multiple comparisons test, (C-J) Kruskal-Wallis with Dunn’s multiple comparisons, or (I-J) Mann-Whitney tests used. (A-H) Data represented as mean ± SD, (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

Article Snippet: Chemicals BPTU, AR-C 118925XX, Bx430, 5-BDBD, MRS2179, MRS2279, MRS2500, 10-Panx, suramin, TAT-Gap19, PPADS (Pyridoxalphosphate-6-azophenyl-2’,4’-disulfonic acid), NOC-7, prostaglandin E2, ARL 67156, and ω-agatoxin were purchased from Tocris Bioscience.

Techniques: Infection, Plaque Assay, Knock-Out, MANN-WHITNEY

Journal: bioRxiv

Article Title: A gated hydrophobic funnel within BAX binds long-chain alkenals to potentiate pro-apoptotic function

doi: 10.1101/2024.12.23.630122

Figure Lengend Snippet: (A–C) MEFs subjected to SPARKL analysis measuring real-time labeling with fluorescently-tagged Annexin V (100 μg/ml) via imaging with an IncuCyte ZOOM. Left panels: kinetics of cell labeling in response to increasing concentrations of 2t–hexadecenal (2t–16) with DMSO vehicle or co-treated with ABT-737 (ABT); black line reports untreated control. Right panels: endpoint data of replicates at 24 hours. Data shown are the mean of technical triplicates and error bars report SEM. (A) WT MEFs (matched to Bax −/− Bak −/− double knockout MEFs) were treated with 2t–16 (10, 20, 40 μM) and DMSO or ABT–737 (1 μM), imaged every 2 hours, and quantified for number of Annexin V-positive objects. (B) Same as in A with Bim −/− Bid −/− double knockout MEFs. (C) Same as in A with Bax −/− Bak −/− double knockout MEFs. (D–I) LUV permeabilization studies with recombinant BAX protein treated as indicated and measured at regular intervals for changes in fluorescence as fluorophores are released from liposomes. Grey data report LUVs solubilized with 1% CHAPS to measure maximal signal. Data shown are the mean of technical replicates. (D) BAX protein (120 nM) was combined with DMSO vehicle or 2t–16 (6.5–50 μM) followed by addition of LUVs and measured by fluorescent spectroscopy. (E) Same as in D with BIM–BH3 peptide (2.5 μM) added to BAX and 2t–16. (F) Heatmap visualization of normalized endpoint LUV permeabilization data from LUVs incubated with BAX (120 nM) treated with 2t–16 (6.5–50 μM) ± BIM–BH3 peptide (0.5, 2.5 μM). Data summarized from Figures S1C . (G–H) LUV permeabilization studies as in D–E with BAX (160 nM) and hexadecanal (6.5–50 μM) ± BIM–BH3 peptide (2.5 μM). (I) Heatmap visualization of normalized endpoint LUV permeabilization data as in F with BAX (160 nM) and 16CHO (6.5−50 μM). Data summarized from Figures S1D . See also .

Article Snippet: Additional recombinant proteins and peptides were purchased from commercial sources: 5-TAMRA labeled BAK-BH3 (Cat. No. AS-64590, AnaSpec); recombinant Bcl–xL ΔC , aa 2-212 (Cat. No. 894-BX, R&D Systems); BIM-BH3, Peptide IV (Cat. No. AS-62279, AnaSpec).

Techniques: Labeling, Imaging, Control, Double Knockout, Recombinant, Fluorescence, Liposomes, Spectroscopy, Incubation

