Journal: CNS Neuroscience & Therapeutics

Article Title: TTBK2‐Driven Ciliogenesis Is Required for Intrinsic Neuronal Regeneration After Spinal Cord Injury

doi: 10.1002/cns.70763

Figure Lengend Snippet: TTBK2 regulates primary cilium formation and axonal growth in spinal neurons. (A) Schematic diagram of the primary cilium. Kinesin‐2 comprises KIF3A; TTBK2 is related to the formation of basal bodies. (B, C) Quantitative RT‐qPCR analysis showing efficient knockdown or overexpression of KIF3A and TTBK2 in spinal neurons via adenoviral infection ( n = 6 from 3 independent experiments). (D, E) Representative immunofluorescence images of spinal neurons labeled with MAP2 (green), ACIII (red), and DAPI (blue) in five experimental groups: NC, shKIF3A, shTTBK2, TTBK2‐OE, and shKIF3A + TTBK2‐OE. White arrows indicate PCs. Compared with that in NC, the cilium length was significantly reduced in the shKIF3A, shTTBK2, and shKIF3A + TTBK2‐OE groups, while TTBK2‐OE overexpression led to elongated cilia ( n = 6 from 3 independent experiments). Scale bars, 20 μm. (F, G) Representative images showing immunolabeling of TUJ1 (green, axons), MAP2 (orange, dendrites), and DAPI (blue, nuclei). Axonal morphology and length were assessed across five groups. KIF3A or TTBK2 knockdown significantly reduced axon length, while TTBK2 overexpression enhanced elongation. shKIF3A + TTBK2‐OE partially rescued axon length compared with that under shKIF3A alone ( n = 5 from 3 independent experiments). Scale bars, 10 μm. Data are presented as mean ± SEM. One‐way ANOVA was performed. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The antibodies were rabbit anti‐GFAP (Abcam, ab7260, 1:5000) mouse anti‐beta III Tubulin (TUJ1) (Proteintech, 66375‐1‐Ig, 1:400), chicken anti‐MAP2 (MAP2) (Abcam, ab5392, 1:1000), rabbit anti‐Calb (Abcam, ab108404, 1:150), mouse monoclonal antibody to ACIII (AC3) (Encorbio, MCA‐1A12, 1:1000), chicken anti‐choline acetyltransferase antibody (ChAT) (Sigma‐Aldrich, AB15468, 1:1000), rabbit anti‐TTBK2 (Sigma‐Aldrich, AB805274 , 1:1000), rabbit anti‐neurofilament‐H (NF200) (Cell Signaling, 30564, 1:400), mouse monoclonal [Rat‐401] to nestin‐neural stem cell marker (Nestin) (Abcam, ab6142, 1:1000), rabbit anti‐PSD95 (Cell Signaling, 3450, 1:400), chicken anti‐GAP43 polyclonal antibody (Thermo Fisher Scientific, PA5‐95660, 1:500), goat anti‐chicken secondary antibody goat anti‐chicken IgY H&L (Alexa Fluor 555) (Abcam, ab150170, 1:2000), goat anti‐rabbit IgG (H + L) (Alexa Fluor 647) (Beyotime, A0468 1:200), goat anti‐rabbit IgG (H + L) (Alexa Fluor 350) (Beyotime, A0408, 1:200), and goat anti‐mouse IgG (H + L) (Alexa Fluor 647) (Beyotime, A0473, 1:200).

Techniques: Quantitative RT-PCR, Knockdown, Over Expression, Infection, Immunofluorescence, Labeling, Immunolabeling

Journal: CNS Neuroscience & Therapeutics

Article Title: TTBK2‐Driven Ciliogenesis Is Required for Intrinsic Neuronal Regeneration After Spinal Cord Injury

