Journal: Cells

Article Title: Inhibiting the P2X4 Receptor Suppresses Prostate Cancer Growth In Vitro and In Vivo, Suggesting a Potential Clinical Target

doi: 10.3390/cells9112511

Figure Lengend Snippet: P2X4R is functionally expressed in PCa cell lines. Total mRNAs were isolated from three human PCa cell lines: PC3, LNCaP, and C4-2B4 cells. Individual quantitative RT-PCRs were performed to reveal the gene expression profile of ( A ) P2X and ( B ) P2Y receptors in these PCa cells. CT values < 35 were regarded as the transcription expression of P2X and P2Y receptors. ( C ) A heatmap showing the gene expression pattern variance was built from reciprocals of CT values, using the ‘GenePattern’ web software. Colour scale = 1/CT values. To understand whether P2X4R was functional, PC3 cells were pretreated with P2X4R specific antagonists, and then 50 μM ATP (to maximally activate P2X4R but not P2X7R)-induced calcium influx was examined using the Fluo-4 Direct™ agent. All data were normalized to both the baseline and ionomycin-induced full calcium influx. The peak fluorescence intensity was compared among the vehicle, ATP, and ATP plus ( D ) 1.0-μM 5-BDBD or ( F ) 1.5-μM PSB-12062. The representative ATP-induced calcium influx curve over a 450 s period with or without ( E ) 5-BDBD or ( G ) PSB-12062 antagonist pretreatments. Data are the mean ± SD, n = six biological repeats, one-way ANOVA with post hoc Tukey test, * p < 0.05, *** p < 0.001.

Article Snippet: The PC3 prostate cancer cell line (Prostate Specific Antigen (PSA) non-expressing, androgen-independent) (ATCC, Manassas, VA, USA) was stably transfected with a firefly luciferase gene luc2 (pGL4.51 [luc2/CMV/Neo] vector, Promega, Madison, WI, USA) using a Gene PulserTM electroporator (Bio-Rad, CA, USA).

Techniques: Isolation, Gene Expression, Expressing, Software, Functional Assay, Fluorescence

Journal: Cells

Article Title: Inhibiting the P2X4 Receptor Suppresses Prostate Cancer Growth In Vitro and In Vivo, Suggesting a Potential Clinical Target

doi: 10.3390/cells9112511

Figure Lengend Snippet: Effects of P2X4R inhibition on PCa proliferation, viability, and apoptosis. The effects of inhibiting P2X4R with specific antagonists (1.0 μM 5-BDBD or 1.5 μM PSB-12062) on the proliferation and viability of PCa cells (PC3 and C4-2B4) up to 96 h were examined by using the ( A – D ) CyQUANT ® cell proliferation assay and ( E – H ) alamarBlue™ cell viability assay. ( I , J ) The apoptotic activity after a 24-h treatment of antagonists or doxorubicin (as a positive control) was measured using the Cell Meter™ Caspase 3/7 apoptosis assay. Data are the mean ± SD, n = three biological repeats, Student’s t -test, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The PC3 prostate cancer cell line (Prostate Specific Antigen (PSA) non-expressing, androgen-independent) (ATCC, Manassas, VA, USA) was stably transfected with a firefly luciferase gene luc2 (pGL4.51 [luc2/CMV/Neo] vector, Promega, Madison, WI, USA) using a Gene PulserTM electroporator (Bio-Rad, CA, USA).

Techniques: Inhibition, CyQUANT Assay, Proliferation Assay, Viability Assay, Activity Assay, Positive Control, Apoptosis Assay

Journal: Cells

Article Title: Inhibiting the P2X4 Receptor Suppresses Prostate Cancer Growth In Vitro and In Vivo, Suggesting a Potential Clinical Target

doi: 10.3390/cells9112511

Figure Lengend Snippet: Inhibiting P2X4R with antagonists impairs PCa cell mobility. Changes in the migration and invasiveness of PC3 and C4-2B4 cells in response to the 1.0 μM 5-BDBD or 1.5 μM PSB-12062 treatment were examined using a scratch test and Transwell assays, respectively. For the scratch test migration assay, percentage areas closed after scratch were compared at 6, 12, and 18 h between ( A ) 5-BDBD- or ( B ) PSB-12062-treated and vehicle groups. ( C ) A representative image of the scratch test assay. Scale bar = 100 μm. For C4-2B4 cells, a Transwell migration assay was adopted with 10% FBS DMEM as the chemoattractant in the lower chamber. Percentage areas that migrated within 16 h were measured and compared between ( D ) 5-BDBD- or ( E ) PSB-12062-treated groups and the appropriate vehicle groups. ( F – I ) The invasion assay was performed by examining the ability of PC3 and C4-2B4 cells invading Matrigel-coated Transwell inserts over a 72-h period and comparing antagonist and vehicle groups. ( J ) A representative comparison image of the PC3 cells invading Matrigel-coated Transwell inserts, with or without P2X4R antagonist treatments. Scale bar = 200 μm. Data are the mean ± SD, n = three biological repeats, Student’s t-test, * p < 0.05, ** p < 0.01.