Journal: bioRxiv

Article Title: A gated hydrophobic funnel within BAX binds long-chain alkenals to potentiate pro-apoptotic function

doi: 10.1101/2024.12.23.630122

Figure Lengend Snippet: (A–D) LUV permeabilization studies with recombinant BAX protein treated as indicated and measured at regular intervals for changes in fluorescence as fluorophores are released from compromised liposomes. Left panels: kinetic fluorescence data; right panels: endpoint data normalized to LUV fluorescence and maximal signal generated by LUVs solubilized with CHAPS detergent (grey data). Data are shown as the mean of technical replicates and error bars report SEM. (A) BAX protein (100 nM) was activated by BIM–BH3 peptide (0.13–2 μM) and added to LUVs. (B) LUVs treated with 2t–16 (16.5–50 μM) in the absence of BAX to confirm no membrane destabilization by 2t–16. (C) Data summarized by . LUVs permeabilized by BAX (120 nM) treated with 2t–16 (6.5– 50 μM) ± BIM–BH3 peptide (0.5, 2.5 μM). (D) Data summarized by . LUVs permeabilized by BAX (160 nM) treated with 16CHO (6.5–50 μM) ± BIM–BH3 peptide (0.5, 2.5 μM).

Article Snippet: Additional recombinant proteins and peptides were purchased from commercial sources: 5-TAMRA labeled BAK-BH3 (Cat. No. AS-64590, AnaSpec); recombinant Bcl–xL ΔC , aa 2-212 (Cat. No. 894-BX, R&D Systems); BIM-BH3, Peptide IV (Cat. No. AS-62279, AnaSpec).

Techniques: Recombinant, Fluorescence, Liposomes, Generated, Membrane

Journal: bioRxiv

Article Title: A gated hydrophobic funnel within BAX binds long-chain alkenals to potentiate pro-apoptotic function

doi: 10.1101/2024.12.23.630122

Figure Lengend Snippet: (A–B) Alexa Fluor 647-labeled recombinant BAX WT (1 nM) was incubated with CHAPS (0.002%) to inhibit oligomerization, treated as indicated, and subjected to MST. Data shown are the mean of replicate data and error bars report SD. (A) Left: Timetrace thermal shift curves of BAX WT titrated with 2t–16 (0.02–40 μM) and subjected to MST. Right: Thermophoresis and temperature jump value for BAX WT treated with a range of 2t–16 concentrations fitted to determine a K D value. (B) BAX WT was treated with 2t–16 or 16CHO (0.04, 1.25, 40 μM) and MST timetrace thermal shift curves were fitted using a one-step exponential function and compared using the decay (K) constants normalized by the untreated BAX curve. Original data in Figure S2C . (C) LC-MS of recombinant BAX WT alone or incubated with 2t–16. Samples were then alkylated with iodoacetamide to identify unmodified cysteine residues and trypsin digested for analysis. Four cysteine-containing peptide fragments were detected. Values denote peptide abundance, calculated as AUC for each peak. (D–I) LUV permeabilization studies with recombinant BAX 2S protein treated as indicated and measured at regular intervals for changes in fluorescence as fluorophores are released from liposomes. Grey data report LUVs solubilized with 1% CHAPS to measure maximal signal. Data shown are the mean of technical replicates. (D) BAX 2S protein (100 nM) was combined with DMSO vehicle or 2t–16 (6.5–50 μM) followed by addition of LUVs and measured by fluorescent spectroscopy. (E) Same as in D with BIM–BH3 peptide (2.5 μM) added to BAX 2S and 2t–16. (F) Heatmap visualization of normalized endpoint LUV permeabilization data from LUVs incubated with BAX 2S (100 nM) treated with 2t–16 (6.5–50 μM) ± BIM–BH3 peptide (0.5, 2.5 μM). Data summarized from Figure S2B . (G–H) LUV permeabilization studies as in D–E with BAX 2S (100 nM) and hexadecanal (6.5–50 μM) ± BIM–BH3 peptide (2.5 μM). (I) Heatmap visualization of normalized endpoint LUV permeabilization data from LUVs incubated with BAX 2S (100 nM) treated with 16CHO (6.5–50 μM) ± BIM–BH3 peptide (0.5, 2.5 μM). Data summarized from Figure S2C . See also .

Article Snippet: Additional recombinant proteins and peptides were purchased from commercial sources: 5-TAMRA labeled BAK-BH3 (Cat. No. AS-64590, AnaSpec); recombinant Bcl–xL ΔC , aa 2-212 (Cat. No. 894-BX, R&D Systems); BIM-BH3, Peptide IV (Cat. No. AS-62279, AnaSpec).

