Journal: bioRxiv

Article Title: Multiple NTS Neuron Populations Synergistically Suppress Physiologic Food Intake but are Dispensable for the Response to VSG

doi: 10.1101/2022.12.23.521804

Figure Lengend Snippet: ( A ) Representative images showing mCherry-IR (hM3Dq, red) and FOS-IR (green) in the NTS of Lepr Dq , Calcr Dq and LC Dq mice following treatment with saline (Lepr Dq VEH) or CNO (Lepr Dq CNO, Calcr Dq CNO and LC Dq CNO IP, 1 mg/kg) for 2 hours before perfusion. NTS: nucleus of the solitary tract, AP: area postrema, cc: central canal. All images were taken at the same magnification; scale bar equals 150 μm. ( B-D ) Food intake in chow-fed Lepr Dq (Lepr), Calcr Dq (Calcr) and LC Dq (LC) mice over the first 4 hours of the dark phase following vehicle (Veh) or CNO injection (IP, 1 mg/kg) when provided with chow ( B , n=25 for Veh group; n=20 for both Lepr and Calcr groups; n=8 for LC group) or HFD ( C , n=21 for Veh group; n=6 for Lepr group; n=7 for Calcr group; n=8 for LC group). ( D ) Food intake for the same groups of mice over the first 6 hours of refeeding during the light cycle following an overnight fast ( D , n=23 for Veh group; n=8 for Lepr group; n=7 for Calcr group; n=7 for LC group) following with CNO (IP, 1 mg/kg) or vehicle (Veh). ( E-F ) Control (Ctrl, n=5) or Lepr Dq (Lepr, n=5), Calcr Dq (Calcr, n=5) and LC Dq (LC, n=8) mice were treated with vehicle for three baseline days, followed by two days of twice daily treatment with CNO (IP 1 mg/kg), followed by two additional days of Veh treatment. Daily food intake ( E ) and body weight (shown as change from baseline) ( F ). Vehicle and CNO treatment are denoted on the graphs. ( G, H ) CTA assays: Mice were treated with vehicle (Veh, n=10), LiCl (IP, 126 mg/kg, n=9), or CNO (IP, 1 mg/kg, n=5) during exposure to a novel tastant (HFD) ( G ) or saccharin ( H ) paired with vehicle (Veh, n=13), LiCl (IP, 126 mg/kg, n=14), or CNO (IP, 1 mg/kg, n=5). Shown is mean +/-SEM; Two-way ANOVA, sidak’s multiple comparisons test was used; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 vs vehicle or Ctrl; # p<0.05, ## p<0.01, ### p<0.001, #### p<0.0001 vs LC.

Article Snippet: Following the 60 min exposure to saccharin, mice were injected intraperitoneally with the desired stimulus (vehicle control (0.9% NaCl), lithium chloride (0.3 M, 203637, Sigma)) in a volume equivalent to 1% of each animal’s body weight (10 mL/g), or CNO (1 mg/kg, 4936, Tocris Bioscience) Access to the two saccharin bottles continued for an additional 1 h, followed by the return of normal water bottles.

Techniques: Saline, Injection

Journal: bioRxiv

Article Title: Multiple NTS Neuron Populations Synergistically Suppress Physiologic Food Intake but are Dispensable for the Response to VSG