Article Snippet: The PC3 prostate cancer cell line (Prostate Specific Antigen (PSA) non-expressing, androgen-independent) (ATCC, Manassas, VA, USA) was stably transfected with a firefly luciferase gene luc2 (pGL4.51 [luc2/CMV/Neo] vector, Promega, Madison, WI, USA) using a Gene PulserTM electroporator (Bio-Rad, CA, USA).

Techniques: Migration, Wound Healing Assay, Transwell Migration Assay, Invasion Assay, Comparison

Journal: Cells

Article Title: Inhibiting the P2X4 Receptor Suppresses Prostate Cancer Growth In Vitro and In Vivo, Suggesting a Potential Clinical Target

doi: 10.3390/cells9112511

Figure Lengend Snippet: P2X4R antagonist 5-BDBD delays the PCa growth in vivo. ( A ) Schematic outline of the in vivo study. Briefly, six-week old BALB/cAnNCrl immunocompromised mice were subcutaneously (s.c) injected with 5 × 10 6 PC3 cell suspension with 50% PBS + 50% Matrigel. After randomization, the mice were daily intraperitoneally (i.p) injected with the P2X4R antagonist 5-BDBD at a dosage of 10 mg/kg or vehicle (20 μL DMSO), starting from day one until being euthanized three weeks post-tumour cell inoculation. ( B ) Tumour sizes were measured by using vernier calipers twice a week, and then tumour volumes were calculated as ellipsoids and compared between treated and vehicle groups. ( C ) Days needed for tumours to reach 200 mm 3 were also compared between treated and vehicle groups. Data are the mean ± SD, n = 7, Student’s t-test, * p < 0.05, ** p < 0.01. Representative images of ( D ) tumour-bearing mice and ( E ) dissected tumours, comparing treated and vehicle groups at endpoint. ( F ) Dissected tumours were then cut in half and revealed a necrotic centre in the tumour from the vehicle control group but not the treated group. ( G ) This was further confirmed by H&E staining of the tumours. Scale bar = 1 cm. ( H ) A statistical analysis with a Chi-square test suggested a significantly reduced presence of necrosis in the 5-BDBD-treated group compared to the vehicle. n = 7, ** p < 0.01.

Article Snippet: The PC3 prostate cancer cell line (Prostate Specific Antigen (PSA) non-expressing, androgen-independent) (ATCC, Manassas, VA, USA) was stably transfected with a firefly luciferase gene luc2 (pGL4.51 [luc2/CMV/Neo] vector, Promega, Madison, WI, USA) using a Gene PulserTM electroporator (Bio-Rad, CA, USA).

Techniques: In Vivo, Injection, Suspension, Control, Staining

Journal: Circulation

Article Title: Detection of Cardiac Allograft Rejection and Response to Immunosuppressive Therapy With Peripheral Blood Gene Expression

doi: 10.1161/01.cir.0000150539.72783.bf

Figure Lengend Snippet: Figure 4. We quantified transcript abundance of 2 candidate markers, CFLAR and SOD2, using qRT-PCR. Data are displayed as fold changes in expression in rejection (n10) and postrejec- tion (n8), each compared with control (n5). In agreement with microarray findings, both CFLAR and SOD2 expression were decreased in rejection. CFLAR expression returned toward control levels in postrejection samples, and SOD2 expression remained low, consistent with persistent partial activation of cir- culating leukocytes after treatment of rejection. *P0.05 com- pared with control by Wilcoxon rank-sum test.

Article Snippet: The specific assays used were Hs00153439_m1 (CFLAR), Hs00167309_m1 (SOD2), and Hs99999905_m1 (GAPDH).

Techniques: Quantitative RT-PCR, Expressing, Control, Microarray, Activation Assay