Techniques: Labeling, Recombinant, Incubation, Liquid Chromatography with Mass Spectroscopy, Fluorescence, Liposomes, Spectroscopy

Journal: bioRxiv

Article Title: A gated hydrophobic funnel within BAX binds long-chain alkenals to potentiate pro-apoptotic function

doi: 10.1101/2024.12.23.630122

Figure Lengend Snippet: (A) Alexa Fluor 647-labeled recombinant BAX WT (1 nM) was incubated with CHAPS (0.002%) to inhibit oligomerization, treated as indicated, and subjected to MST. Timetrace thermal shift curves of BAX WT titrated with 2t–16 or 16CHO (0.04, 1.25, 40 μM) report the mean of replicate data. (B−C) The melting temperature of BAX WT and BAX 2S ± 2t–16 (6.5−50 μM) was measured by thermal shift assay using SYPRO orange and compared. Statistical significance was determined by two-way ANOVA; ns, not significant ( P > 0.05). (D–E) LUV permeabilization studies with recombinant BAX 2S protein treated as indicated and measured at regular intervals for changes in fluorescence as fluorophores are released from compromised liposomes. Left panels: kinetic fluorescence data; right panels: endpoint data normalized to LUV fluorescence and maximal signal generated by LUVs solubilized with CHAPS detergent (grey data). Data are shown as the mean of technical replicates and error bars report SEM. (D) Data summarized by . LUVs permeabilized by BAX 2S (100 nM) treated with 2t–16 (6.5–50 μM) ± BIM–BH3 peptide (0.5, 2.5 μM). (E) Data summarized by . LUVs permeabilized by BAX 2S (100 nM) treated with 16CHO (6.5–50 μM) ± BIM–BH3 peptide (0.5, 2.5 μM).

Article Snippet: Additional recombinant proteins and peptides were purchased from commercial sources: 5-TAMRA labeled BAK-BH3 (Cat. No. AS-64590, AnaSpec); recombinant Bcl–xL ΔC , aa 2-212 (Cat. No. 894-BX, R&D Systems); BIM-BH3, Peptide IV (Cat. No. AS-62279, AnaSpec).

Techniques: Labeling, Recombinant, Incubation, Thermal Shift Assay, Fluorescence, Liposomes, Generated

Journal: bioRxiv

Article Title: A gated hydrophobic funnel within BAX binds long-chain alkenals to potentiate pro-apoptotic function