doi: 10.1002/cns.70763

Figure Lengend Snippet: TTBK2 regulates axonal regeneration via the primary cilium–SHH pathway. (A–C) Volcano plots showing differentially expressed proteins between shTTBK2 vs. NC, shKIF3A vs. NC, and shKIF3A + TTBK2‐OE vs. shKIF3A. The x ‐axis represents log2 (fold change), and the y ‐axis represents −log10( p ‐value). Gray dots indicate proteins that did not meet significance thresholds ( p > 0.05). Blue and red dots indicate downregulated and upregulated proteins, respectively. (D) The heatmap displays differentially expressed proteins identified in each of the four experimental groups relative to the NC control group, with color intensity representing expression levels. (E–H) Western blotting analysis demonstrating significant reductions in MAP2, Gli1, and Smo protein expression in the shTTBK2 group ( n = 3 from 3 independent experiments). (I, J) Treatment with the SHH pathway agonist SAG restored MAP2 expression in shTTBK2 neurons ( n = 3, from 3 independent experiments * p < 0.05). (K, L) Representative immunofluorescence images of spinal neurons stained for TUJ1 (green), PSD95 (red), and DAPI (blue). TTBK2‐OE group showed markedly increased PSD95 expression compared to other groups ( n = 6 from 3 independent experiments). Scale bar, 5 μm. Data are presented as mean ± SEM. One‐way ANOVA was used for statistical analysis. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The antibodies were rabbit anti‐GFAP (Abcam, ab7260, 1:5000) mouse anti‐beta III Tubulin (TUJ1) (Proteintech, 66375‐1‐Ig, 1:400), chicken anti‐MAP2 (MAP2) (Abcam, ab5392, 1:1000), rabbit anti‐Calb (Abcam, ab108404, 1:150), mouse monoclonal antibody to ACIII (AC3) (Encorbio, MCA‐1A12, 1:1000), chicken anti‐choline acetyltransferase antibody (ChAT) (Sigma‐Aldrich, AB15468, 1:1000), rabbit anti‐TTBK2 (Sigma‐Aldrich, AB805274 , 1:1000), rabbit anti‐neurofilament‐H (NF200) (Cell Signaling, 30564, 1:400), mouse monoclonal [Rat‐401] to nestin‐neural stem cell marker (Nestin) (Abcam, ab6142, 1:1000), rabbit anti‐PSD95 (Cell Signaling, 3450, 1:400), chicken anti‐GAP43 polyclonal antibody (Thermo Fisher Scientific, PA5‐95660, 1:500), goat anti‐chicken secondary antibody goat anti‐chicken IgY H&L (Alexa Fluor 555) (Abcam, ab150170, 1:2000), goat anti‐rabbit IgG (H + L) (Alexa Fluor 647) (Beyotime, A0468 1:200), goat anti‐rabbit IgG (H + L) (Alexa Fluor 350) (Beyotime, A0408, 1:200), and goat anti‐mouse IgG (H + L) (Alexa Fluor 647) (Beyotime, A0473, 1:200).

Techniques: Control, Expressing, Western Blot, Immunofluorescence, Staining

Journal: CNS Neuroscience & Therapeutics

Article Title: TTBK2‐Driven Ciliogenesis Is Required for Intrinsic Neuronal Regeneration After Spinal Cord Injury

doi: 10.1002/cns.70763

Figure Lengend Snippet: The TTBK2–SHH–MAP2 axis regulates endogenous neuronal repair following SCI. (A) Representative immunofluorescence images of frozen spinal cord sections stained with GFAP (green), MAP2 (orange), TUJ1 (red), and DAPI (blue). Scale bars: Left, 200 μm; right, 50 μm ( n = 6 from six mice in each group). (B–D) The quantification of immunostaining showed increased GFAP in all injured groups. MAP2 levels in WT‐SCI were comparable to those in uninjured controls, while Ttbk2 fl/fl ‐SCI showed a marked reduction. TUJ1 staining indicated significantly higher immature neuron proportion in WT‐SCI than in other groups ( n = 6 from six mice in each group). (E, F) Co‐staining of NF200 (green) and Nestin (red) revealed elevated neural progenitor marker Nestin in injured groups. Ttbk2 fl/fl ‐SCI mice exhibited a higher Nestin/NF200 ratio than did WT‐SCI mice ( n = 6 from six mice in each group). (G–J) Western blotting results confirmed that MAP2, Smo, and Gli1 protein levels were significantly reduced in Ttbk2 fl/fl ‐SCI mice, indicating SHH pathway suppression ( n = 3 from three mice in each group). Bars and errors represent mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001 (one‐way ANOVA).

Article Snippet: The antibodies were rabbit anti‐GFAP (Abcam, ab7260, 1:5000) mouse anti‐beta III Tubulin (TUJ1) (Proteintech, 66375‐1‐Ig, 1:400), chicken anti‐MAP2 (MAP2) (Abcam, ab5392, 1:1000), rabbit anti‐Calb (Abcam, ab108404, 1:150), mouse monoclonal antibody to ACIII (AC3) (Encorbio, MCA‐1A12, 1:1000), chicken anti‐choline acetyltransferase antibody (ChAT) (Sigma‐Aldrich, AB15468, 1:1000), rabbit anti‐TTBK2 (Sigma‐Aldrich, AB805274 , 1:1000), rabbit anti‐neurofilament‐H (NF200) (Cell Signaling, 30564, 1:400), mouse monoclonal [Rat‐401] to nestin‐neural stem cell marker (Nestin) (Abcam, ab6142, 1:1000), rabbit anti‐PSD95 (Cell Signaling, 3450, 1:400), chicken anti‐GAP43 polyclonal antibody (Thermo Fisher Scientific, PA5‐95660, 1:500), goat anti‐chicken secondary antibody goat anti‐chicken IgY H&L (Alexa Fluor 555) (Abcam, ab150170, 1:2000), goat anti‐rabbit IgG (H + L) (Alexa Fluor 647) (Beyotime, A0468 1:200), goat anti‐rabbit IgG (H + L) (Alexa Fluor 350) (Beyotime, A0408, 1:200), and goat anti‐mouse IgG (H + L) (Alexa Fluor 647) (Beyotime, A0473, 1:200).

