Journal: PLoS ONE

Article Title: Effects of Melanocortin 1 Receptor Agonists in Experimental Nephropathies

doi: 10.1371/journal.pone.0087816

Figure Lengend Snippet: (A) Five different doses of adriamycin, in the range of 5–10 mg/kg, were intravenously injected and the level of albuminuria was measured after 7 days. The urinary albumin-to-creatinine ratio (UACR) increased with increasing levels of adriamycin. (B) Albuminuria at day 7. Treatment with the MC1R agonist BMS-470539 did not reduce the level of albuminuria for the 8 mg/kg adriamycin dose, on the contrary it slightly increased albuminuria for the 10 mg/kg dose (n = 17–19, p<0.05). The unspecific melanocortin receptor agonist α-MSH did not have any significant effect (n = 9, n.s.) compared with untreated adriamycin mice. Results are presented as mean ± SEM.

Article Snippet: An MC1R control peptide antigen (Alomone Labs, Ltd., Israel) was separately added at a 1∶1 weight ratio to the MC1R antibody solution before incubation to confirm antibody specificity.

Techniques: Injection

Journal: PLoS ONE

Article Title: Effects of Melanocortin 1 Receptor Agonists in Experimental Nephropathies

doi: 10.1371/journal.pone.0087816

Figure Lengend Snippet: Gene expression of MC1R in human, rat and mouse kidney tissue.

Article Snippet: An MC1R control peptide antigen (Alomone Labs, Ltd., Israel) was separately added at a 1∶1 weight ratio to the MC1R antibody solution before incubation to confirm antibody specificity.

Techniques: Expressing

Journal: PLoS ONE

Article Title: Effects of Melanocortin 1 Receptor Agonists in Experimental Nephropathies

doi: 10.1371/journal.pone.0087816

Figure Lengend Snippet: Western blotting was performed in order to detect MC1R protein expression in mouse and human tissue. Left panel: incubation with a rabbit polyclonal MC1R antibody (Alomone Labs). Right panel: incubation with MC1R antibody and control blocking peptide antigen (BP) in order to show antibody specificity. A) mouse whole glomerular lysate, B) cultured wild type mouse podocytes and C) a human malignant melanoma cell line A375. MC1R protein expression was detected at the expected size of 37 kDa (left panel).

Article Snippet: An MC1R control peptide antigen (Alomone Labs, Ltd., Israel) was separately added at a 1∶1 weight ratio to the MC1R antibody solution before incubation to confirm antibody specificity.

Techniques: Western Blot, Expressing, Incubation, Blocking Assay, Cell Culture

Journal: Oxidative Medicine and Cellular Longevity

Article Title: BMS-470539 Attenuates Oxidative Stress and Neuronal Apoptosis via MC1R/cAMP/PKA/Nurr1 Signaling Pathway in a Neonatal Hypoxic-Ischemic Rat Model

doi: 10.1155/2022/4054938

Figure Lengend Snippet: Temporal expression of endogenous MC1R and Nurr1 in the ipsilateral brain hemisphere post-HI. (a) Representative Western blot bands of the time course. (b) Western blot data showed that the endogenous expression levels of MC1R significantly increased from 12 h reaching peak at 48 h post-HI. (c) Nurr1 expression levels significantly increased over time, reached highest at the 48 h post-HI. n = 6 per group. Data were represented as mean ± SD. ∗ p < 0.05 versus sham, # p < 0.05 versus 6 h HI, and & p < 0.05 versus 48 h HI; one-way ANOVA, Tukey's post hoc test.

Article Snippet: MC1R CRISPR KO plasmid (Santa Cruz Biotechnology, USA), Nurr1 CRISPR KO plasmid (Santa Cruz Biotechnology, USA), or control CRISPR plasmid (Santa Cruz Biotechnology, USA) was given via intracerebroventricular injection at 48 h before HI induction according to our previous study [ ].