doi: 10.1101/2024.12.23.630122

Figure Lengend Snippet: (A) Illustration of BAX and BAK–BH3 interactions within FLAMBE. The reporter is a fluorescently-labeled BAK–BH3 peptide that exhibits changes in Polarization measurements as it is bound by BAX. Over time, the BAX population binds BAK–BH3 peptides resulting in increased Polarization, which eventually plateaus if the entire population forms heterodimers (dotted line). In conditions that activate BAX, activation-induced intramolecular rearrangements within BAX result in the dissociation of the BAK–BH3 peptide and a concomitant decrease in Polarization over time as the population of unbound BAK–BH3 peptide increases (solid line). (B) Illustration of FLAMBE data parameterization and analysis. Left: Kinetic Polarization data is collected for a treatment causing BAX activation and exhibiting accelerated kinetics of BAK TAMRA dissociation (blue lines, depicted as a titration exhibiting dose-dependent BAX activation). Kinetic data is parameterized by extracting endpoint Polarization (EP) and time-to-maximum peak (Tmax) for each condition. Right: Parameterized data are normalized to BAK TAMRA and BAX controls (grey and black, respectively) and titrations or separate conditions can be visualized as a two-dimensional plot. Generally, conditions exhibiting minimal or robust activation of the BAX population cluster in the upper-right or lower-left regions, respectively. Conditions plotted above BAX (i.e., EP > 1) form stable non-activating complexes with the BAX:BAK TAMRA heterodimer (red region). (C) BAX WT (60 nM) was treated with BIM–BH3 peptide (0.25–2 μM) and subjected to FLAMBE to visualize dose-dependent activation-induced dissociation of BAK TAMRA . BIM–BH3 at a low concentration (0.25 μM, dark blue data) demonstrated a stable, non-activating interaction with the BAX:BAK TAMRA complex and exhibited increased Polarization. (D) BAX WT (60 nM) was treated with 16CHO (2–50 μM), combined with BAK TAMRA (50 nM), and subjected to FLAMBE. (E) Same as in D with BAX 2S (60 nM). (F) Left: BAX 2S (60 nM) was treated with three non-activating concentrations of 2t–16 (green: 4.5 μM; orange: 6.5 μM; red: 10 μM). Parameterization of this data is included in . Right: AUC calculated for each condition was normalized to the BAX and BAK TAMRA controls and reported as a percent change from the vehicle-treated BAX condition. (G) Left: BAX WT (60 nM) was combined with a non-activating concentrations of BIM–BH3 peptide (0.15 μM) and 2t–16 (4.5 μM), followed by BAK TAMRA (50 nM), and subjected to FLAMBE. Middle: Parameterized FLAMBE data including three concentrations of 2t–16 (green: 4.5 μM; orange: 6.5 μM; red: 10 μM) in the absence or presence of BIM–BH3 (circle and square datapoints, respectively). Annotations report the magnitude of shift between data with and without BIM–BH3. Right: AUC calculated for each condition was normalized to the BAX and BAK TAMRA controls and reported as a percent change from the vehicle-treated BAX condition. (H) Fluorescence polarization competition assay with recombinant BCL-xL ΔC protein treated with 2t–16 (2–50 μM) and combined with BAK TAMRA .

Article Snippet: Additional recombinant proteins and peptides were purchased from commercial sources: 5-TAMRA labeled BAK-BH3 (Cat. No. AS-64590, AnaSpec); recombinant Bcl–xL ΔC , aa 2-212 (Cat. No. 894-BX, R&D Systems); BIM-BH3, Peptide IV (Cat. No. AS-62279, AnaSpec).

Techniques: Labeling, Activation Assay, Titration, Concentration Assay, Fluorescence, Competitive Binding Assay, Recombinant

Journal: bioRxiv

Article Title: A gated hydrophobic funnel within BAX binds long-chain alkenals to potentiate pro-apoptotic function

doi: 10.1101/2024.12.23.630122

Figure Lengend Snippet: (A) Alexa Fluor 647-labeled recombinant BAX WT (1 nM) was incubated with CHAPS (0.002%) to inhibit oligomerization, treated with the indicated 2t–alkenals (0.16–5 μM), and subjected to MST. Timetrace data are shown as the mean of replicates. Thermophoresis metrics for each 2t–alkenal are summarized in . (B) BAX 2S (60 nM) was treated with the indicated 2t–alkenal (3–50 μM), combined with BAK TAMRA (50 nM), and subjected to FLAMBE. Data are shown as the mean of replicates. Parameterized data reporting EP and Tmax for each experiment are provided in Figures 5D –E . (C) LUVs permeabilized by BAX 2S (100 nM) treated with the indicated 2t–alkenal (6.5–50 μM). Data are shown as the mean of replicates. Normalized endpoint permeabilization data summarized in .

Article Snippet: Additional recombinant proteins and peptides were purchased from commercial sources: 5-TAMRA labeled BAK-BH3 (Cat. No. AS-64590, AnaSpec); recombinant Bcl–xL ΔC , aa 2-212 (Cat. No. 894-BX, R&D Systems); BIM-BH3, Peptide IV (Cat. No. AS-62279, AnaSpec).

