Journal: Arthritis & rheumatology (Hoboken, N.J.)

Article Title: Monocarboxylate transporter 4, associated with the acidification of synovial fluid, is a novel therapeutic target for inflammatory arthritis.

doi: 10.1002/art.39270

Figure Lengend Snippet: Figure 2. Monocarboxylate transporter 4 (MCT4) mRNA and protein are up-regulated in rheumatoid arthritis synovial fibroblasts (RASFs). A, Lev- els of mRNA transcripts of Na1/H1 exchanger 1 (NHE-1), vacuolar-type H1ATPase (V-ATPase), MCT1, and MCT4 relative to that of b-actin in nor- mal SFs, 4 osteoarthritis SF (OASF) lines, and 4 RASF lines. B, Western blot analysis of MCT4 and b-actin protein in normal SFs, OASF lines, and RASF lines. C, Quantification depiction of the findings shown in B. Shown is MCT4 protein expression relative to b-actin protein levels in the 4 OASF lines and the 4 RASF lines. Values in A and C are the mean 6 SD. * 5 P , 0.05; ** 5 P , 0.01 by Student’s t-test. D, RA synovial tissue specimens incubated with rabbit anti-MCT4 antibodies (left) and with control normal rabbit serum (middle), and OA synovial tissue specimens incubated with anti-MCT4 antibodies (right). Boxed areas at the top are shown at higher magnification at the bottom. Results are representative of 8 RA synovial tis- sue specimens and 5 OA synovial tissue specimens. Bars 5 100 mm (top); 10 mm (bottom). Original magnification 3 100 (top); 3 400 (bottom).

Article Snippet: A rabbit anti-human MCT4 antibody (1:100) or a rabbit anti-mouse MCT4 antibody (1:100; Santa Cruz Biotechnology) served as the primary antibody, and normal rabbit serum (1:100; Wako) served as the control.

Techniques: Western Blot, Expressing, Incubation, Control

Journal: Arthritis & rheumatology (Hoboken, N.J.)

Article Title: Monocarboxylate transporter 4, associated with the acidification of synovial fluid, is a novel therapeutic target for inflammatory arthritis.

doi: 10.1002/art.39270

Figure Lengend Snippet: Figure 3. Knockdown of monocarboxylate transporter 4 (MCT4) with small interfering RNA (siRNA). A, Rheumatoid arthritis syno- vial fibroblast (RASF) lines were transfected with MCT4 siRNA or with control siRNA (mock). MCT4 siRNA transfection caused the number of MCT4 mRNA transcripts to fall relative to the number of b-actin mRNA transcripts. Values are the mean 6 SD of 3 RASF lines. ** 5 P , 0.01 by Student’s unpaired t-test. B, Western blot analysis of MCT4 and b-actin showed that MCT4 siRNA transfec- tion abrogated MCT4 expression in RASFs. Results are representa- tive of 3 independent experiments.

Article Snippet: A rabbit anti-human MCT4 antibody (1:100) or a rabbit anti-mouse MCT4 antibody (1:100; Santa Cruz Biotechnology) served as the primary antibody, and normal rabbit serum (1:100; Wako) served as the control.

Techniques: Knockdown, Small Interfering RNA, Transfection, Control, Western Blot, Expressing

Journal: Arthritis & rheumatology (Hoboken, N.J.)

Article Title: Monocarboxylate transporter 4, associated with the acidification of synovial fluid, is a novel therapeutic target for inflammatory arthritis.