Journal: Nature Communications

Article Title: Microglial cannabinoid receptor type 1 mediates social memory deficits in mice produced by adolescent THC exposure and 16p11.2 duplication

doi: 10.1038/s41467-023-42276-5

Figure Lengend Snippet: a Experimental flow of Fluorescence-Activated Cell Sorting (FACS)-based microglia (CD45 + CD11b + TMEM119 + ), astrocyte (ACSA-2 + ), and neuron (NeuN + ) isolation using specific cell markers. b Relative mRNA expression levels (arbitrary units: a.u.) of Cnr1 in microglia, astrocytes, and neurons isolated from wild type mice were measured by quantitative real time PCR (qPCR) using the TaqMan assay protocol. n = 6 mice per group. *** p < 0.001, ** p < 0.01 ( p values are Microglia versus Astrocytes: p = 0.0010, Microglia versus Neurons: p < 0.0001, Astrocytes versus Neurons: p < 0.0001), determined by one-way ANOVA with post hoc Tukey test. c Relative mRNA expression levels (arbitrary units: a.u.) of Cnr1 in microglia, astrocytes, and neurons isolated from Cx3cr1 CreER/+ ; Cnr1 +/+ and Cx3cr1 CreER/+ ; Cnr1 flox/flox mice were measured by qPCR. n = 6 mice per group. *** p < 0.001 ( p values are p < 0.0001), determined by unpaired two-tailed Student’s t test. d Microglia-enriched CD11b + cells, ACSA-2 + astrocytes, and remaining cells including neurons were collected from the cerebral cortex of Cx3cr1 CreER/+ ; Cnr1 flox/flox mice and littermate controls ( Cx3cr1 CreER/+ ; Cnr1 +/+ ) by magnetic activated cell sorting (MACS). For each cell type, expression of Cnr1, marker proteins (Iba1, GFAP, NeuN) and a loading control (GAPDH) in the total protein (arbitrary units: a.u.) were analyzed with SDS-PAGE followed by Western blotting with 10 μg of protein sample loaded in each well. n = 3 mice per group. * p < 0.05 ( p values are Cnr1 +/+ Cd11b + cells versus Cnr1 +/+ Astrocytes: p = 0.0495, Cnr1 +/+ Cd11b + cells versus Cnr1 +/+ Remaining cells: p = 0.0204, Cnr1 +/+ Cd11b + cells versus Cnr1 flox/flox Cd11b + cells: p = 0.0479), determined by two-way ANOVA with post hoc Tukey test. b – d Each symbol represents one animal. Data are presented as the mean ± s.e.m.

Article Snippet: Real-time PCR reactions contained diluted cDNA from the synthesis reaction and 200 nM of forward and reverse TaqMan primers specific to targets of interest (Assay IDs for Cnr1: Mm01212171_s1, Iba1: Mm00479862_g1, and Trp53: Mm01731290_g1, Applied Biosystems).

Techniques: Fluorescence, FACS, Isolation, Expressing, Real-time Polymerase Chain Reaction, TaqMan Assay, Two Tailed Test, Marker, Control, SDS Page, Western Blot

Journal: Nature Communications

Article Title: Microglial cannabinoid receptor type 1 mediates social memory deficits in mice produced by adolescent THC exposure and 16p11.2 duplication

doi: 10.1038/s41467-023-42276-5

Figure Lengend Snippet: a Apoptosis assay of primary microglia cultures produced from WT and 16p11dup male mice. Quantification of signal intensity (arbitrary units: a.u.) of apoptosis marker apopxin ( n = 12 fields in 3 mice per condition). b Necrosis assay of primary microglia cultures produced from WT and 16p11dup male mice. Quantification of signal intensity (arbitrary units: a.u.) of necrosis marker 7-AAD ( n = 12 fields in 3 mice per condition). c Quantification of cellular process area of phalloidin-stained microglia cultures produced from WT and 16p11dup male mice ( n = 12 fields in 3 mice per condition). d Quantification of cellular process number of phalloidin-stained microglia cultures produced from WT and 16p11dup male mice ( n = 12 fields in 3 mice per condition). e Representative images of microglia cell cultures produced from WT and 16p11dup male mice in apoptosis and necrosis assays. Apopxin (green) and 7-AAD (red) are shown. Scale bar, 50 μm. f Immunohistochemistry with antibody against phalloidin (green) of primary microglia cultures produced from WT and 16p11dup male mice. Scale bar, 25 μm. g Apoptosis assay of primary microglia cultures produced from genetic deletion of Cnr1 (Cnr1 KO) and genetic deletion of Cnr2 (Cnr2 KO) male mice. Quantification of signal intensity (arbitrary units: a.u.) of apopxin ( n = 12 fields in 3 mice per condition). h Necrosis assay of primary microglia cultures produced from Cnr1 KO and Cnr2 KO male mice. Quantification of signal intensity (arbitrary units: a.u.) of 7-AAD ( n = 12 fields in 3 mice per condition). i Quantification of cellular process area of microglia cultures produced from Cnr1 KO and Cnr2 KO male mice. ( n = 12 fields in 3 mice per condition). j Quantification of cellular process number of microglia cultures of Cnr1 KO and Cnr2 KO male mice ( n = 12 fields in 3 mice per condition). k Representative images of microglia cell cultures produced from Cnr1 KO and Cnr2 KO male mice in apoptosis and necrosis assays. Apopxin (green) and 7-AAD (red) are shown. Scale bar, 100 μm. l Immunohistochemistry with antibody against phalloidin (green) of primary microglia cultures of Cnr1 KO and Cnr2 KO male mice. Scale bar, 25 μm. m Representative images of immunohistochemistry of Iba1 (green) and Casp3-p17 (red) (top left) as well as TUNEL signals (red) and DAPI (blue) (bottom left) in the mPFC at P51. Scale bar, 50 μm. Quantification of signal intensity (arbitrary units: a.u.) of Casp3-p17 and TUNEL (right) ( n = 6 mice per group). *** p < 0.001, ** p < 0.01, * p < 0.05 ( p values are ( a ) all: p < 0.0001, ( c ) all: p < 0.0001, ( d ) WT-vehicle (Veh) versus WT-THC: p < 0.0001, WT-THC versus 16p11dup-THC: p = 0.0410, 16 p 11dup-Veh versus 16p11dup-THC: p < 0.0001, ( m ) (Left) WT-Veh versus WT-THC: p = 0.0230, WT-Veh versus 16p11dup-Veh: p = 0.0471, WT-THC versus 16p11dup-THC: p = 0.0249, 16p11dup-Veh versus 16p11dup-THC: p = 0.0119, (Right) WT-Veh versus WT-THC: p < 0.0001, WT-Veh versus 16p11dup-Veh: p = 0.0376, WT-THC versus 16p11dup-THC: p < 0.0001, 16p11dup-Veh versus 16p11dup-THC: p < 0.0001), determined by two-way ANOVA with post hoc Tukey test. *** p < 0.001 ( p values are ( g ) p < 0.0001, ( i ) p < 0.0001, ( j ) p < 0.0001), determined by unpaired two-tailed Student’s t test. Each symbol represents one field ( a – d , g – j ) and one animal ( m ). Data are presented as the mean ± s.e.m.