Techniques: Labeling, Recombinant, Incubation

Journal: bioRxiv

Article Title: CCR1 Mediates Müller Cell Activation and Photoreceptor Cell Death in Macular and Retinal Degeneration

doi: 10.1101/2023.01.03.522541

Figure Lengend Snippet: ( A ) Albino BALB/c mice were subjected to photic injury followed by subcutaneous injections of the CCR1 inhibitor BX471 or vehicle for 5 days. ERG recordings were then performed, and the amplitude of the b-wave was measured and is plotted against flash intensity (n=12 eyes for each group, Student’s t -test). ( B ) The number of photoreceptor nuclei was measured at the indicated distances from the optic nerve (n=14 eyes for each group, Student’s t -test). ( C ) Retinal sections were prepared from vehicle-treated mice and BX471-treated mice and immunostained for the microglial cell marker IBA-1; The asterisks indicate amoeboid-shaped cells in the ONL and subretinal layer, and the arrows indicate elongated cells in the GCL and IPL, with one cell shown in a magnified view (inset). ( D ) Real-time qPCR analysis of retinal Adgre1 mRNA (which encodes the macrophage marker F4/80) in control mice, vehicle-treated photic-injured mice, and BX471-treated photic-injured mice; (n=6 mice for each group, one-way ANOVA with multiple comparisons). ( E ) Retinal sections were prepared from vehicle-treated photic-injured mice and BX471-treated photic-injured mice and immunostained for CCR1. ( F ) Real-time qPCR analysis of retinal Ccr1 mRNA measured in vehicle-treated photic-injured mice and BX471-treated photic-injured mice; (n=6 mice for each group, Student’s t -test). ( G ) Real-time qPCR analysis of retinal Ccl2 , Cxcl1 , and Cxcl10 mRNA measured in control mice, vehicle-treated photic-injured mice, and BX471-treated photic mice; (n=6 mice for each group, one-way ANOVA with multiple comparisons). GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; ONL, outer nuclear layer. Data shown as mean ± SEM. p-Values indicated by ns, not significant and * p <0.05, ** p <0.01, # p <0.001. Scale bars: 50 µm (C and E) and 20 µm (inset in C).

Article Snippet: Immediately after photic injury was induced, the mice received subcutaneous injections of either the CCR1-specific antagonist BX471 (50 mg/kg body weight; Tocris, Bristol, UK) or vehicle (40% cyclodextrin in saline) every 12 hours for 7 days.

Techniques: Marker, Control

Journal: bioRxiv

Article Title: CCR1 Mediates Müller Cell Activation and Photoreceptor Cell Death in Macular and Retinal Degeneration

doi: 10.1101/2023.01.03.522541

Figure Lengend Snippet: ( A ) M1 and M2a hMdɸs were immunostained for CCR1 (green); magnified views are shown below. ( B ) Summary of the percentage of CCR1-postive cells measured using cell sorting analysis of CCR1-stained M1 hMdɸ and M2a hMdɸs; (n=5 per group, t Student’s t -test). ( C ) Summary of ROS levels measured in untreated M2a hMdɸs and M2a hMdɸs treated with 0.5 or 5 µM BX471; (n=4 per group, one-way ANOVA with multiple comparisons). ( D ) Summary of the migration of monocytes treated or not with 10 µM BX471 that migrated toward the M2a hMdɸs using a Boyden chamber; (n=3 per group, one-way ANOVA with multiple comparisons). Data shown as mean ± SEM. p-Values indicated by * p <0.05 and ** p <0.01. Scale bars: 50 µm (A) and 20 µm (insets in A).

Article Snippet: Immediately after photic injury was induced, the mice received subcutaneous injections of either the CCR1-specific antagonist BX471 (50 mg/kg body weight; Tocris, Bristol, UK) or vehicle (40% cyclodextrin in saline) every 12 hours for 7 days.

Techniques: FACS, Staining, Migration

Journal: Transplant international : official journal of the European Society for Organ Transplantation

Article Title: Intracellular sirolimus concentration is reduced by tacrolimus in human pancreatic islets in vitro.

doi: 10.1111/tri.12617

Figure Lengend Snippet: Figure 1 Intracellular concentrations of TAC in human islets. Human islets were cultured with TAC (10 or 30 lg/l), SRL (10 or 30 lg/l), or the combi- nation thereof for 24–48 h before the intracellular concentration of TAC was measured in islet lysate and normalized to total protein as detailed in methods. Data are presented as the mean SD, n = 6 for each group. TAC, tacrolimus; SRL, sirolimus; *P < 0.04; ** P < 0.007; *** P < 0.0006.