Techniques: Immunofluorescence, Staining, Immunostaining, Marker, Western Blot

Journal: Journal of neuroinflammation

Article Title: Activation of MC1R with BMS-470539 attenuates neuroinflammation via cAMP/PKA/Nurr1 pathway after neonatal hypoxic-ischemic brain injury in rats.

doi: 10.1186/s12974-021-02078-2

Figure Lengend Snippet: Fig. 1 Expression levels of α-MSH, MC1R, and Nurr1 post-HI. a Representative pictures of Western blot data. b Western blot data showed that the expression levels of α-MSH significantly increased at 12 h post-HI, and peaked at 24 h. c, d The endogenous expression levels of MC1R and Nurr1 increased over time and peaked at 48 h post-HI. Data were represented as mean ± SD. Statistical differences between groups were analyzed using one-way ANOVA, followed by Tukey’s post hoc test (*p < 0.05 versus sham, #p < 0.05 versus 6 h HI, @p < 0.05 versus 24 h HI, &p < 0.05 versus 48 h HI; n = 4 per group). e Representative microphotographs of double immunofluorescence of α-MSH (green) with microglia (Iba-1, red), astrocyte (GFAP, red), and neuron (NeuN, red) in the peri-infarcted area at 24 h post-HI. DAPI was stained blue. Merged images showed that α- MSH was colocalized with microglia, astrocytes, and neurons. n = 2 per group. Scale bar = 100 μm

Article Snippet: Forty-eight hours before HI induction, 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 administered via intracerebroventricular injection at 1.5 mm posteriors, 1.5 mm lateral to the bregma, and 1.7 mm deep into the ipsilateral hemisphere.

Techniques: Expressing, Western Blot, Immunofluorescence, Staining

Journal: Journal of neuroinflammation

Article Title: Activation of MC1R with BMS-470539 attenuates neuroinflammation via cAMP/PKA/Nurr1 pathway after neonatal hypoxic-ischemic brain injury in rats.

doi: 10.1186/s12974-021-02078-2

Figure Lengend Snippet: Fig. 2 Effect of MC1R activation on brain infarct area, body weight, and short-term neurological function at 48 h post-HI. a, b TTC staining showed that medium (160 μg/kg) and high (500 μg/kg) doses of BMS-470539 treatment significantly reduced brain infarct area compared to the vehicle. c Rats in the vehicle group showed to lose significant weight compared to the sham group, medium (160 μg/kg) and high (500 μg/kg) doses of BMS-470539 treatment groups. d Geotaxis reflex showed that medium (160 μg/kg) and high (500 μg/kg) doses of BMS-470539 significantly improved neurological function compared to the vehicle group. Data were represented as mean ± SD. *p < 0.05 versus sham, #p < 0.05 versus vehicle, @p < 0.05 versus BMS-470539 (50 μg/kg). n = 6 per group

Article Snippet: Forty-eight hours before HI induction, 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 administered via intracerebroventricular injection at 1.5 mm posteriors, 1.5 mm lateral to the bregma, and 1.7 mm deep into the ipsilateral hemisphere.

Techniques: Activation Assay, Staining

Journal: Journal of neuroinflammation

Article Title: Activation of MC1R with BMS-470539 attenuates neuroinflammation via cAMP/PKA/Nurr1 pathway after neonatal hypoxic-ischemic brain injury in rats.

doi: 10.1186/s12974-021-02078-2

Figure Lengend Snippet: Fig. 3 Immunofluorescence staining of MC1R and Nurr1 with microglia in the brain at 48 h post-HI. Immunofluorescence staining showed an increase expression of MC1R (a) and Nurr1 (b) on microglia in the vehicle group when compared with the sham group, and further increased expression of MC1R (a) and Nurr1 (b) in the BMS-470359 treatment group. Merge showed the colocalization of MC1R and Nurr1 on microglia. Microglia were stained red. MC1R and Nurr1 were stained green. DAPI was stained blue. n = 2 per group. Scale bar = 100 μm

Article Snippet: Forty-eight hours before HI induction, 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 administered via intracerebroventricular injection at 1.5 mm posteriors, 1.5 mm lateral to the bregma, and 1.7 mm deep into the ipsilateral hemisphere.