doi: 10.1101/2022.12.23.521804

Figure Lengend Snippet: ( A ) Representative images showing mCherry-IR (hM3Dq, red) and FOS-IR (green) in the NTS of LCK Dq mice following treatment with CNO (IP, 1 mg/kg) 2 hours before perfusion. Scale bar equals 150 μm. NTS: nucleus of the solitary tract, AP: area postrema, cc: central canal. ( B-D ) Food intake in LCK Dq mice over the first 4 hours of the dark phase during expose to chow ( B ) or HFD ( C ) (n=9 per group), and during the first 6 hours of refeeding in the light cycle following an overnight fast ( D , n=7 per group) during treatment with CNO (IP, 1 mg/kg) or vehicle. ( E-F ) Control (Ctrl, n=7) or LCK Dq mice (n=7) mice were treated vehicle (Veh) for one days, followed by two days with CNO (1 mg/kg, IP, twice per day) and daily food intake ( E ) and body weight (change from baseline) ( F ) were determined. Veh and CNO treatment are denoted on the graphs. ( G, H ) Mice were treated with Veh, LiCl, or CNO (IP, 1 mg/kg) during exposure to a novel tastant (HFD ( G ) or saccharin ( H )); n=6 per group. Shown is mean +/-SEM; Two-way ANOVA, sidak’s multiple comparisons test was used in all figures, except panels G and H where unpaired T test was used; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 vs Veh or Ctrl.

Article Snippet: Following the 60 min exposure to saccharin, mice were injected intraperitoneally with the desired stimulus (vehicle control (0.9% NaCl), lithium chloride (0.3 M, 203637, Sigma)) in a volume equivalent to 1% of each animal’s body weight (10 mL/g), or CNO (1 mg/kg, 4936, Tocris Bioscience) Access to the two saccharin bottles continued for an additional 1 h, followed by the return of normal water bottles.

Techniques:

Journal: American Journal of Cancer Research

Article Title: Shikonin enhances the antitumor effect of cabazitaxel in prostate cancer stem cells and reverses cabazitaxel resistance by inhibiting ABCG2 and ALDH3A1

doi:

Figure Lengend Snippet: Shikonin reverses the resistant-state of caba-DU145 through inhibiting the expression of ABCG2 and ALDH3A1. A and B. Pretreatment of caba-DU145 cells with ABCG2 inhibitor Ko143 for 7 days led to an increased cabazitaxel sensitivity as shown by the viability assay and the proliferation assay. C and D. Pretreatment of caba-DU145 cells with ALDH3A1 inhibitor CB29 for 7 days also led to an increased sensitivity against cabazitaxel. The caba-DU145 cells were treated with cabazitaxel for another 72 hours after pretreatment with Ko143 and CB29. E. Annexin V and 7-AAD staining demonstrated an enhanced rate of apoptosis in caba-DU145 cells following treatment with Ko143 and CB29 compared to cabazitaxel (3 nM) alone. Ko143 and CB29 alone cannot induce apoptosis. F. Combination of shikonin with cabazitaxel increased the rate of apoptosis in caba-DU145 cells based on the annexin V and 7-AAD staining. G. Proposed model for apoptosis induction by shikonin and the mechanism of reversing the cabazitaxel-related resistance. The results are presented as the means ± SEM of values obtained in three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: Two inhibitors of ABCG2 and ALDH3A1 were used namely Ko143 (Tocris Biosciences, Bio-Techne GmbH, Wiesbaden-Neuenstadt, Germany) at 1 μM, and CB29 (Sigma Aldrich) at 32 μM, respectively.

Techniques: Expressing, Viability Assay, Proliferation Assay, Staining

Journal: Frontiers in Immunology

Article Title: Retinal response to systemic inflammation differs between sexes and neurons

doi: 10.3389/fimmu.2024.1340013

Figure Lengend Snippet: Antagonism of TNFR1 and P2X7R rescues RGCs from systemic inflammation. (A) Isodensity maps showing the distribution of Brn3a + RGCs in retinas of intact male mice and mice treated with LPS+vehicle, LPS and TNFR1 antagonist (αTNFR1), LPS and P2X7R antagonist (αP2X7R), and LPS and αP2X7R + αTNFR1. Retinas were analysed 7 days after the injection of LPS. (B) Column graph showing the mean total number ± SD of Brn3a + RGCs the same groups. *Significant vs. intact (*** p <0.001; **** p <0.0001); σ Significant between groups ( σ p <0.05; σσσ p <0.001; σσσσ p <0.0001). One-way ANOVA within sexes, post-hoc Tukey’s test. (C) Column graph showing the averaged percentage ± SD of Brn3a + RGCs in the same groups as before with respect to intact retinas (100%). *Significant differences between females and males (** p <0.01; *** p <0.001; Two-way ANOVA Šidák’s multiple comparison test (treatment p <0.0001; sex p <0.0001). F: females, M: males. I: intact, V: vehicle.