Article Snippet: The islets were exposed to 10 and 30 lg/l of tacrolimus (Santa Cruz Biotechnology, Dallas, TX, USA) or sirolimus (Toronto Research Chemicals, Toronto, Ontario, Canada) or the combination thereof for 24 h and 48 h at 37 °C (5% CO2).

Techniques: Cell Culture, Concentration Assay

Journal: Transplant international : official journal of the European Society for Organ Transplantation

Article Title: Intracellular sirolimus concentration is reduced by tacrolimus in human pancreatic islets in vitro.

doi: 10.1111/tri.12617

Figure Lengend Snippet: Figure 2 Intracellular concentrations of SRL in human islets. Human islets were cultured with TAC (10 or 30 lg/l), SRL (10 or 30 lg/l), or the combi- nation thereof for 24–48 h before the intracellular concentration of SRL was measured in islet lysate and normalized to total protein as detailed in methods. Data are presented as the mean SD, n = 6 for each group. TAC: tacrolimus; SRL: sirolimus; *** P < 0.001; **** P < 0.0001.

Article Snippet: The islets were exposed to 10 and 30 lg/l of tacrolimus (Santa Cruz Biotechnology, Dallas, TX, USA) or sirolimus (Toronto Research Chemicals, Toronto, Ontario, Canada) or the combination thereof for 24 h and 48 h at 37 °C (5% CO2).

Techniques: Cell Culture, Concentration Assay

Journal: Transplant international : official journal of the European Society for Organ Transplantation

Article Title: Intracellular sirolimus concentration is reduced by tacrolimus in human pancreatic islets in vitro.

doi: 10.1111/tri.12617

Figure Lengend Snippet: Figure 3 Effect of CsA on intracellular concentration of SRL in human islets. Human islets were cultured with the combination of SRL (30 lg/l) and CsA (5 lg/ml), or the drug alone for 24 h before the intracellular concentration of SRL (a) or CsA (b) was measured in islet lysate and nor- malized to total protein as detailed in methods. Data are calculated as percentages of control and are presented as mean SD, n = 6 for each group. CsA, cyclosporine A; SRL, sirolimus.

Article Snippet: The islets were exposed to 10 and 30 lg/l of tacrolimus (Santa Cruz Biotechnology, Dallas, TX, USA) or sirolimus (Toronto Research Chemicals, Toronto, Ontario, Canada) or the combination thereof for 24 h and 48 h at 37 °C (5% CO2).

Techniques: Concentration Assay, Cell Culture, Control

Journal: Transplant international : official journal of the European Society for Organ Transplantation

Article Title: Intracellular sirolimus concentration is reduced by tacrolimus in human pancreatic islets in vitro.

doi: 10.1111/tri.12617

Figure Lengend Snippet: Figure 4 The effect of SRL, TAC, or CsA on phosphorylation of p70S6k in islets. Human islets were cultured with TAC (30 lg/l), SRL (30 lg/l), or the combination thereof for 24 h before the presence of p- p70s6k was assessed by the cell-signaling Bio-Plex assay in human islet cell lysate and normalized to total protein (a). In a parallel experiment, human islets were cultured with SRL (30 lg/l), CsA (5 lg/ml), or the combination thereof for 24 h before p-p70S6k was detected in the lysate and normalized to total protein. Data are calculated as ratio to control and are presented as mean SD, n = 3–6 for each group. TAC, tacrolimus; SRL, sirolimus; CsA, cyclosporine A, ** P < 0.01, **** P < 0.0001.

Article Snippet: The islets were exposed to 10 and 30 lg/l of tacrolimus (Santa Cruz Biotechnology, Dallas, TX, USA) or sirolimus (Toronto Research Chemicals, Toronto, Ontario, Canada) or the combination thereof for 24 h and 48 h at 37 °C (5% CO2).