Article Snippet: Real-time PCR reactions contained diluted cDNA from the synthesis reaction and 200 nM of forward and reverse TaqMan primers specific to targets of interest (Assay IDs for Cnr1: Mm01212171_s1, Iba1: Mm00479862_g1, and Trp53: Mm01731290_g1, Applied Biosystems).

Techniques: Apoptosis Assay, Produced, Marker, Staining, Immunohistochemistry, TUNEL Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Microglial cannabinoid receptor type 1 mediates social memory deficits in mice produced by adolescent THC exposure and 16p11.2 duplication

doi: 10.1038/s41467-023-42276-5

Figure Lengend Snippet: a Schematic diagram of the experimental design. b Immunohistochemical analysis of Iba1 (green) in the mPFC at P51. (Left) Representative images of the mPFC, representative tracing images (red), and images of cellular processes (green) and cell bodies (yellow) of Iba1 + cells. Scale bar, 50 μm (left) and 10 μm (middle and right). c The number of Iba1 + P2ry12 + cells (left) and Iba1 + P2ry12 − cells (middle) in the mPFC, presented as % of control. (Right) The percentage of Iba1 + P2ry12 − cells among all Iba1 + cells in the mPFC. ( n = 6 slices in 3 mice per condition). d Quantification of the ratio of cellular process area (left) and cell body size to total cell size (right) of Iba1 + cells. ( n = 50 cells in 5 mice per condition). e Representative images of immunohistochemistry of Iba1 (green) and Casp3-p17 (red) (top left) as well as TUNEL signals (red) and DAPI (blue) (bottom left) in the mPFC at P51. Scale bar, 50 μm. f Quantification of signal intensity of Casp3-p17 in Iba1 + cells (left) and TUNEL (right). n = 6 slices in 3 mice per condition. c , d , f *** p < 0.001, ** p < 0.01, * p < 0.05 ( p values are ( c ) (Left) 16p11 wt ; Cnr1 +/+ versus 16p11 wt ; Cnr1 flox/flox : p = 0.0281, 16p11 wt ; Cnr1 +/+ versus 16p11 dup ; Cnr1 +/+ : p = 0.0001, 16p11 wt ; Cnr1 +/+ versus 16p11 dup ; Cnr1 flox/flox : p = 0.0221, 16p11 dup ; Cnr1 +/+ versus 16p11 dup ; Cnr1 flox/flox : p < 0.0001, (Middle) 16p11 wt ; Cnr1 +/+ versus 16p11 wt ; Cnr1 flox/flox : p = 0.0285, 16p11 wt ; Cnr1 +/+ versus 16p11 dup ; Cnr1 +/+ : p < 0.0001, 16p11 wt ; Cnr1 +/+ versus 16p11 dup ; Cnr1 flox/flox : p = 0.0337, 16p11 dup ; Cnr1 +/+ versus 16p11 dup ; Cnr1 flox/flox : p < 0.0001, (Right) all: p < 0.0001, ( d ) (Left) 16p11 wt ; Cnr1 +/+ versus 16p11 wt ; Cnr1 flox/flox : p < 0.0001, 16p11 wt ; Cnr1 +/+ versus 16p11 dup ; Cnr1 +/+ : p = 0.0017, 16p11 wt ; Cnr1 +/+ versus 16p11 dup ; Cnr1 flox/flox : p < 0.0001, 16p11 dup ; Cnr1 +/+ versus 16p11 dup ; Cnr1 flox/flox : p < 0.0001, (Right) 16p11 wt ; Cnr1 +/+ versus 16p11 wt ; Cnr1 flox/flox : p = 0.0225, 16p11 wt ; Cnr1 +/+ versus 16p11 dup ; Cnr1 +/+ : p = 0.0183, 16p11 wt ; Cnr1 +/+ versus 16p11 dup ; Cnr1 flox/flox : p = 0.0330, 16p11 dup ; Cnr1 +/+ versus 16p11 dup ; Cnr1 flox/flox : p < 0.0001, ( f ) (Left) 16p11 wt ; Cnr1 +/+ versus 16p11 wt ; Cnr1 flox/flox : p = 0.0016, 16p11 wt ; Cnr1 +/+ versus 16p11 du p ; Cnr1 +/+ : p < 0.0001, 16p11 wt ; Cnr1 +/+ versus 16p11 dup ; Cnr1 flox/flox : p = 0.0032, 16p11 dup ; Cnr1 +/+ versus 16p11 dup ; Cnr1 flox/flox : p < 0.0001, (Right) all: p < 0.0001), determined by two-way ANOVA with p ost hoc Tukey test. Each symbol represents one slice ( a , f ) and one animal ( d ). Data are presented as the mean ± s.e.m.