Article Snippet: Madrid, Spain] and TNFR1 antagonist [12 mg/kg i.p. in 5% of DMSO-saline; R7050, Tocris Bioscience; Bio-Techne R&D Systems, Madrid, Spain], as previously published , were both injected intraperitoneally in a final volume of 200 μL.

Techniques: Injection, Comparison

Journal: Frontiers in Immunology

Article Title: Retinal response to systemic inflammation differs between sexes and neurons

doi: 10.3389/fimmu.2024.1340013

Figure Lengend Snippet: Transient impairment of retinal functionality after systemic inflammation: effect of TNFR1 and P2X7R antagonism. (A) Electroretinographic waves from female and male mice recorded before (PRE) and 3 and 7 days after being treated with LPS + vehicle, LPS and TNFR1 antagonist (αTNFR1), LPS and P2X7R antagonist (αP2X7R), and LPS and αP2X7R + αTNFR1. (B) ERG quantification bar graphs showing the mean wave amplitude (µV ± SD). Control amplitudes are baseline recordings (pre). * vs. baseline values (* p <0.05; ** p <0.01***; p <0.001; **** p <0.0001); φ 3 rd vs. 7 th day within the same group ( φφφ p <0.001; φφφφ p <0.0001). σ Between different groups ( σ p <0.05; σσ p <0.01; σσσ p <0.001; σσσσ p <0.0001). † p <0.001 females vs. males at the same time point and treatment. Two-way ANOVA Šidák’s multiple comparison test (treatment p <0.0001; sex p >0.05).

Article Snippet: Madrid, Spain] and TNFR1 antagonist [12 mg/kg i.p. in 5% of DMSO-saline; R7050, Tocris Bioscience; Bio-Techne R&D Systems, Madrid, Spain], as previously published , were both injected intraperitoneally in a final volume of 200 μL.

Techniques: Control, Comparison

Journal: Journal of Lipid Research

Article Title: Ligand-dependent interactions between SR-B1 and S1PR1 in macrophages and atherosclerotic plaques

doi: 10.1016/j.jlr.2024.100541

Figure Lengend Snippet: The S1PR1 selective agonist, SEW2871, stimulates and the selective antagonist Ex26 inhibits the interaction between SR-B1 and S1PR1. Thioglycolate-elicited peritoneal macrophages from S1pr1 eGFP/eGFP mice were (A) pre-treated with either the anti-SR-B1 antiserum (1.5 μg/ml) or the SR-B1 inhibitor BLT-1 (150 nM) for 45 min or with control non-immune rabbit serum and DMSO vehicle (where not indicated). After the pre-treatment period, HDL (100 μg protein/ml) or the S1PR1 selective agonist, SEW2871 (1 μM) or no stimulus (untreated) was added for an additional 30 min before cells were fixed. B: Cells were treated with either Ex26 (10 μM, added as a 1,000× stock in DMSO) or DMSO vehicle control for 60 min, at which point HDL (100 μg protein/ml) or vehicle was added and cells were incubated for a further 30 min. After the 30 min incubation with HDL, cells were washed, fixed, and subjected to Duolink PLA and DAPI staining, imaging, and analysis as in the legend to Figs. 1 and . Each symbol represents cells isolated from a different mouse. Data were analyzed using Kruskal-Wallis test; P -values are indicated above each graph. C: Thioglycolate-elicited peritoneal macrophages from S1pr1 eGFP/eGFP mice were incubated for 60 min with 10 μM Ex26 or DMSO vehicle control followed by treatment without or with HDL (100 μg protein/ml) for 30 min as indicated. Cells were then washed and fixed, and PLA staining (red) was carried out followed by staining for caveolin-1 using a mouse anti-caveolin-1 antibody and Alexa488-conjugated anti-mouse secondary antibody (green) and DAPI staining (blue). Panels on the right show zoomed-in views of the boxed areas of the merged images of HDL-treated control and Ex26-treated cells. eGFP, enhanced green fluorescent protein; PLA, proximity ligation assay; S1PR, sphingosine-1-phosphate receptor; SR-B1, scavenger receptor class B, type I.