Techniques: Expressing, Western Blot, Hi-C

Journal: Oxidative Medicine and Cellular Longevity

Article Title: BMS-470539 Attenuates Oxidative Stress and Neuronal Apoptosis via MC1R/cAMP/PKA/Nurr1 Signaling Pathway in a Neonatal Hypoxic-Ischemic Rat Model

doi: 10.1155/2022/4054938

Figure Lengend Snippet: Immunofluorescence staining showed MC1R and Nurr1 colocalization with neurons in the ipsilateral brain hemisphere at 48 h post-HI. Immunofluorescence staining showed that MC1R (a) and Nurr1 (b) expressions on neurons were seen to be higher in the vehicle-treated pup rats compared to the sham group, and a higher expression of MC1R (a) and Nurr1 (b) on neurons after BMS-470359 treatment compared with vehicle. Merge showed the colocalization of MC1R and Nurr1 on neurons. n = 2 per group. Neurons were stained red. MC1R and Nurr1 were stained green. DAPI was stained blue. Scale bar = 100 μ m.

Article Snippet: MC1R CRISPR KO plasmid (Santa Cruz Biotechnology, USA), Nurr1 CRISPR KO plasmid (Santa Cruz Biotechnology, USA), or control CRISPR plasmid (Santa Cruz Biotechnology, USA) was given via intracerebroventricular injection at 48 h before HI induction according to our previous study [ ].

Techniques: Immunofluorescence, Staining, Expressing

Journal: Oxidative Medicine and Cellular Longevity

Article Title: BMS-470539 Attenuates Oxidative Stress and Neuronal Apoptosis via MC1R/cAMP/PKA/Nurr1 Signaling Pathway in a Neonatal Hypoxic-Ischemic Rat Model

doi: 10.1155/2022/4054938

Figure Lengend Snippet: Effects of BMS-470539 treatment on neuronal apoptosis at 48 h post-HI, and the knockout efficiency of MC1R or Nurr1 knockout CRISPR in naive and HI rats. (a, b) Representative microphotographs and quantitative analysis of C-Cas 3-positive neurons in the ipsilateral hemisphere at 48 h post-HI. The number of C-Cas 3-positive neurons significantly increased in the vehicle group compared with the sham group and BMS-470539 treatment group. n = 4 per group. C-Cas 3 was green. Neurons were red. Blue was for DAPI. Scale bar = 100 μ m. (c, d) Representative Western blot bands and quantitative analysis of MC1R and Nurr1 protein levels in the ipsilateral hemisphere at 48 h post-HI. The expression of MC1R or Nurr1 was significantly reduced by MC1R or Nurr1 knockout CRISPR in naive and HI rats. n = 4 per group (data were represented as mean ± SD; ∗ p < 0.05 versus sham; # p <0.05 versus HI+vehicle; @ p < 0.05 versus naive+control CRISPR, & p < 0.05 versus HI+control CRISPR; one-way ANOVA, Tukey's post hoc test).

Article Snippet: MC1R CRISPR KO plasmid (Santa Cruz Biotechnology, USA), Nurr1 CRISPR KO plasmid (Santa Cruz Biotechnology, USA), or control CRISPR plasmid (Santa Cruz Biotechnology, USA) was given via intracerebroventricular injection at 48 h before HI induction according to our previous study [ ].

Techniques: Knock-Out, CRISPR, Western Blot, Expressing, Control

Journal: Oxidative Medicine and Cellular Longevity

Article Title: BMS-470539 Attenuates Oxidative Stress and Neuronal Apoptosis via MC1R/cAMP/PKA/Nurr1 Signaling Pathway in a Neonatal Hypoxic-Ischemic Rat Model

doi: 10.1155/2022/4054938

Figure Lengend Snippet: BMS-470539 treatment exerted its antioxidative stress and antiapoptosis effects via MC1R/cAMP/PKA/Nurr1 signaling pathway at 48 h post-HI. (a) Representative Western blot bands. (b–i) Quantification of MC1R, cAMP, p-PKA, Nurr1, 4-HNE, HO-1, Bax, and Bcl-2 expression levels in the ipsilateral hemisphere at 48 h post-HI. BMS-470539 treatment significantly increased the protein levels of MC1R, cAMP, p-PKA, Nurr1, HO-1, and Bcl-2 but significantly decreased the expression of 4-HNE and Bax compared to the HI+vehicle group. Knockout of MC1R or Nurr1 with CRISPR interventions significantly abolished such effects of BMS-470539. n = 6 per group. Data was represented as mean ± SD. ∗ p < 0.05 versus sham, # p < 0.05 versus HI+vehicle, @ p < 0.05 HI+BMS-470539 or HI+BMS-470539+control CRISPR; one-way ANOVA, Tukey's post hoc test.