Article Snippet: Real-time PCR reactions contained diluted cDNA from the synthesis reaction and 200 nM of forward and reverse TaqMan primers specific to targets of interest (Assay IDs for Cnr1: Mm01212171_s1, Iba1: Mm00479862_g1, and Trp53: Mm01731290_g1, Applied Biosystems).

Techniques: Immunohistochemical staining, Control, Immunohistochemistry, TUNEL Assay

Journal: Nature Communications

Article Title: Microglial cannabinoid receptor type 1 mediates social memory deficits in mice produced by adolescent THC exposure and 16p11.2 duplication

doi: 10.1038/s41467-023-42276-5

Figure Lengend Snippet: a Schematic diagram of the experimental design. b (Left) Representative voltage traces recorded from PT neurons in response to current step injections. (Right) The intrinsic excitability of PT neurons, as quantified by input resistance (left), rheobase (middle), and spike frequency (right). 16p11 wt ; Cnr1 +/+ ( n = 9 cells in 2 mice), 16p11 wt ; Cnr1 flox/flox ( n = 5 cells in 3 mice), 16p11 dup ; Cnr1 +/+ ( n = 6 cells in 2 mice), and 16p11 dup ; Cnr1 flox/flox ( n = 8 cells in 3 mice). *** p < 0.001, ** p < 0.01, * p < 0.05 ( p values are (Left) 16p11 wt ; Cnr1 +/+ versus 16p11 dup ; Cnr1 +/+ : p = 0.0042, 16p11 wt ; Cnr1 +/+ versus 16p11 dup ; Cnr1 flox/flox : p = 0.0001, 16p11 dup ; Cnr1 +/+ versus 16p11 dup ; Cnr1 flox/flox : p < 0.0001, (Right) 16p11 wt ; Cnr1 +/+ versus 16p11 dup ; Cnr1 +/+ : p = 0.0016, 16p11 wt ; Cnr1 +/+ versus 16p11 dup ; Cnr1 flox/flox : p = 0.0396, 16p11 dup ; Cnr1 +/+ versus 16p11 du p ; Cnr1 flox/flox : p < 0.000 determined by two-way ANOVA with post hoc Tukey test. c (Left) Representative voltage traces recorded from IT neurons in response to current step injections. (Right) The intrinsic excitability of IT neurons, as quantified by input resistance (left), rheobase (middle), and spike frequency (right). 16p11 wt ; Cnr1 +/+ ( n = 4 cells in 2 mice), 16p11 wt ; Cnr1 flox/flox ( n = 4 cells in 2 mice), 16p11 dup ; Cnr1 +/+ ( n = 6 cells i n 2 mice), and 16p11 dup ; Cnr1 flox/flox ( n = 5 cells in 2 mice). d Sociability phenotypes (left) and preference of social novelty (right) as indicated by the discrimination index in the three-chamber social interaction test. e, Time spent with an ovariectomized female mouse in the 5-trial social memory test. d , e 16p11 wt ; Cnr1 +/+ ( n = 6 mice), 16p11 wt ; Cnr1 flox/flox ( n = 5 mice), 16p11 dup ; Cnr1 +/+ ( n = 6 mice), 16p11 dup ; Cnr1 flox/flox ( n = 6 mice). d , e *** p < 0.001, ** p < 0.01 ( p values are ( d ) 16p11 wt ; Cnr1 +/+ versus 16p11 dup ; Cnr1 +/+ : p < 0.0001, 16p11 wt ; Cnr1 +/+ versus 16p11 dup ; Cnr1 flox/flox : p = 0.0004, 16p11 du p ; Cnr1 +/+ versus 16p11 dup ; Cnr1 flox/flox : p = 0.0026, ( e ) Cnr1 flox/flox x 16p11dup interaction for Trial 4 [ F 1,19 = 11.30, p = 0.0033], Cnr1 flox/flox x 16p11dup interaction for Trial 5 [ F 1,19 = 4.760, p = 0.0419]), determined by two-way ANOVA with post hoc Tukey test. Each symbol represents one cell ( b , c ) and one animal ( d , e ). Data are presented as the mean ± s.e.m.