Techniques: Immunofluorescence, Staining, Expressing

Journal: Journal of neuroinflammation

Article Title: Activation of MC1R with BMS-470539 attenuates neuroinflammation via cAMP/PKA/Nurr1 pathway after neonatal hypoxic-ischemic brain injury in rats.

doi: 10.1186/s12974-021-02078-2

Figure Lengend Snippet: Fig. 5 Effects of knockout MC1R and Nurr1 on infarct area, body weight, and neurological function at 48 h post-HI. a, b The infarct area was significantly increased in both treatment groups with CRISPR compared to the HI + BMS-470539 group or HI + BMS-470539 + control CRISPR group. c Activation of MC1R with BMS-470539 significantly reduced body weight loss compared to the vehicle. However, both treatment groups with CRISPR interventions significantly reversed these effects compared to the HI + BMS-470539 group or HI + BMS-470539 + control CRISPR group. d The geotaxis test showed that the rat pups treated with BMS-470539 and either MC1R CRISPR or Nurr1 CRISPR had significantly impaired neurological function compared to the corresponding controls. 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. n = 6 per group

Article Snippet: Forty-eight hours before HI induction, 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 administered via intracerebroventricular injection at 1.5 mm posteriors, 1.5 mm lateral to the bregma, and 1.7 mm deep into the ipsilateral hemisphere.

Techniques: Knock-Out, CRISPR, Control, Activation Assay

Journal: Journal of neuroinflammation

Article Title: Activation of MC1R with BMS-470539 attenuates neuroinflammation via cAMP/PKA/Nurr1 pathway after neonatal hypoxic-ischemic brain injury in rats.

doi: 10.1186/s12974-021-02078-2

Figure Lengend Snippet: Fig. 6 MC1R activation increased the expression of CD206 at 48 h post-HI. Representative microphotographs of immunofluorescence staining and quantification of CD206 with microglia (CD11 b/c) at 48 h post-HI. a, b The number of CD206+ CD11 b/c+ cells significantly increased in the vehicle group compared to the sham group. Activation of MC1R with BMS-470539 further increased the number of CD206+ CD11 b/c+ cells compared to the vehicle group, while knockout CRISPR interventions significantly reversed these effects. CD206 was green. CD11 b/c was red. Blue was for DAPI. Scale bar = 100 μm. Data was represented as mean ± SD. The one-way ANOVA was followed by Tukey’s post hoc test (*p < 0.05 versus sham; #p < 0.05 versus HI + vehicle; @p < 0.05 HI + BMS-470539 or HI + BMS-470539 + control CRISPR; n = 6 per group)

Article Snippet: Forty-eight hours before HI induction, 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 administered via intracerebroventricular injection at 1.5 mm posteriors, 1.5 mm lateral to the bregma, and 1.7 mm deep into the ipsilateral hemisphere.

Techniques: Activation Assay, Expressing, Immunofluorescence, Staining, Knock-Out, CRISPR, Control

Journal: Journal of neuroinflammation

Article Title: Activation of MC1R with BMS-470539 attenuates neuroinflammation via cAMP/PKA/Nurr1 pathway after neonatal hypoxic-ischemic brain injury in rats.

doi: 10.1186/s12974-021-02078-2

Figure Lengend Snippet: Fig. 7 MC1R activation on immunofluorescence staining of IL-1β and MPO at 48 h post-HI. Representative microphotographs of immunofluorescence staining and quantification of a, c IL-1β-positive cells and b, d MPO-positive cells. The number of IL-1β-positive cells or MPO-positive cells was significantly increased in the vehicle group compared to the sham group. BMS-470539 treatment significantly suppressed neuroinflammation, while these effects were reversed by knockout of MC1R or Nurr1 with CRISPR. IL-1β and MPO were green. Blue was for DAPI. Scale bar = 100 μm. Data was represented as mean ± SD. The one-way ANOVA was followed by Tukey’s post hoc test (*p < 0.05 versus sham; #p < 0.05 versus HI + vehicle; @p < 0.05 HI + BMS-470539 or HI + BMS-470539 + control CRISPR; n = 6 per group)

Article Snippet: Forty-eight hours before HI induction, 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 administered via intracerebroventricular injection at 1.5 mm posteriors, 1.5 mm lateral to the bregma, and 1.7 mm deep into the ipsilateral hemisphere.

Techniques: Activation Assay, Immunofluorescence, Staining, Knock-Out, CRISPR, Control

Journal: Journal of neuroinflammation

Article Title: Activation of MC1R with BMS-470539 attenuates neuroinflammation via cAMP/PKA/Nurr1 pathway after neonatal hypoxic-ischemic brain injury in rats.