Article Snippet: MC1R CRISPR KO plasmid (Santa Cruz Biotechnology, USA), Nurr1 CRISPR KO plasmid (Santa Cruz Biotechnology, USA), or control CRISPR plasmid (Santa Cruz Biotechnology, USA) was given via intracerebroventricular injection at 48 h before HI induction according to our previous study [ ].

Techniques: Western Blot, Expressing, Knock-Out, CRISPR, Control

Journal: Oxidative Medicine and Cellular Longevity

Article Title: BMS-470539 Attenuates Oxidative Stress and Neuronal Apoptosis via MC1R/cAMP/PKA/Nurr1 Signaling Pathway in a Neonatal Hypoxic-Ischemic Rat Model

doi: 10.1155/2022/4054938

Figure Lengend Snippet: BMS-470539 administration reduced neuronal degeneration at 48 h post-HI, which was reversed by MC1R or Nurr1 knockout CRISPR. (a, b) Representative microphotographs and quantitative analysis of FJC-positive neurons in the ipsilateral hemisphere at 48 h post-HI. n = 6 per group. FJC was green. Blue was for DAPI. Scale bar = 100 μ m. Data was represented as mean ± SD. ∗ p < 0.05 versus sham; # p < 0.05 versus HI+vehicle; @ p < 0.05 HI+BMS-470539 or HI+BMS-470539+control CRISPR; one-way ANOVA, Tukey's post hoc test.

Article Snippet: MC1R CRISPR KO plasmid (Santa Cruz Biotechnology, USA), Nurr1 CRISPR KO plasmid (Santa Cruz Biotechnology, USA), or control CRISPR plasmid (Santa Cruz Biotechnology, USA) was given via intracerebroventricular injection at 48 h before HI induction according to our previous study [ ].

Techniques: Knock-Out, CRISPR, Control

Journal: Oxidative Medicine and Cellular Longevity

Article Title: BMS-470539 Attenuates Oxidative Stress and Neuronal Apoptosis via MC1R/cAMP/PKA/Nurr1 Signaling Pathway in a Neonatal Hypoxic-Ischemic Rat Model

doi: 10.1155/2022/4054938

Figure Lengend Snippet: BMS-470539 administration suppressed oxidative stress at 48 h post-HI, which was abolished by MC1R or Nurr1 knockout CRISPR. (a, b) Representative microphotographs of MitoSox (red) and 8-OHdG (green) staining in the ipsilateral hemisphere at 48 h post-HI. Blue was for DAPI. (c, d) Quantitative analysis of MitoSox- and 8-OHdG-positive cells. n = 6 per group. Scale bar = 100 μ m. Data was represented as mean ± SD. ∗ p < 0.05 versus sham; # p < 0.05 versus HI+vehicle; @ p < 0.05 HI+BMS-470539 or HI+BMS-470539+control CRISPR; one-way ANOVA, Tukey's post hoc test.

Article Snippet: MC1R CRISPR KO plasmid (Santa Cruz Biotechnology, USA), Nurr1 CRISPR KO plasmid (Santa Cruz Biotechnology, USA), or control CRISPR plasmid (Santa Cruz Biotechnology, USA) was given via intracerebroventricular injection at 48 h before HI induction according to our previous study [ ].