Techniques: Phospho-proteomics, Cell Culture, Plex Assay, Control

Journal: Transplant international : official journal of the European Society for Organ Transplantation

Article Title: Intracellular sirolimus concentration is reduced by tacrolimus in human pancreatic islets in vitro.

doi: 10.1111/tri.12617

Figure Lengend Snippet: Figure 5 Oxygen consumption rates (OCR) in human islets after treatment of TAC, SIR, or SIR+TAC. Human islets were cultured with TAC (30 lg/l), SIR (30 lg/l), or a combination thereof for 24 h before the glucose-stimulated OCR was measured as indicated in methods. OCR is expressed as percentage of baseline and is presented as the mean SD, n = 6 for each group. TAC, tacrolimus; SRL, sirolimus, ** P < 0.01.

Article Snippet: The islets were exposed to 10 and 30 lg/l of tacrolimus (Santa Cruz Biotechnology, Dallas, TX, USA) or sirolimus (Toronto Research Chemicals, Toronto, Ontario, Canada) or the combination thereof for 24 h and 48 h at 37 °C (5% CO2).

Techniques: Cell Culture

Journal: Transplant international : official journal of the European Society for Organ Transplantation

Article Title: Intracellular sirolimus concentration is reduced by tacrolimus in human pancreatic islets in vitro.

doi: 10.1111/tri.12617

Figure Lengend Snippet: Figure 6 Expression of ABCB1 (Pgp), OATP1B1, and CYP3A4 in human islets. Human islets were cultured for 24 h before the expression of the drug transporter (ABCB1(Pgp) and OATP1B1), and the metabolic enzyme CYP3A4 was evaluated. RNA was prepared and subjected to qPCR as detailed in methods. The reference gene index is calculated by the mean of ALAS1, B2M, and RPL13A expression and used to normalize the expression of target genes in isolated hepatocytes relative to the mRNA level of ABCB1(Pgp), OATP1B1, and CYP3A4 in human islets (a). OATB1 mRNA expression in human islets was normalized to reference gene index after exposure to TAC (30 lg/l), SRL (30 lg/l), or a combination thereof for 24 h (b). Represen- tative immunofluorescence image of dispersed human islets stained for insulin (green), ABCB1(Pgp) (red) and nuclear staining with DAPI (blue) (c), or glucagon (green), ABCB1(Pgp) (red) and nuclear staining wit DAPI (blue) (d). Data are presented as mean SD, n = 4–5 for each group. TAC, tacroli- mus; SRL, sirolimus; *P < 0.05; ** P < 0.01.

Article Snippet: The islets were exposed to 10 and 30 lg/l of tacrolimus (Santa Cruz Biotechnology, Dallas, TX, USA) or sirolimus (Toronto Research Chemicals, Toronto, Ontario, Canada) or the combination thereof for 24 h and 48 h at 37 °C (5% CO2).

Techniques: Expressing, Cell Culture, Isolation, Staining

Journal: Cells

Article Title: Photochemotherapy Induces Interferon Type III Expression via STING Pathway

doi: 10.3390/cells9112452

Figure Lengend Snippet: The Stimulator of Interferon Genes (STING) pathway is activated in Hut78 by 8–MOP + UVA treatment. Downregulation of alleged pathway elements by specific small interfering RNA (siRNA) or by a chemical inhibitor result in decreased IFNL1 expression. Expression of IFNL1 following 8–MOP + UVA treatment combined with ( A ) STING downregulation by siRNA, ( B ) cyclic GMP-AMP synthase (cGAS) downregulation by siRNA, ( C ) TBK1 inhibition by BX795 chemical inhibitor, ( D ) IRF3 downregulation by siRNA and ( E ) IRF1 downregulation by siRNA. Cell viability for respective treatments is presented in ( F ) for STING-siRNA, ( G ) for cGAS-siRNA, ( H ) for BX795-mediated TBK1 inhibition, ( I ) for IRF3-siRNA and ( J ) for IRF1-siRNA. ( K ) Transfection efficiencies for various siRNAs. Error bars represent ± SEM of the indicated N repeats. Statistics—normal distribution, paired t -test: ( A – E ) and skewed distribution, paired Wilcoxon: (F–J) . Choice of the statistical test was made based on the type of data distribution (see Methods). * p < 0.1, ** p < 0.05, ns–not significant.