doi: 10.1186/s12974-021-02078-2

Figure Lengend Snippet: Fig. 8 MC1R activation on neuroinflammation via cAMP/PKA/Nurr1 signaling pathway at 48 h post-HI. a Representative picture of Western blot data showing bands of the expression levels of MC1R, cAMP, p-PKA, Nurr1, IL-1β, TNFα, and IL-6 either with BMS-470539 treatment alone, BMS- 470539 + MC1R KO CRISPR, BMS-470539 + Nurr1 KO CRISPR, and BMS-470539 + control CRISPR groups. b–h Western blot data quantification of bands showed that BMS-470539 treatment significantly increased the expression of MC1R, cAMP, p-PKA, and Nurr1 compared to the HI + vehicle group. Knockout of MC1R using CRISPR significantly decreased MC1R, cAMP, p-PKA, and Nurr1 expression levels compared to the HI + BMS- 470539 group or HI + BMS-470539 + control CRISPR group. Furthermore, knockout of Nurr1 using CRISPR significantly decreased the levels of Nurr1, but did not affect MC1R, cAMP, and p-PKA expression compared to the corresponding controls. Activation of MC1R with BMS-470539 showed significantly decreased levels of IL-1β, TNFα, and IL-6, while both treatment groups with CRISPR interventions significantly reversed these effects. Data was represented as mean ± SD. The one-way ANOVA was followed by Tukey’s post hoc test (*p < 0.05 versus sham; #p < 0.05 versus HI + vehicle; @p < 0.05 HI + BMS-470539 or HI + BMS-470539 + control CRISPR; n = 6 per group)

Article Snippet: Forty-eight hours before HI induction, 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 administered via intracerebroventricular injection at 1.5 mm posteriors, 1.5 mm lateral to the bregma, and 1.7 mm deep into the ipsilateral hemisphere.

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

Journal: Journal of neuroinflammation

Article Title: Activation of MC1R with BMS-470539 attenuates neuroinflammation via cAMP/PKA/Nurr1 pathway after neonatal hypoxic-ischemic brain injury in rats.

doi: 10.1186/s12974-021-02078-2

Figure Lengend Snippet: Fig. 9 The potential molecular mechanisms of anti-inflammatory and neuroprotective effects through the MC1R/cAMP/PKA/Nurr1 signaling pathway post-HI

Article Snippet: Forty-eight hours before HI induction, 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 administered via intracerebroventricular injection at 1.5 mm posteriors, 1.5 mm lateral to the bregma, and 1.7 mm deep into the ipsilateral hemisphere.

Techniques:

Journal: NPJ precision oncology

Article Title: MC1R signaling through the cAMP-CREB/ATF-1 and ERK-NFκB pathways accelerates G1/S transition promoting breast cancer progression.

doi: 10.1038/s41698-023-00437-1

Figure Lengend Snippet: Fig. 1 MC1R expression is associated with breast cancer. a The MC1R R% allele frequency across cancers in The Cancer Genome Atlas (TCGA). The orange dashed line indicates the R% allele frequency in control ExAC non-TCGA non-finnish European (NFE) Ancestry population. *p < 0.05 (Fisher’s exact test with Benjamini–Hochberg (BH) multiple comparisons adjustment) compared to the control population. The cancer type abbreviations are expanded and listed in Supplementary Table 2. b MC1R mRNA expression in normal breast, primary, and metastatic breast cancers. Data obtained from TCGA. The dashed lines in the violin plot show the median and the 25th and 75th percentiles. *p < 0.05 (one-way ANOVA with Dunnett’s multiple comparisons). c–h Disease-free survival (DFS) (c, e, g) and progression-free survival (PFS) (d, f, h) of all patients with breast cancer in TCGA (DFS: low expression n = 462, high expression n = 477; PFS: low expression n = 539, high expression n = 541) (c, d), patients carrying active MC1R variants (DFS: low expression n = 395, high expression n = 408; PFS, low expression n = 461, high expression n = 462) (e, f), and patients carrying a disruptive MC1R variant (DFS, low expression n = 67, high expression n = 69; PFS, low expression n = 78, high expression n = 79) (g, h) based on MC1R expression. p-Value calculated using the Log-rank test.

Article Snippet: For transient MC1R overexpression, HEK 293 T cells, plated in 6-well plates in complete DMEM, were transfected with an MC1R overexpression plasmid (OriGene; Cat#: RC203218) using Lipofectamine 3000 (Thermo Fisher).

Techniques: Expressing, Control, Variant Assay

Journal: NPJ precision oncology

Article Title: MC1R signaling through the cAMP-CREB/ATF-1 and ERK-NFκB pathways accelerates G1/S transition promoting breast cancer progression.