Techniques: Knock-Out, CRISPR, Staining, Control

Journal: bioRxiv

Article Title: Farnesyl Transferase Inhibition for the Treatment of Tauopathies

doi: 10.1101/500801

Figure Lengend Snippet: Cortical micrographs of transgenic rTg4510 (Tg) aged 12 (A-B; G-H; M-N), 16 (C-D; I-J; O-P) or 20 (E-F; K-L; Q-R) weeks-old, displaying the progression of tau pathology, showing no reactivity in control (Ctr) littermates (A, C, E) and age-related increase of tau immunoreactivity with MC1 antibody starting at ~16 weeks (B, D, F). Cortical astrocyte reactivity and density also increased in Tg mice starting at ~12 weeks (H, J, L) in contrast to age-matched Ctr (G, I, K). Microglia reactivity and density in cortex of Tg mice also increased in an age-related manner in transgenic mice (N, P, R), in contrast to Ctr (M, O, Q). Scale bar 100 μm.

Article Snippet: Sections were immersed in normal donkey serum 1:20 in PBS containing 0.5% BSA, 0.1% Triton-X 100, and 0.1% sodium azide (PBTA) at 4°C on a rotator for continuous overnight agitation followed by immersion MC1 (provided by Peter Davies, Feinstein Institute for Medical Research, Manhasset, NY; mouse monoclonal, 1:200), anti-Iba1 (Wako laboratory chemicals, Richmond, VA; rabbit polyclonal, 1:200), anti-GFAP (abcam, San Francisco, CA; chicken polyclonal, 1:500), anti-ubiquitin (abcam, ab7780; rabbit polyclonal), and anti-sumo 1 (abcam, ab11672; rabbit monoclonal) diluted in PBTA.

Techniques: Transgenic Assay

Journal: bioRxiv

Article Title: Farnesyl Transferase Inhibition for the Treatment of Tauopathies

doi: 10.1101/500801

Figure Lengend Snippet: (A) Representative image of brain coronal section tiling 800 x 800 pixels Z-Stacks mosaic labeled with MC1 (Green), GFAP (Red) and Hoechst (cell nuclei, blue) is shown. (B) The coronal section area of the micrographs was computed. At five weeks, rTg4510 transgenic mice (Tg) did not differ in brain size from their non-transgenic littermates (p=0.942), however at 20 weeks, the area was significantly reduced in transgenic mice compared to both age-matched controls and younger transgenic (p=6.94×10 −4 ). (C) 20-week-old control littermates (Ctr) are non-reactive to MC1 staining; (D) whereas 20 week old transgenic mice have a high density of strongly labeled neurons in both cerebral cortex (CTX) and hippocampal formation (HPF). (E) Quantification of NFTs/mm 2 in CTX and HPF. Representative micrographs showing microglia (Iba1 + cells) in 20 week old Ctr (F) and Tg (G) mice, and (H) quantification of microglia/mm 2 density, revealing that microglia lacks an age-related decline in rTg4510 transgenic mice. At 20-weeks, in the Ctr CTX there were 178.45±66.19 microglia/mm 2 and in the Tg CTX there were 444.20±55.91 microglia/mm 2 (p=1.9×10 −3 ). In the Ctr HPF, there were 236.74±17.68 microglia/mm 2 and in the Tg HPF there were 470.99±41.97 microglia/mm 2 (p=5.11×10 −3 ). (I) Astrocytes labeled with GFAP antibody in non-transgenic (Ctr) and (J) 20 week-old transgenic (Tg) mice show cortical astrogliosis accompanying the MC1 immunoreactivity in the rTg4510 mouse model. Activated hypertrophied astrocytes are shown in the inset. (K) GFAP + percentage area in the coronal sections was quantified. The cortical GFAP signal (ANOVA p=9.14×10 −4 ) quantified in Tg at 20 weeks increased in comparison of age-matched Ctr (p=9.2×10 −3 ). The cortical GFAP signal also increased in 20-week-old transgenic (Tg) compared to five-week-old Tg (p=0.020). The hippocampal GFAP signal between Tg and Ctr mice at 5 and 20 weeks did not differ (ANOVA p=0.676). Statistics shown for post-hoc Tukey-HSD test at *p<0.05, **p<0.01, ***p<1×10 −3 . Scale bar scale 1 mm.