Article Snippet: The following compounds were used: anti-SR-B1 blocking antibody, KKB-1, 1.5 μg/ml; originally generated by Karen Kozarsky, SwanBio Therapeutics and provided by Monty Krieger, Massachusetts Institute of Technology) ( ); BLT-1 (150 nM, Millipore Sigma Canada, Oakville, ON, Canada; catalog #SML0059); S1P (10 nM, Avanti Polar Lipids, Inc, Birmingham AL; catalog # 860492P); HDL (100 μg/ml; Athens Research And Technology, Athens, GA; catalog #12-16-080412); SEW2871 (1 μM, Cayman Chemicals, Ann Arbor, MI; catalog #10006440), and Ex26 (10 μM, Tocris Bioscience, Bio-Techne Canada, Toronto, ON, Canada; catalog #5833/10).

Techniques: Control, Incubation, Staining, Imaging, Isolation, Proximity Ligation Assay

Journal: Journal of Lipid Research

Article Title: Ligand-dependent interactions between SR-B1 and S1PR1 in macrophages and atherosclerotic plaques

doi: 10.1016/j.jlr.2024.100541

Figure Lengend Snippet: The SR-B1 inhibitor, BLT-1, the S1PR1 antagonist Ex26, and S1PL treatment of HDL all reduce HDL-mediated cholesterol efflux from macrophages. Thioglycolate-elicited peritoneal macrophages were collected from wild type C57BL/6J mice, cultured in DMEM containing 10% FBS, 2 mM L-glutamine, 50 μg/ml penicillin, and 50 U/ml streptomycin, for 16 h prior to changing the medium to phenol red-free DMEM containing 3% newborn calf lipoprotein-deficient serum, 2 mM L-glutamine, 50 μg/ml penicillin, and 50 U/ml streptomycin, for an additional 16 h as described above. Cholesterol efflux was measured using the cell-based fluorescent Cholesterol Efflux Assay kit. After loading cells with the fluorescent tracer for 1 h, cells were washed and efflux was initiated by addition of the efflux acceptor medium. Efflux was monitored using either no acceptor or control- or S1PL-treated HDL (each at 100 μg protein/ml) as cholesterol acceptors. Some cells were also treated with Ex26 (10 μM) or BLT-1 (150 nM) during efflux. Cholesterol efflux was measured as the amount of cholesterol tracer appearing in the medium at the end of 3 h, as a % of the total cholesterol tracer (cells + medium). Each symbol represents cells isolated from a different mouse (n = 4); bars represent means and error bars represent standard errors. Data was analyzed by the Kruskal-Wallis test; P value is indicated. S1PL, S1PL-lyase; S1PR, sphingosine-1-phosphate receptor; SR-B1, scavenger receptor class B, type I.

Article Snippet: The following compounds were used: anti-SR-B1 blocking antibody, KKB-1, 1.5 μg/ml; originally generated by Karen Kozarsky, SwanBio Therapeutics and provided by Monty Krieger, Massachusetts Institute of Technology) ( ); BLT-1 (150 nM, Millipore Sigma Canada, Oakville, ON, Canada; catalog #SML0059); S1P (10 nM, Avanti Polar Lipids, Inc, Birmingham AL; catalog # 860492P); HDL (100 μg/ml; Athens Research And Technology, Athens, GA; catalog #12-16-080412); SEW2871 (1 μM, Cayman Chemicals, Ann Arbor, MI; catalog #10006440), and Ex26 (10 μM, Tocris Bioscience, Bio-Techne Canada, Toronto, ON, Canada; catalog #5833/10).