doi: 10.1038/s41698-023-00437-1

Figure Lengend Snippet: Fig. 2 MC1R signaling is active in breast cancer cells and promotes breast cancer cell progression in vitro. a Mean ± SEM cAMP levels in T-47d and MCF7 cells treated with or without 0.2 μM NDP-MSH from three independent experiments. *p < 0.05 (unpaired Student’s t-test). b, c Mean ± SEM percentage fold change in cAMP levels in (b) T-47d and (c) MCF7 cells treated with or without 20 μM MSG-606 and stimulated with 0.2 μM NDP-MSH compared to those left untreated (UT) from three independent experiments. *p < 0.05 (one-way ANOVA with Tukey’s multiple comparisons). d T-47d and MCF7 cells were treated with 0.2 μM NDP-MSH or pretreated with 20 μM MSG-606, followed by stimulation with 0.2 μM NDP-MSH or left untreated. Representative western blot showing MC1R expression and downstream signaling in T-47d, MCF7 cells. GAPDH is shown as the loading control. Western blot quantification plots are shown in Supplementary Fig. 3a. e WT T-47d (WT) and MC1R knockdown (KD) T-47d (MC1R Sh1 and MC1R Sh2) cell lines were treated with 0.2 μM NDP-MSH. Representative western blot showing MC1R expression and downstream signaling. GAPDH is shown as the loading control. Western blot quantification plots are shown in Supplementary Fig. 3b. f Mean ± SEM fold change in cAMP levels in MC1R-KD T-47D cells treated with 0.2 μM NDP-MSH from 3 independent experiments. *p < 0.05 (one-way ANOVA with Tukey’s multiple comparisons). g, h Mean ± SEM fold change in cell number in (g) T-47d and (h) MCF7 cells treated either with 0.2 μM NDP-MSH or 20 μM MSG-606 or left untreated (UT) over time from three independent experiments. *p < 0.05 (two-way ANOVA with Dunnett’s multiple comparisons) compared to UT at the indicated time points. i Mean ± SEM fold change in cell number of WT T-47d cells and MC1R-KD T-47d cells (MC1R Sh1 and MC1R Sh2) treated with or without 0.2 μM NDP-MSH. (VC, vector control) over time from three independent experiments. *p < 0.05 (two-way ANOVA with Dunnett’s multiple comparisons) compared to T-47d WT at the indicated time points.

Article Snippet: For transient MC1R overexpression, HEK 293 T cells, plated in 6-well plates in complete DMEM, were transfected with an MC1R overexpression plasmid (OriGene; Cat#: RC203218) using Lipofectamine 3000 (Thermo Fisher).

Techniques: In Vitro, Western Blot, Expressing, Control, Knockdown, Plasmid Preparation

Journal: NPJ precision oncology

Article Title: MC1R signaling through the cAMP-CREB/ATF-1 and ERK-NFκB pathways accelerates G1/S transition promoting breast cancer progression.

doi: 10.1038/s41698-023-00437-1

Figure Lengend Snippet: Fig. 3 MC1R promotes tumorigenicity and breast cancer progression in vitro and in vivo. a, b Soft agar colony formation for WT T-47d and MC1R-KD T-47d (MC1R Sh1 and MC1R Sh2) cells. a Representative images of the soft agar wells. b Mean ± SEM fold change in the number of soft agar colonies from three independent experiments. *p < 0.05 (one-way ANOVA with Dunnett’s multiple comparisons). c, d Athymic nude mice carrying 17β-estradiol pellets were subcutaneously injected with WT T-47d or MC1R-KD T-47d cells, and tumor development was monitored. c Mice pictured on day 24 post-implant. d Tumor volume on day 24 post-implant in mice that received the WT T-47d (n = 7) or MC1R-KD T-47d (n = 6) cells are shown; the error bar shows mean ± SEM. *p < 0.05 (unpaired Student’s t-test). e Representative micrograph (40×) showing breast cancer tissue samples with different MC1R staining scores (0, 2, 6) and Ki67 expression in the breast cancer tissue microarray. Scale bar = 50 μm. f Plot comparing MC1R expression and Ki67 expression. The dashed lines in the violin plot show the median and the 25th and 75th percentiles. *p < 0.05 unpaired Student’s t-test.

Article Snippet: For transient MC1R overexpression, HEK 293 T cells, plated in 6-well plates in complete DMEM, were transfected with an MC1R overexpression plasmid (OriGene; Cat#: RC203218) using Lipofectamine 3000 (Thermo Fisher).

Techniques: In Vitro, In Vivo, Injection, Staining, Expressing, Microarray

Journal: NPJ precision oncology

Article Title: MC1R signaling through the cAMP-CREB/ATF-1 and ERK-NFκB pathways accelerates G1/S transition promoting breast cancer progression.

doi: 10.1038/s41698-023-00437-1

Figure Lengend Snippet: Fig. 4 MC1R downregulation delays G1–S progression in breast cancer cells. a–c Wild-type (WT) T-47d and MC1R-Knockdown (KD) T-47d cells were synchronized to the G1 phase by a double-thymidine block and then released. a Mean ± SEM percentage of cells in the G1, S, and G2 phases at 0, 3, 6, 9, and 12 h post-release. *p < 0.05 (unpaired Student’s t-test). b Mean ± SEM percentage of cells in the S phase across time. *p < 0.05 (one-way ANOVA with Dunnett’s multiple comparisons). c Representative western blot showing Cyclin D1, Cyclin E1, Ser780 p-RB, Rb, and GAPDH (loading control). Western blot quantification plots are shown in Supplementary Fig. 4a. d–f WT T-47d cells were treated with 0.2 μM NDP-MSH or 20 μM MSG-606 or left untreated after releasing from a double-thymidine block. d Mean ± SEM percentage of cells in the G1, S, and G2 phases at 0, 3, 6, 9, and 12 h post-release. *p < 0.05 (unpaired Student’s t-test). e Mean ± SEM percentage of cells in the S phase across time. *p < 0.05 (one-way ANOVA with Dunnett’s multiple comparisons). f Representative western blot showing Cyclin D1, Cyclin E1, Ser780 p-RB, Rb, and GAPDH (loading control). Western blot quantification plots are shown in Supplementary Fig. 4b.