Article Snippet: Sections were immersed in normal donkey serum 1:20 in PBS containing 0.5% BSA, 0.1% Triton-X 100, and 0.1% sodium azide (PBTA) at 4°C on a rotator for continuous overnight agitation followed by immersion MC1 (provided by Peter Davies, Feinstein Institute for Medical Research, Manhasset, NY; mouse monoclonal, 1:200), anti-Iba1 (Wako laboratory chemicals, Richmond, VA; rabbit polyclonal, 1:200), anti-GFAP (abcam, San Francisco, CA; chicken polyclonal, 1:500), anti-ubiquitin (abcam, ab7780; rabbit polyclonal), and anti-sumo 1 (abcam, ab11672; rabbit monoclonal) diluted in PBTA.

Techniques: Labeling, Transgenic Assay, Staining

Journal: bioRxiv

Article Title: Farnesyl Transferase Inhibition for the Treatment of Tauopathies

doi: 10.1101/500801

Figure Lengend Snippet: (A) Brain coronal section area in 20-week old rTg4510 transgenic mice (Tg) that received chronic oral administration of Lonafarnib (L80) is larger than that of untreated (Unt) or vehicle alone (Veh) Tg mice. Untreated and vehicle-treated mice measured 40.43±0.83 mm 2 and 40.02±0.73 mm 2 , respectively. Coronal section areas from lonafarnib-treated Tg animals averaged 44.55±0.95 mm 2 (untreated p=5.6×10 −3 , vehicle-treated p=2.4×10 −3 ). (B-C) Reduction of the extent of MC1 immunoreactivity in lonafarnib (L80) treated transgenic mice compared to untreated mice (Unt). Scale bar 1 mm. (D-G) Detail of insets of panels B and C showing representative MC1 immunoreactivity on cortex (CTX) and hippocampus (HPF) of either untreated (Unt) or lonafarnib treated (L80) in 20 week-old Tg mice; (H) large-scale coronal section mosaics quantified for MC1 + /mm 2 indicates a significant reduction of tau pahology after lonafarnib treatment (L80) when compared to untreated animals (Unt) or animals treated with vehicle alone (Veh). (I-L) Density of microglia in the CTX and HPF of transgenic mice treated with lonafarnib (L80) or left untreated (Unt) is shown by Iba1 immunolabeling. Hippocampal microglial reactivity declined upon lonafarnib treatment. (M) Microglia quantification of coronal section mosaics in both CTX and HPF of transgenic mice treated accordingly. In the HPF (ANOVA p=0.040), lonafarnib-treated Tg mice at 20 weeks had 343.33±13.24 Iba1 + cells/mm 2 compared to vehicle treated animals (488.38±11.53 Iba1+ cells/mm 2 ) or untreated controls (526.95±12.02 Iba1 + cells/mm 2 , p=0.05) and represents a significant reduction in microgliosis. No statistically significant differences were observed in the CTX (ANOVA p=0.211) of lonafarnib-treated animals (442.51±10.73 Iba1 + cells/mm 2 ) when compared to vehicle (425.30±7.94 Iba1 + cells/mm 2 ) or untreated animals (515.45±11.03 Iba1 + cells/mm 2 ). (N-Q) Astrocytes immunostained with GFAP in CTX or HPF of untreated and lonafarnib treated Tg mice, and (R) quantification of GFAP signal in full coronal slices. Neither lonafarnib (L80) nor vehicle alone (Veh) altered astrocytes in 20 weeks-old Tg mice (cortex ANOVA p=0.411, hippocampus ANOVA p=0.111). Statistics shown for post-hoc Tukey-HSD test at *p<0.05, **p<0.01, ***p<1×10 −3 . (S) Lonafarnib treatment (L80) reduced sumoylated cells compared to rTg4510 mice (Tg; p=2.55×10 −3 ). (T) Lonafarnib reduced ubiquitin labeled cells compared to rTg4510 mice (Tg; p=0.05). Counts of double-labeled cells in the micrographs were used to compute Bayesian probabilities of any cell to be tau immunoreactive (MC1 + ) given sumo + (U) or ubiquitin + (V) immunoreactivity. Lonafarnib also decreased these probabilities of double-labeling, in comparison to age-matched rTg4510 mice (Tg). Statistics shown for post-hoc Tukey-HSD test at *p<0.05, **p<0.01, ***p<1×10 −3 .Scale bar 100 μm.