doi: 10.1002/art.39270

Figure Lengend Snippet: Figure 4. Knockdown of monocarboxylate transporter 4 (MCT4) inhibits growth of rheumatoid arthritis synovial fibroblasts (RASFs). A, Num- bers of RASFs and osteoarthritis SFs (OASFs) after transfection with MCT4 small interfering RNA (siRNA) or control siRNA (mock). B, Flow cytometry data showing dilution of 5,6-carboxyfluorescein succinimidyl ester (CFSE) in MCT4 siRNA– and control siRNA–transfected RASFs on day 3. Shown are representative histograms (left) and percentages of divided RASFs (n 5 3 cell lines) (right). C, Flow cytometric analysis of annexin V and propidium iodide (PI) staining of RASFs and OASFs transfected with MCT4 siRNA or control siRNA, and of RASFs treated with cycloheximide (CHX; final concentration 100 mg/ml) as a positive control. Shown are representative dot plots (top) and percentages of PI- negative/annexin V–positive early apoptotic cells (bottom left) and PI-positive/annexin V–positive late apoptotic cells (bottom right). D, Left, Intracellular pH and L-lactate concentrations in RASFs transfected with MCT4 siRNA or control siRNA. Right, Extracellular pH and L-lactate concentrations in medium of RASFs transfected with MCT4 siRNA or control siRNA. Values are the mean 6 SD. * 5 P , 0.05; ** 5 P , 0.01 versus control siRNA–transfected RASFs, by Student’s t-test.

Article Snippet: A rabbit anti-human MCT4 antibody (1:100) or a rabbit anti-mouse MCT4 antibody (1:100; Santa Cruz Biotechnology) served as the primary antibody, and normal rabbit serum (1:100; Wako) served as the control.

Techniques: Knockdown, Transfection, Small Interfering RNA, Control, Flow Cytometry, Staining, Concentration Assay, Positive Control

Journal: Arthritis & rheumatology (Hoboken, N.J.)

Article Title: Monocarboxylate transporter 4, associated with the acidification of synovial fluid, is a novel therapeutic target for inflammatory arthritis.

doi: 10.1002/art.39270

Figure Lengend Snippet: Figure 5. Knockdown of monocarboxylate transporter 4 (MCT4) induces intrinsic apoptosis of rheumatoid arthritis synovial fibroblasts (RASFs). A, Western blot analysis of RASF lines transfected with MCT4 small interfering RNA (siRNA) or control siRNA (mock) to determine caspase 3, caspase 9, and caspase 8 protein expression. B, Levels of cleaved caspase 3 and caspase 9 expression relative to pro- caspase 3 and procaspase 9 expression levels, respectively, for all 3 RASF lines. Values are the mean 6 SD. * 5 P , 0.05 versus control siRNA–transfected RASFs, by Student’s t-test.

Article Snippet: A rabbit anti-human MCT4 antibody (1:100) or a rabbit anti-mouse MCT4 antibody (1:100; Santa Cruz Biotechnology) served as the primary antibody, and normal rabbit serum (1:100; Wako) served as the control.

Techniques: Knockdown, Western Blot, Transfection, Small Interfering RNA, Control, Expressing

Journal: Arthritis & rheumatology (Hoboken, N.J.)

Article Title: Monocarboxylate transporter 4, associated with the acidification of synovial fluid, is a novel therapeutic target for inflammatory arthritis.

doi: 10.1002/art.39270

Figure Lengend Snippet: Figure 6. Knockdown of monocarboxylate transporter 4 (MCT4) reduces severity of collagen-induced arthritis (CIA) in mice. A, Synovial tissue from the knee joint of a mouse with CIA and a control naive mouse after incubation with a rabbit anti-MCT4 antibody. Bars 5 100 mm. B, Left, Expression of 6-FAM in the knee joint 24 hours after electroporation, as observed under a fluorescence stereomicroscope. Right, Brightfield image of the same joint. C, Synovial tissue from the left knee joints of mice with CIA after electrotransfer of MCT4-specific small interfering RNA (siRNA) or control siRNA (mock) and incubation with a rabbit anti-MCT4 antibody. Bars 5 100 mm. D, Top, Hematoxylin and eosin (H&E)–stained synovial tissue from the knee joints of mice with CIA 72 hours after electrotransfer of MCT4-specific siRNA or control siRNA. Bars 5 100 mm. Middle, Histologic scores (n 5 10 mice per group). Values are the mean 6 SD. * 5 P , 0.05 by Mann-Whitney U test. Bottom, TUNEL assay performed in synovial tissue from the knee joints of mice with CIA transfected with MCT4-specific siRNA or control siRNA. Bars 5 10 mm.