Article Snippet: For transient MC1R overexpression, HEK 293 T cells, plated in 6-well plates in complete DMEM, were transfected with an MC1R overexpression plasmid (OriGene; Cat#: RC203218) using Lipofectamine 3000 (Thermo Fisher).

Techniques: Knockdown, Blocking Assay, Western Blot, Control

Journal: NPJ precision oncology

Article Title: MC1R signaling through the cAMP-CREB/ATF-1 and ERK-NFκB pathways accelerates G1/S transition promoting breast cancer progression.

doi: 10.1038/s41698-023-00437-1

Figure Lengend Snippet: Fig. 5 MC1R signaling through MC1R-cAMP-CREB contributes to the accelerated G1-S transition in breast cancer cells. a–c. MC1R-KD T- 47d cells were synchronized to the G1 phase by a double-thymidine block and then released with or without treatment with 25 μM FSK. (a) Mean ± SEM percentage of cells in the G1, S, and G2 phases at 0, 3, 6, 9, and 12 h post-release. *p < 0.05 (unpaired Student’s t-test). (b) Mean ± SEM percentage of cells in the S phase across time. *p < 0.05 (one-way ANOVA with Dunnett’s multiple comparisons). (c) Representative western blot showing Cyclin D1, Cyclin E1, Ser780 p-Rb, Rb, and GAPDH (loading control). Western blot quantification plots are shown in Supplementary Fig. 4d. d–f. WT T-47d cells were treated with 0.2 μM NDP-MSH or 0.2 μM NDP-MSH + 5 μM 666-15 (CREBi) or left untreated after releasing from a double-thymidine block. (d) Mean ± SEM percentage of cells in the G1, S, and G2 phases at 0, 3, 6, 9, and 12 h post-release. *p < 0.05 (unpaired Student’s t-test). (e) Mean ± SEM percentage of cells in the S phase across time. *p < 0.05 (one-way ANOVA with Dunnett’s multiple comparisons). (f) Representative western blot showing Cyclin D1, Cyclin E1, Ser780 p-Rb, Rb, and GAPDH (loading control). Western blot quantification plots are shown in Supplementary Fig. 4e.

Article Snippet: For transient MC1R overexpression, HEK 293 T cells, plated in 6-well plates in complete DMEM, were transfected with an MC1R overexpression plasmid (OriGene; Cat#: RC203218) using Lipofectamine 3000 (Thermo Fisher).

Techniques: Blocking Assay, Western Blot, Control

Journal: NPJ precision oncology

Article Title: MC1R signaling through the cAMP-CREB/ATF-1 and ERK-NFκB pathways accelerates G1/S transition promoting breast cancer progression.

doi: 10.1038/s41698-023-00437-1

Figure Lengend Snippet: Fig. 6 MC1R signaling through MC1R-cAMP-CREB/ATF and MC1R-ERK-NFκB axes promote G1/S transition. a WT T-47d (WT) and MC1R knockdown (KD) T-47d (MC1R Sh1 and MC1R Sh2) cell lines were stimulated with 0.2 μM NDP-MSH. Representative western blot showing p- ERK, t-ERK, p-p65 NFκB, and p65 NFκB, and GAPDH (loading control) in T-47d WT (wild-type), VC (vector control), and MC1R-KD T-47d (Sh1 and Sh2) cells. Western blot quantification plots are shown in Supplementary Fig. 5a. b T-47d and MCF7 cells were treated with different concentrations of MSG-606, as indicated, before stimulation with 0.2 μM NDP-MSH. Representative western blot showing p-CREB, p-ATF-1, t-CREB, p-ERK, t-ERK, p-p65 NFκB, p65 NFκB, and GAPDH (loading control). Western blot quantification plots are shown in Supplementary Fig. 5b. c HEK 293 T VC (vector control) and HEK 293 T cells transiently overexpressing MC1R (HEK 293 T MC1R) were serum-starved overnight and then released with 0.2 μM NDP-MSH with or without treatment with a MEK inhibitor, 5 μM U0126, a CREB inhibitor, 5 μM 666-15, or 20 μM MSG- 606. Representative western blot showing p-CREB, p-ATF-1, t-CREB, p-ERK, t-ERK, p-p65 NFκB, and p65 NFκB, Cyclin D1, Cyclin E1, Ser780 p-Rb, Rb, and GAPDH (loading control). Western blot quantification plots are shown in Supplementary Fig. 6c. d HEK 293 T VC (vector control) and HEK 293 T cells transiently overexpressing wild-type MC1R (MC1R WT) or the MC1R variants (MC1R R151C and MC1R R160W) were serum- starved overnight and then released with or without 0.2 μM NDP-MSH. Representative western blot showing p-CREB, p-ATF-1, t-CREB, p-ERK, t-ERK, p-p65 NFκB, p65 NFκB, and GAPDH (loading control). Western blot quantification plots are shown in Supplementary Fig. 6d.