Article Snippet: Sections were immersed in normal donkey serum 1:20 in PBS containing 0.5% BSA, 0.1% Triton-X 100, and 0.1% sodium azide (PBTA) at 4°C on a rotator for continuous overnight agitation followed by immersion MC1 (provided by Peter Davies, Feinstein Institute for Medical Research, Manhasset, NY; mouse monoclonal, 1:200), anti-Iba1 (Wako laboratory chemicals, Richmond, VA; rabbit polyclonal, 1:200), anti-GFAP (abcam, San Francisco, CA; chicken polyclonal, 1:500), anti-ubiquitin (abcam, ab7780; rabbit polyclonal), and anti-sumo 1 (abcam, ab11672; rabbit monoclonal) diluted in PBTA.

Techniques: Transgenic Assay, Immunolabeling, Labeling

Journal: bioRxiv

Article Title: Farnesyl Transferase Inhibition for the Treatment of Tauopathies

doi: 10.1101/500801

Figure Lengend Snippet: Cortical micrographs showing double immunostaining using anti-sumo and MC1, or anti-ubiquitin and MC1 were used to compute Bayesian probabilities of any cell to be (A) sumo + or (B) ubiquitin + given tau immunoreactivity (MC1 + ). Lonafarnib had no statistically significant effect on either of these probabilities, whereas Rhes-miR altered sumo double-labelling of MC1 + cells (sumo + given MC1 + ANOVA p=1.61×10 −3 , ubiquitin + given MC1 + ANOVA p=0.174). Statistics shown for post-hoc Tukey-HSD test at *p<0.05, **p<0.01.

Article Snippet: Sections were immersed in normal donkey serum 1:20 in PBS containing 0.5% BSA, 0.1% Triton-X 100, and 0.1% sodium azide (PBTA) at 4°C on a rotator for continuous overnight agitation followed by immersion MC1 (provided by Peter Davies, Feinstein Institute for Medical Research, Manhasset, NY; mouse monoclonal, 1:200), anti-Iba1 (Wako laboratory chemicals, Richmond, VA; rabbit polyclonal, 1:200), anti-GFAP (abcam, San Francisco, CA; chicken polyclonal, 1:500), anti-ubiquitin (abcam, ab7780; rabbit polyclonal), and anti-sumo 1 (abcam, ab11672; rabbit monoclonal) diluted in PBTA.