Article Snippet: TBK1 inhibitor BX795 (Tocris, Abingdon, UK) and ataxia-telangiectasia and Rad3-related (ATR) kinase inhibitor AZD6738 (Selleckchem, Houston, TX, USA) were added immediately after UVA irradiation.

Techniques: Small Interfering RNA, Expressing, Inhibition, Transfection

Journal: Developmental Cell

Article Title: Selective Autophagy of Mitochondria on a Ubiquitin-Endoplasmic-Reticulum Platform

doi: 10.1016/j.devcel.2019.06.016

Figure Lengend Snippet: Inducers of Mitophagy in Mammalian Cells and IVM Action (A–C) HEK293 cells treated for 2 h with 15 μM IVM, for 8 h with 10 μM oligomycin and 10 μM antimycin A (OA), or for 8 h with 4 μM CCCP and stained for LC3 (A) or immunoblotted for LC3. (B) Cells treated as above, stained for TOMM20 (MITO) and WIPI2. (D) Live-cell imaging of HEK293 cells expressing CFP-LC3 and mCherry-MITO and treated with 15 μM IVM. Shown are selected time points; arrows mark mitochondrial fragments targeted by LC3. See for the whole sequence. Scale bar, 10 μm. (E–G) OCR of HEK293 cells treated with IVM. Time course and percent inhibition are plotted as shown. (H) HEK293 cells treated with 15 μM IVM and 40 μm mdiv-1 as indicated for 45 min, stained for ubiquitin, and puncta per cell determined. Means of two experiments done in duplicate are shown. (I and J) HEK293 cells treated with siRNA against DNM1L or with a non-targeting (NT) control for 72 h. After incubation with 15 μM IVM, cells were stained for ubiquitin and puncta per cell were determined. Means of two experiments done in duplicate are shown. (K) HEK-293 cells untreated or treated with 15 μM IVM for 45 min, lysed, and immunoprecipitated with ubiquitin antibodies. Samples were analyzed by mass spectrometry and the top 11 hits enriched after IVM treatment are shown. (L) Samples as in (K) were blotted for CIAP1, TRAF2, or β-COP (a loading control). (M) HEK293 cells treated with siRNA against TRAF2 or NT control for 72 h were treated as in (K) and immunoblotted for TRAF2. (N) HEK-293 cells treated with siRNA against CIAP1, CIAP2 and TRAF2 or with NT control for 96 h. After treatment with 15 μM IVM and staining for ubiquitin, puncta per cell were determined. Means of three experiments done in duplicate are shown. (O) Parallel samples were lysed and blotted for CIAP1, TRAF2 or β-COP. (P) Cells downregulated for CIAP1, CIAP2, and TRAF2 as in (N) and (O) above were incubated with IVM and the levels of mitochondrial proteins TOMM20 and MITOFUSIN 2 were determined by immunoblots and quantitated.

Article Snippet: BX-795, oligomycin, and CCCP were purchased from Tocris Biosciences.

Techniques: Staining, Live Cell Imaging, Expressing, Sequencing, Inhibition, Ubiquitin Proteomics, Control, Incubation, Immunoprecipitation, Mass Spectrometry, Western Blot

Journal: Developmental Cell

Article Title: Selective Autophagy of Mitochondria on a Ubiquitin-Endoplasmic-Reticulum Platform

doi: 10.1016/j.devcel.2019.06.016

Figure Lengend Snippet:

Article Snippet: BX-795, oligomycin, and CCCP were purchased from Tocris Biosciences.

Techniques: Recombinant, Software