Article Snippet: A rabbit anti-human MCT4 antibody (1:100) or a rabbit anti-mouse MCT4 antibody (1:100; Santa Cruz Biotechnology) served as the primary antibody, and normal rabbit serum (1:100; Wako) served as the control.

Techniques: Knockdown, Control, Incubation, Expressing, Electroporation, Fluorescence, Electrotransfer, Small Interfering RNA, Staining, MANN-WHITNEY, TUNEL Assay, Transfection

Journal: European neuropsychopharmacology : the journal of the European College of Neuropsychopharmacology

Article Title: Pimavanserin exhibits serotonin 5-HT 2A receptor inverse agonism for G αi1 - and neutral antagonism for G αq/11 -proteins in human brain cortex.

doi: 10.1016/j.euroneuro.2020.05.004

Figure Lengend Snippet: Fig. 2 Modulation of specific [ 35 S]GTP γ S binding to G αq/11 - , G αi1 -, G αi2 -, G αi3 - and G αo -proteins by 10 μM pimavanserin in prefrontal human brain cortex. Basal values of specific [ 35 S]GTP γ S binding to the different G-proteins are expressed as 0% and stimulatory/inhibitory effects on the respective basal are shown . Each box spans the 25–75 interquartile range and whiskers extend to the 5% −95% observations. The line inside the box indicates median value. Independent experi- ments were carried out by triplicate in samples of six subjects. ∗∗∗p < 0.001 vs basal values (one-sample t -test).

Article Snippet: Antibodies, drugs and reagents onoclonal antibodies against G αq/11 - (sc-515689), G αi1 - (sc-56536), αi2 - (sc-13534), G αi3 - (sc-365422) and G αo - (sc-393874) proteins ere obtained from Santa Cruz Biotechnologies (USA).

Techniques: Binding Assay

Journal: Science Advances

Article Title: Top2a promotes the development of social behavior via PRC2 and H3K27me3

doi: 10.1126/sciadv.abm7069

Figure Lengend Snippet: ( A ) Screening workflow. ( B ) Fluoroquinolones induce social deficits. Comparing DMSO ( n = 68, 22, and 82, from left to right) with flumequine (Flum; 25 μM; n = 56), lomefloxacin (Lome; 100 μM; n = 60), ofloxacin (Ofl; 200 μM; n = 22), and oxolinic acid (OA; 200 μM; n = 62). SS, sodium salicylate. ( C ) The catalytic cycle of Top2 and inhibitors targeting different steps of the cycle. ATP, adenosine 5′-triphosphate; ADP, adenosine 5′-diphosphate. ( D to F ) Eukaryotic Top2 inhibitors induce social deficits (D). Comparing fish treated with vehicle control (0 μM in each panel); Top2 poisons ellipticine (0 μM: n = 48; 2 μM: n = 31), amsacrine (0 μM: n = 21; 200 μM: n = 20), etoposide (0 μM: n = 24; 10 μM: n = 11), and mitoxantrone (0 μM: n = 38; 5 μM: n = 29) (E); and Top2 catalytic inhibitors aclarubicin (0 μM: n = 22; 8 μM: n = 6), ICRF-193 (0 μM: n = 65; 100 μM: n = 29), and sobuzoxane (0 μM: n = 27; 100 μM: n = 8) (F). * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: Other compounds were purchased from the following sources: from Cayman Chemical: flumequine, oxolinic acid, etoposide, mitoxantrone (hydrochloride), ellipticine, bleomycin (sulfate), hydroxyurea, actinomycin D, triptolide, and UNC1999; from Sigma-Aldrich: lomefloxacin hydrochloride, ofloxacin, and sodium salicylate; from ApexBio: amsacrine; from Santa Cruz Biotechnology: ICRF-193, sobuzoxane, and aclarubicin (aclacinomycin A); from Tocris: UNC2400.