Article Snippet: Real-time PCR reactions contained diluted cDNA from the synthesis reaction and 200 nM of forward and reverse TaqMan primers specific to targets of interest (Assay IDs for Cnr1: Mm01212171_s1, Iba1: Mm00479862_g1, and Trp53: Mm01731290_g1, Applied Biosystems).

Techniques:

Journal: PLoS ONE

Article Title: Effects of a High Fat Diet and Voluntary Wheel Running Exercise on Cidea and Cidec Expression in Liver and Adipose Tissue of Mice

doi: 10.1371/journal.pone.0130259

Figure Lengend Snippet: Cidea and Cidec gene expression for liver (Panels A and B) and epididymal white adipose tissue (Panels C and D) were normalized to GAPDH and expressed relative to LFD-SED values. *Denotes statistically significant difference from all other groups by Fisher’s LSD post-hoc test. + Denotes statistically significant difference from LFD-SED and LFD-WR groups by Fisher’s LSD post-hoc test. The number of mice per group is 4–5.

Article Snippet: The resultant cDNA (20 ng cDNA/sample in duplicate) was then subjected to quantitative polymerase chain reaction (qPCR) using standard target specific TaqMan gene expression assays for Cidea (Assay ID: Mm00432554_m1), Cidec (Assay ID: Mm00617672_m1), peroxisome proliferator activated receptor gamma (PPARγ) (Assay ID:), PPARγ coactivator-1 alpha (PGC-1α) (Assay ID: Mm01208835_m1) and a real time PCR system (StepOne Plus Real-Time PCR System, Applied Biosystems, Foster City, CA).

Techniques: Gene Expression

Journal: PLoS ONE

Article Title: Effects of a High Fat Diet and Voluntary Wheel Running Exercise on Cidea and Cidec Expression in Liver and Adipose Tissue of Mice

doi: 10.1371/journal.pone.0130259

Figure Lengend Snippet: Mice were fed a LFD or HFD and housed in standard cages or wheel running cages for weeks. Liver was excised and rapidly frozen in liquid nitrogen for subsequent Western blot analyses. (A) Representative immunoblots for Cidea and Cidec as well as β-actin as a loading control. Quantification of (B) Cidea and (C) Cidec protein, each normalized to β-actin. *, Denotes statistically significant difference from HFD-Vehicle. N = 5 mice per group.

Article Snippet: The resultant cDNA (20 ng cDNA/sample in duplicate) was then subjected to quantitative polymerase chain reaction (qPCR) using standard target specific TaqMan gene expression assays for Cidea (Assay ID: Mm00432554_m1), Cidec (Assay ID: Mm00617672_m1), peroxisome proliferator activated receptor gamma (PPARγ) (Assay ID:), PPARγ coactivator-1 alpha (PGC-1α) (Assay ID: Mm01208835_m1) and a real time PCR system (StepOne Plus Real-Time PCR System, Applied Biosystems, Foster City, CA).

Techniques: Western Blot, Control

Journal: PLoS ONE

Article Title: Effects of a High Fat Diet and Voluntary Wheel Running Exercise on Cidea and Cidec Expression in Liver and Adipose Tissue of Mice

doi: 10.1371/journal.pone.0130259

Figure Lengend Snippet: Mice were fed a LFD or HFD and housed in standard cages or wheel running cages for weeks. Adipose tissue was excised and rapidly frozen in liquid nitrogen for subsequent Western blot analyses. (A) Representative immunoblots for Cidea and Cidec as well as β-actin as a loading control. Quantification of (B) Cidea and (C) Cidec protein, each normalized to β-actin. *, Denotes statistically significant difference from HFD-Vehicle. N = 5 mice per group.