Techniques: Double Immunostaining

Journal: bioRxiv

Article Title: Farnesyl Transferase Inhibition for the Treatment of Tauopathies

doi: 10.1101/500801

Figure Lengend Snippet: Ten-week old transgenic rTg4510 mice were intracranially infected with AAV 2/5 viral vectors containing either U6, Rhes-miR or Rhes-WT constructs and sacrificed at 20 weeks of age. (A-C) Full mosaic immunohistochemistry of MC1 immunoreactivity following Rhes suppression by Rhes-miR have fewer NFTs than transduction with U6 or Rhes. (D-F) Full-mosaic immunohistochemistry of Iba1 was also reduced in mice treated with Rhes-miR AAV. Scale bar 1 mm. Quantification of micrographs (N=3), indicates (G) significantly increased coronal section area, Rhes-miR (40.53±0.70 mm 2 ) compared to U6 (37.33±0.36 mm 2 ; p=5.0×10 −3 ) or Rhes injected transgenic mice (36.41±0.79 mm 2 ; p=3.98×10 −4 ). (H) reduced number of MC1 + cells/mm 2 both in cerebral cortex (CTX) and hippocampal formation (HPF). The cortical density of MC1 + cells/mm 2 in animals injected with Rhes-miR was 78.76±8.875 MC1 + cells/mm 2 compared to U6 (157.81±12.56 MC1 + cells/mm 2 ; p=1.7×10 −3 ) or Rhes overexpression (172.15±13.120 MC1+ cells/mm 2 ; p=2.86×10 −4 ). In the HPF, Rhes-miR injected mice had 49.15±7.01 MC1+ cells/mm 2 compared to U6 (86.09±9.28 MC1+ cells/mm 2 ; p=0.013) or Rhes overexpression (92.86±9.64 MC1 + cells/mm 2 ; p=3.3×10 −3 ). (I) Reduced number of microglia/mm2 in CTX and HPF in Rhes-miR injected mice. Rhes-miR injected transgenic mice were significantly reduced in the CTX (U6 control p= 5.85×10 −5 , Rhes overexpression, p=0.031) and in the HPF (U6 control p=9.48×10 −4 , Rhes over-expression, p=5.0×10 −3 ). (J) Astrocytic immunoreactivity: In CTX, Rhes-miR significantly reduced GFAP immunoreactivity compared to Rhes overexpression (p=6.9×10 −3 ), but not from U6 controls (p=0.357). In HPF, Rhes-miR significantly reduced GFAP immunostaining compared to U6 controls (p=0.024), but not when compared to Rhes overexpression (p=0.248); (K-M) Double immunostaining of CTX using MC1 and anti-sumo, or (O-Q) anti-ubiquitin in 20-week old rTg4510 mice and immunostained cell densities quantified as sumo + /mm 2 or ubiquitin + /mm 2 . (M) Rhes-miR reduced sumoylated cells compared to Rhes overexpression (p=5.4×10 −3 ). (Q) Rhes-miR injected mice did not show a significant reduction in ubiquitin labeled cells compared to either Rhes overexpression or U6 injected mice (p=0.615 or p=0.889, respectively). Counts of double-labeled cells in the micrographs were used to compute Bayesian probabilities of any cell to be tau immunoreactive (MC1 + ) given sumo + (N) or ubiquitin + (R) immunoreactivity. Rhes-miR treatments decreased these probabilities of double-labeling: (N) Rhes-miR (0.159±0.045 p=4.7×10 −3 ) significantly reduced the likelihood of sumoylated cells to double-label compared to Rhes overexpression. (R) Rhes-miR (0.0907±0.0112; p=2.5×10 −3 ) significantly reduced the likelihood of ubiquitinated cells to co-label compared to mice injected with U6. Statistics shown for post-hoc Tukey-HSD test at *p<0.05, **p<0.01, ***p<1×10 −3 . Scale bar 100 μm.

Article Snippet: Sections were immersed in normal donkey serum 1:20 in PBS containing 0.5% BSA, 0.1% Triton-X 100, and 0.1% sodium azide (PBTA) at 4°C on a rotator for continuous overnight agitation followed by immersion MC1 (provided by Peter Davies, Feinstein Institute for Medical Research, Manhasset, NY; mouse monoclonal, 1:200), anti-Iba1 (Wako laboratory chemicals, Richmond, VA; rabbit polyclonal, 1:200), anti-GFAP (abcam, San Francisco, CA; chicken polyclonal, 1:500), anti-ubiquitin (abcam, ab7780; rabbit polyclonal), and anti-sumo 1 (abcam, ab11672; rabbit monoclonal) diluted in PBTA.

Techniques: Transgenic Assay, Infection, Construct, Immunohistochemistry, Transduction, Injection, Over Expression, Immunostaining, Double Immunostaining, Labeling

Journal: Bioconjugate chemistry

Article Title: Synthesis and Characterization of a Melanoma-Targeted Fluorescence Imaging Probe by Conjugation of a Melanocortin 1 Receptor (MC1R) Specific Ligand

doi: 10.1021/bc300549s

Figure Lengend Snippet: ICC of MC1R expression on the surface of A375 parental and engineered cells. Confocal micrographs of cells incubated with the nuclear marker DAPI (blue), the plasma- and plasma-membrane marker, WGA (green) and MC1R antibody-Alexa 555 (red). To inhibit cellular uptake, cells were incubated with antibodies and dyes at 4°C for 10 min. The merged image shows co-localization of MC1R (red) with membrane marker (green) indicating accumulation of the receptor on the cell-surface (yellow).