Techniques:

Journal: Science Advances

Article Title: Top2a promotes the development of social behavior via PRC2 and H3K27me3

doi: 10.1126/sciadv.abm7069

Figure Lengend Snippet: ( A ) Top2a-MO (0.05 mM; n = 44) induces social deficits compared to vehicle (H 2 O, n = 44), which is rescued by hTOP2A mRNA (250 ng/μl; Top2a-MO + A, n = 44). ( B ) Top2b-MO (0.05 mM; n = 50) does not induce social deficits compared to vehicle (H 2 O, n = 71). ( C ) Top2a-selective inhibitor sodium salicylate (100 μM) induces social deficits. ( D ) Heterozygous can4 mutant ( can4 +/− , n = 20) exhibits social deficits compared to WT ( n = 20). Both mutant and WT were acquired from a heterozygous ( can4 +/− ) incross and genotyped individually after Fishbook assay. ( E ) Heterozygous noto mutant ( noto +/− , n = 32) shows no social deficits compared to WT ( n = 21). Both mutant and WT were acquired from a heterozygous ( noto +/− ) incross and genotyped individually after Fishbook assay. ( F ) ICRF-193 (I; 0.1 mM; n = 23) induced social deficits compared to the vehicle control ethanol (E; 1%; n = 50), which is rescued by hTOP2AY165S mRNA (250 ng/μl; I + A Y165S , n = 31). ns, not significant; * P < 0.05, ** P < 0.01, and **** P < 0.0001.

Article Snippet: Other compounds were purchased from the following sources: from Cayman Chemical: flumequine, oxolinic acid, etoposide, mitoxantrone (hydrochloride), ellipticine, bleomycin (sulfate), hydroxyurea, actinomycin D, triptolide, and UNC1999; from Sigma-Aldrich: lomefloxacin hydrochloride, ofloxacin, and sodium salicylate; from ApexBio: amsacrine; from Santa Cruz Biotechnology: ICRF-193, sobuzoxane, and aclarubicin (aclacinomycin A); from Tocris: UNC2400.

Techniques: Mutagenesis

Journal: Science Advances

Article Title: Top2a promotes the development of social behavior via PRC2 and H3K27me3

doi: 10.1126/sciadv.abm7069

Figure Lengend Snippet: ( A ) Experimental design for prenatal ICRF-193 injection and subsequent experiments for the offspring. L, left; R, right. ( B to D ) ICRF-193 (I, n = 16) reduces the total duration (B), total number (C), and mean duration (D) of social investigation compared to vehicle control ethanol (E, n = 20). ( E to G ) ICRF-193 ( n = 16) does not reduce the total duration (E), total number (F), and mean duration (G) of novel object investigation compared to ethanol ( n = 19). ( H and I ) ICRF-193 ( n = 16) does not change the percentage of time spent in the center zone (H) and velocity (I) of mice in the open-field assay compared to ethanol ( n = 18). ( J and K ) ICRF-193 ( n = 16) reduces the total number (J) but increases the mean peak frequency (K) of USV compared to ethanol ( n = 20). ( L and M ) ICRF-193 ( n = 16) reduces the total number of alterations (the number of times a mouse enters an alternative arm compared to the previous trial) (L) while increasing the number of trials before the first alteration (M) compared to ethanol ( n = 20) during a T-maze assay that consisted of eight trials. ( N ) ICRF-193 ( n = 16) increases the coefficient of variation in the hole-board assay compared to ethanol ( n = 20). * P < 0.05, ** P < 0.01, and **** P < 0.0001.

Article Snippet: Other compounds were purchased from the following sources: from Cayman Chemical: flumequine, oxolinic acid, etoposide, mitoxantrone (hydrochloride), ellipticine, bleomycin (sulfate), hydroxyurea, actinomycin D, triptolide, and UNC1999; from Sigma-Aldrich: lomefloxacin hydrochloride, ofloxacin, and sodium salicylate; from ApexBio: amsacrine; from Santa Cruz Biotechnology: ICRF-193, sobuzoxane, and aclarubicin (aclacinomycin A); from Tocris: UNC2400.

Techniques: Injection