Article Snippet: The resultant cDNA (20 ng cDNA/sample in duplicate) was then subjected to quantitative polymerase chain reaction (qPCR) using standard target specific TaqMan gene expression assays for Cidea (Assay ID: Mm00432554_m1), Cidec (Assay ID: Mm00617672_m1), peroxisome proliferator activated receptor gamma (PPARγ) (Assay ID:), PPARγ coactivator-1 alpha (PGC-1α) (Assay ID: Mm01208835_m1) and a real time PCR system (StepOne Plus Real-Time PCR System, Applied Biosystems, Foster City, CA).

Techniques: Western Blot, Control

Journal: PLoS ONE

Article Title: Effects of a High Fat Diet and Voluntary Wheel Running Exercise on Cidea and Cidec Expression in Liver and Adipose Tissue of Mice

doi: 10.1371/journal.pone.0130259

Figure Lengend Snippet: CIDE protein expression in adipose tissue and liver from CON-SED, CON-WR, HFD-SED, and HFD-WR mice is related to measures of adiposity and insulin resistance.

Article Snippet: The resultant cDNA (20 ng cDNA/sample in duplicate) was then subjected to quantitative polymerase chain reaction (qPCR) using standard target specific TaqMan gene expression assays for Cidea (Assay ID: Mm00432554_m1), Cidec (Assay ID: Mm00617672_m1), peroxisome proliferator activated receptor gamma (PPARγ) (Assay ID:), PPARγ coactivator-1 alpha (PGC-1α) (Assay ID: Mm01208835_m1) and a real time PCR system (StepOne Plus Real-Time PCR System, Applied Biosystems, Foster City, CA).

Techniques: Expressing

Journal: Foods

Article Title: Structure Characterization, In Vitro Antioxidant and Anti-Tumor Activity of Sulfated Polysaccharide from Siraitia grosvenorii

doi: 10.3390/foods12112133

Figure Lengend Snippet: Reactive oxygen content and apoptosis-related gene expression in A549 cancer cell lines after S-SGP treatment. ( A – E ) The content of ROS in A549 cells from each group was assessed using DCFH-DA staining; ( F ) the effect of S-SGP on the content of reactive oxygen species in A549 cells; ( G – K ) relative mRNA expression levels of apoptosis-related genes in A549 cancer cell line treated by S-SGP.

Article Snippet: Chlorosulfonic acid, concentrated hydrochloric acid, trifluoroacetic acid, sodium hydroxide and pyridine were obtained from Sinopharm Chemical Reagent Company; Annexin V-FITC apoptosis kit, BCA protein concentration assay kit and reactive oxygen species (ROS) detection kit were obtained from Beyotime Biotechnology (Shanghai, China); the mitochondrial membrane potential assay (JC-1) kit, 2× SYBR Green and qPCR Master Mix (Low ROX) were purchased from Servicebio (Wuhan, China); rabbit anti-Cyt-C monoclonal antibody, rabbit anti-Bcl-2 monoclonal antibody, rabbit anti-Bax monoclonal antibody, rabbit anti-Caspase-3 monoclonal antibody and β -action antibody were obtained from Cell Signaling Technology (CST, Boston, MA, USA).

Techniques: Gene Expression, Staining, Expressing

Journal: Foods

Article Title: Structure Characterization, In Vitro Antioxidant and Anti-Tumor Activity of Sulfated Polysaccharide from Siraitia grosvenorii

doi: 10.3390/foods12112133

Figure Lengend Snippet: Mechanism of action of S-SGP in inducing apoptosis in A549 cells. Upon receiving apoptotic signals (such as ROS) stimulation, cells initiate the mitochondrial apoptotic pathway. S-SGP facilitates the relocalization of Bax to the surface of mitochondria, where it forms pores in the mitochondrial outer membrane. This event leads to a reduction of membrane potential and an increase in membrane permeability, resulting in the release of cytochrome C from mitochondria. Consequently, cytochrome C activates Casp-9, which subsequently activates Casp-3, ultimately culminating in cell apoptosis and exerting an anti-tumor effect. Bax: Bcl-2-associated protein X; ROS: reactive oxygen species; Bcl-2: B-cell lymphoma 2; C: cytochrome c; Casp: caspase.

Article Snippet: Chlorosulfonic acid, concentrated hydrochloric acid, trifluoroacetic acid, sodium hydroxide and pyridine were obtained from Sinopharm Chemical Reagent Company; Annexin V-FITC apoptosis kit, BCA protein concentration assay kit and reactive oxygen species (ROS) detection kit were obtained from Beyotime Biotechnology (Shanghai, China); the mitochondrial membrane potential assay (JC-1) kit, 2× SYBR Green and qPCR Master Mix (Low ROX) were purchased from Servicebio (Wuhan, China); rabbit anti-Cyt-C monoclonal antibody, rabbit anti-Bcl-2 monoclonal antibody, rabbit anti-Bax monoclonal antibody, rabbit anti-Caspase-3 monoclonal antibody and β -action antibody were obtained from Cell Signaling Technology (CST, Boston, MA, USA).