Article Snippet: Vector pCMV6 containing homo sapiens MC1R and neomycin as a selection marker was purchased (Origene).

Techniques: Expressing, Incubation, Marker

Journal: Bioconjugate chemistry

Article Title: Synthesis and Characterization of a Melanoma-Targeted Fluorescence Imaging Probe by Conjugation of a Melanocortin 1 Receptor (MC1R) Specific Ligand

doi: 10.1021/bc300549s

Figure Lengend Snippet: In vitro uptake studies of the MC1RL-Cy5 targeted imaging probe. (A) Fluorescence microscopy images at 15 s, 1 min and 5 min following addition of probe to an incubation chamber containing A375/MC1R cells grown on a coverslip. Probe binding is observed by 15 s and internalization by 5 mins. (B) Fluorescence microscopy images of probe uptake by B16 cells at the 5 min time-point showing internalization of probe into the cells. For both panels A and B, images are included for blocking studies at the 5 min time-point, where cells were pre-incubated with the unlabeled melanocortin-receptor ligand, NDP-α-MSH, prior to addition of probe. Pre-incubation with unlabeled ligand completely blocked binding and uptake of probe. For both panels A and B, the image on the left of each pair shows the overlay of the fluorescence image on the visible light (differential interference contrast) image of the corresponding cells and the left panel shows only the fluorescence.

Article Snippet: Vector pCMV6 containing homo sapiens MC1R and neomycin as a selection marker was purchased (Origene).

Techniques: In Vitro, Imaging, Fluorescence, Microscopy, Incubation, Binding Assay, Blocking Assay

Journal: Bioconjugate chemistry

Article Title: Synthesis and Characterization of a Melanoma-Targeted Fluorescence Imaging Probe by Conjugation of a Melanocortin 1 Receptor (MC1R) Specific Ligand

doi: 10.1021/bc300549s

Figure Lengend Snippet: In vivo targeting of MC1R specific probe. (A) Representative images of normalized fluorescence intensity maps overlayed on mice bearing xenograft tumors. A375 cells that constitutively express low levels of MC1R were used to form the low-expressing tumor (left flank) and A375/MC1R cells were used to form the high expressing tumor (right flank). The control image (left mouse) shows lower fluorescence signal in the left flank tumor relative to the right flank tumor 4 hours after intravenous injection of 3 nmol/kg of the MC1RL-800 probe. A blocking experiment (right mouse) was performed by co-injection of 0.25 μg unlabeled NDP-α-MSH and 3 nmol/kg of the MC1RL-800 probe to demonstrate specific binding. Inset on the far right graphs normalized fluorescence counts that significantly vary among low and high expressing tumors, and among tumors in control experiments compared to blocking experiments. (B) Representative image at 4 hours after injection of 3 nmol/kg of the MC1RL-Cy5 probe. Graph shows relative quantification of fluorescence in low and high expressing tumors. For panels A & B, images were acquired using the Optix-MX3 imaging system (Advanced Research Technologies, Inc.), graphed data represent the mean ± s.d., and NC = Normalized Counts. (C) Representative tomographic image of a mouse bearing a B16F10 xenograft tumor at 4 hours after injection of 3 nmol/kg of the MC1RL-800 imaging probe using the FMT 2500 LX quantitative fluorescence tomography in vivo imaging system (PerkinElmer). (D) MC1R IHC staining of A375 and A375/MC1R xenograft tumors. The bar graph shows the quantification of MC1R staining. Data represent mean ± s.d..

Article Snippet: Vector pCMV6 containing homo sapiens MC1R and neomycin as a selection marker was purchased (Origene).

Techniques: In Vivo, Fluorescence, Expressing, Injection, Blocking Assay, Binding Assay, Imaging, Tomography, In Vivo Imaging, Immunohistochemistry, Staining