Article Snippet: For transient MC1R overexpression, HEK 293 T cells, plated in 6-well plates in complete DMEM, were transfected with an MC1R overexpression plasmid (OriGene; Cat#: RC203218) using Lipofectamine 3000 (Thermo Fisher).

Techniques: Knockdown, Western Blot, Control, Plasmid Preparation

Journal: Frontiers in physiology

Article Title: Agouti Signaling Protein and Its Receptors as Potential Molecular Markers for Intramuscular and Body Fat Deposition in Cattle.

doi: 10.3389/fphys.2018.00172

Figure Lengend Snippet: FIGURE 7 | Localization of ASIP in bovine tissues. The polyclonal rabbit anti-ASIP antibody was detected with an Alexa Fluor 488 labeled goat anti rabbit IgG secondary antibody in bovine liver (A), MLD (C), and SCF (E). Nuclei were stained with Hoechst 33248 and overlaid to the brightfield image of liver (B), MLD (D), and SCF (F), respectively. Arrows indicate specific staining in stellate cells (A) or in and around adipocytes (C,E). SCF, subcutaneous fat; MLD, M. longissimus dorsi.

Article Snippet: The polyclonal antibodies against MC1R (ELA-ENT2673, Elabscience) and ATRN (BYT-ORB155769, Biorbyt) were also generated in rabbit and purchased from Biozol (Eching, Germany).

Techniques: Labeling, Staining

Journal: Annals of Translational Medicine

Article Title: Protective effect of hydrogen sulfide on endothelial cells through Sirt1-FoxO1-mediated autophagy

doi: 10.21037/atm-20-3647

Figure Lengend Snippet: Primary antibodies used

Article Snippet: Concentration used RRIDs Anti-GAPDH GAPDH fusion protein Ag0766 Proteintech, rabbit polyclonal antibody, 10494-1-AP 1:10,000 (WB) AB_2263076 Anti-LC3 Residues surrounding Leu44 of human LC3B protein (conserved in LC3A) Cell Signalling Technology, rabbit monoclonal antibody, #12741 1:1,000 (WB) AB_2617131 Anti-p62 P62/SQSTM1 fusion protein Ag13131 Proteintech, rabbit polyclonal antibody, 18420-1-AP 1:1,000 (WB) AB_10694431 Anti-ATG5 Residues surrounding Leu265 of human Atg5 protein Cell Signaling Technology, rabbit monoclonal antibody, #12994 1:1,000 (WB) AB_2630393 Anti-beclin-1 Beclin 1 fusion protein Ag1843 Proteintech, rabbit polyclonal antibody, 11306-1-AP 1:1,000 (WB) AB_2259061 Anti-Sirt1 A recombinant protein representing the central region of human SirT1 protein Cell Signaling Technology, mouse monoclonal antibody, #8469 1:1,000 (WB) AB_10999470 Anti-cleaved-caspase 3 Synthesized peptide derived from human caspase 3 Affinity Biosciences, rabbit polyclonal antibody, AF7022 1:1,000 (WB) AB_2835326 Anti-cleaved-PARP Synthetic peptide within human cleaved PARP1 aa 150–250 Abcam, rabbit monoclonal antibody, ab32064 1:1,000 (WB) AB_777102 FoxO1 Synthetic peptide mapping to a region between residues 600 and the C-terminus (residue 652) of human FOXO1A Abcam, rabbit polyclonal antibody, ab70382 1:500 (IF), 1:1,000 (WB) AB_1268912 Anti-acetylated-lysine A synthetic acetylated lysine-containing peptide Cell Signalling Technology, rabbit antibody, #9441 1:1,000 (WB) AB_331805 Anti-histone-H3 Histone-H3 fusion protein Ag10644 Proteintech, rabbit polyclonal antibody, 17168-1-AP 1:3,000 (WB) AB_2716755 Anti-tubulin Beta-tubulin fusion protein Ag0117 Proteintech, rabbit polyclonal antibody, 10068-1-AP 1:3,000 (WB) AB_2303998 Open in a separate window caption a8 Primary antibodies used

Techniques: Concentration Assay, Recombinant, Synthesized, Derivative Assay, Residue