Techniques: Membrane, Permeability

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Gradient-Elution Nanoflow Liquid Chromatography Without a Binary Pump: Smoothed Step Gradients Enable Reproducible, Sensitive, and Low-Cost Separations for Single-Cell Proteomics

doi: 10.1016/j.mcpro.2024.100880

Figure Lengend Snippet: Analysis of two colonies of ATG-KO HeLa cells analyzed with 20-min active gradients. A , Western blot of HeLa WT, ATG5 KO responsive clone (R), and ATG5 KO nonresponsive clone (NR), treated with or without 1 μg/ml poly dA:dT for 24 h. B , protein identifications analyzed with match between runs using a 10-ng bulk HeLa digest library. C , PCA plot, color coded as green (R) and yellow (NR). The red line shows separation between the two colonies. D , volcano plot of differentially expressed proteins with an alpha of 0.01 and a minimum fold change of 1.25. E , enriched gene ontology terms for each population. F , STRING analysis showing a cluster of ER/ERAD-associated proteins that are upregulated in the NR clone. G , a model to explain how autophagy-adapted cancer cells can suppress STING- and IRF3-mediated inflammatory signaling by increasing expression of ERAD-associated proteins. ATG5, autophagy protein 5; ERAD, ER-associated degradation; IRF3, interferon regulatory factor 3; PCA, principal component analysis; STING, stimulator of interferon genes.

Article Snippet: Autophagy protein 5 (ATG5) KO HeLa cells (American Type Culture Collection) were generated using single-guide RNA (gRNA) 5′- AAGAGTAAGTTATTTGACGT-3′ against human ATG5 (ENST00000369076.8).

Techniques: Western Blot, Expressing

Journal: Oncotarget

Article Title: Dual inhibition of HDAC and EGFR signaling with CUDC-101 induces potent suppression of tumor growth and metastasis in anaplastic thyroid cancer.

doi: 10.18632/oncotarget.3268

Figure Lengend Snippet: Figure 3: CUDC-101 inhibits ATC cell proliferation, and induces cell cycle arrest and apoptosis. (A) Basal expression of HDAC1, HDAC2 and EGFR in ATC cell lines. (B) Cell proliferation assay. Error bars are mean ± SD. (C) Cell cycle analysis after 24 hours of treatment. (D) The Caspase-Glo 3/7 assay after 48 hours of treatment with CUDC-101. *p < 0.05, **p < 0.01, ***p < 0.001. NS, no significant difference.

Article Snippet: Phospho-kinase and apoptosis protein arrays Human phospho-kinase (Catalog # ARY003B) array (detecting site-specific phosphorylation of 43 kinases and 2 related total proteins, and apoptosis array (Catalog # ARY009 detecting the expression level of 35 apoptosisrelated proteins) were purchased from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Proliferation Assay, Cell Cycle Assay, Caspase-Glo Assay

Journal: Oncotarget

Article Title: Dual inhibition of HDAC and EGFR signaling with CUDC-101 induces potent suppression of tumor growth and metastasis in anaplastic thyroid cancer.

doi: 10.18632/oncotarget.3268

Figure Lengend Snippet: Figure 5: Protein targets of CUDC-101 in ATC cells. (A) CUDC-101 inhibits HDACs and MAPK in ATC cells. (B) Targets of CUDC-101 identified by phospho-kinase array. Phospho-kinase arrays were performed using cell lysates treated with and without CUDC-101. Shown are those proteins that were altered with CUDC-101 treatment in both ATC cell lines with > 1.5-fold difference. (C) Differentially expressed apoptotic proteins with and without CUDC-101 treatment. Apoptosis arrays were performed. Shown are those proteins that were altered with CUDC-101 treatment in both ATC cell lines with > 1.5-fold difference. For A–C, ATC cells were treated with the vehicle or CUDC-101 at 1.1 μM for 24 hours. (D) CUDC-101 reduces the expression of survivin, XIAP, and β-catenin. Cells were treated with the vehicle or CUDC-101 for 24 hours.

Article Snippet: Phospho-kinase and apoptosis protein arrays Human phospho-kinase (Catalog # ARY003B) array (detecting site-specific phosphorylation of 43 kinases and 2 related total proteins, and apoptosis array (Catalog # ARY009 detecting the expression level of 35 apoptosisrelated proteins) were purchased from R&D Systems (Minneapolis, MN).

Techniques: Expressing