Techniques: Cell Culture, Control, Isolation

Journal: Journal of Lipid Research

Article Title: Ligand-dependent interactions between SR-B1 and S1PR1 in macrophages and atherosclerotic plaques

doi: 10.1016/j.jlr.2024.100541

Figure Lengend Snippet: SR-B1 and S1PR1 interact in atherosclerotic plaques of high-fat diet-fed ApoE KO/KO mice. Control ApoE KO/KO mice in which S1PR1 was not GFP tagged ( S1pr1 WT/WT / ApoE KO/KO mice), and S1pr1 eGFP/eGFP ApoE KO/KO mice were fed a high fat, high cholesterol diet for 8 weeks, beginning at 15 weeks of age. S1pr1 eGFP/eGFP / ApoE KO/KO mice were then treated with Ex26 (30 mg/kg in DMSO) or DMSO vehicle and mice were euthanized, and tissues were harvested 12 h later. Adjacent cross-sections in the aortic sinus were collected and stained with oil red O (lipid) and hematoxylin (nuclei) to detect lipid-rich atherosclerotic plaques or were fixed, permeabilized, and subjected to Duolink PLA staining using antibodies against SR-B1 and GFP as described in the ‘ ’ section. PLA-stained sections were counterstained with DAPI for nuclear DNA. A: Representative images of oil red O and hematoxylin-stained atherosclerotic plaques in the aortic sinuses. B: Images of adjacent PLA-stained sections of plaques showing DAPI, PLA signal, and merged DAPI and PLA signals. Scale bars (A, B) represent 20 μm. C: Quantification of PLA signal in the atherosclerotic plaques of S1pr1 WT/WT /ApoE KO/KO and DMSO vehicle or Ex26-treated S1pr1 eGFP/eGFP /ApoE KO/KO mice was performed by counting the average of PLA signal punctae within atherosclerotic plaques across three fields of view per plaque of three sections for each mouse and dividing by the number of DAPI-stained nuclei. Each data point represents data from a different mouse. Data were analyzed using the Kruskal-Wallis test; P value is indicated above the graph. D: Confocal images of PLA (red) and anti-Mac3 immunofluorescence (green) and DAPI (blue) co-stained images of atherosclerotic plaques from control S1pr1 WT/WT /ApoE KO/KO and DMSO vehicle or Ex26-treated S1pr1 eGFP/eGFP /ApoE KO/KO mice. Images in the bottom row correspond to zoomed-in views of the boxed areas. eGFP, enhanced green fluorescent protein; PLA, proximity ligation assay; S1PR, sphingosine-1-phosphate receptor; SR-B1, scavenger receptor class B, type I.

Article Snippet: The following compounds were used: anti-SR-B1 blocking antibody, KKB-1, 1.5 μg/ml; originally generated by Karen Kozarsky, SwanBio Therapeutics and provided by Monty Krieger, Massachusetts Institute of Technology) ( ); BLT-1 (150 nM, Millipore Sigma Canada, Oakville, ON, Canada; catalog #SML0059); S1P (10 nM, Avanti Polar Lipids, Inc, Birmingham AL; catalog # 860492P); HDL (100 μg/ml; Athens Research And Technology, Athens, GA; catalog #12-16-080412); SEW2871 (1 μM, Cayman Chemicals, Ann Arbor, MI; catalog #10006440), and Ex26 (10 μM, Tocris Bioscience, Bio-Techne Canada, Toronto, ON, Canada; catalog #5833/10).

Techniques: Control, Staining, Immunofluorescence, Proximity Ligation Assay