Journal: bioRxiv

Article Title: HDAC6 promotes self-renewal and migration/invasion of rhabdomyosarcoma

doi: 10.1101/823864

Figure Lengend Snippet: (A) Western blots against acetylated alpha-Tubulin and alpha-Tubulin in RD and Rh 5 cells with targeted disruption of HDAC6 by CRISPR or treated with DMSO (vehicle) and Tubastatin A (200 nM). (B) Scratch assay assessing effects of adding back wild-type (wt) HDAC6 and catalytically-dead (cd) HDAC6 in RD cells with HDAC6 knockout. Results are shown from one representative experiment of at least 3 repeats. (C) Phalloidin staining in RD cells with safe-harbor control region and HDAC6 CRISPR targeting following serum starvation and 15-minute EGF (50 ng/mL) treatment. Arrowheads point to representative areas of membrane ruffles and filopodia formation. Green = phalloidin, Blue = DAPI. (D) Double IF against HDAC6 (green) and RAC1 (red) in RD cells. (E) RAC1 GTP pulldown assay in RD cells harboring safe-harbor control and HDAC6 CRISPR targeting. (F) Summary of scratch assays assessing the effects of overexpressing GFP as a control, RAC1V12, RAC1N17 and RHOAV14 in the presence of CRISPR-mediated targeted disruption of HDAC6 in RD cells following 24 hours of serum starvation and 15 minutes of EGF (50 ng/mL) treatment. (G) Summary of cell growth change by cell counts over 6 days assessing the effects of overexpressing GFP as a control, RAC1V12 and RAC1N17 on cell growth of RD cells harboring targeted disruption of HDAC6 . Results for the average of 4 replicates for each condition in one of 3 independent experiments are shown. (H) RAC1-GTP pulldown assay in RD cells overexpressing GFP, RAC1V12 and RAC1N17 following 24 hours of serum starvation and 15 minutes of EGF (50 ng/mL) treatment. Each error bar in B, F and G represents standard deviation. NS = no significance, p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001.

Article Snippet: The following antibodies including dilutions were used: rabbit polyclonal anti human mouse monoclonal anti human Ki- 67, (1:100, clone: MIB1, Dako), rabbit monoclonal anti human HDAC6 (1:100, clone, D2E5; Cell Signaling), mouse monoclonal anti human RAC1 (1:100, clone 102; BD Biosciences), mouse monoclonal anti acetylated alpha-tubulin (1:200; clone 6-11B-1; Santa Cruz Biotechnology) and mouse monocloncal anti-alpha-tubulin (1:200; B-5-1-2; Santa Cruz Biotechnology).

Techniques: Western Blot, CRISPR, Wound Healing Assay, Knock-Out, Staining, Standard Deviation

Journal: PLoS ONE

Article Title: Cannabinoids Reduce Markers of Inflammation and Fibrosis in Pancreatic Stellate Cells

doi: 10.1371/journal.pone.0001701

Figure Lengend Snippet: (A) IL-6 and MCP-1 secretion were significantly reduced by WIN (p = 0.001 and p = 0.0002, respectively), independent of TGFbeta (B; unchanged TGFbeta levels). The reduction in IL-6 and MCP-1 levels was partially reversed by a combination of the CB1-receptor and CB2-receptor antagonists AM251 and AM630 (A). While control PSC secreted significant amounts of fibronectin and collagen 1 (as seen by an intense signal at 220 and 190 kDa, respectively), treatment with WIN reduced the signal at the respective molecular weights (B; immunoblots of cell culture supernatants, pooled from three independent experiments). AlphaSMA levels were also suppressed by WIN (B; immunoblot of PSC cell lysates; gamma-tubulin: equal loading control). A combination of both antagonists AM251 and AM630 partially reversed the suppressive effects elicited by incubation with WIN (B, immunoblots of fibronectin, collagen 1 and alphaSMA). White bars: control; black bars: WIN55,212-2±AM251/AM630. Data are shown as mean±SEM.

Article Snippet: Primary antibodies for collagen type-1 (diluted 1∶200; sc-28657, Santa Cruz biotechnology, Santa Cruz, CA, USA), fibronectin (1∶10,000; F3648, Sigma Aldrich, Taufkirchen, Germany), alphaSMA (1∶10,000, M0851, DAKO Cytomation, Hamburg, Germany) and gamma-tubulin (1∶5,000; sc-7396, Santa Cruz biotechnology) were applied overnight at 4°C; secondary antibodies (diluted correspondingly 1∶2,000, 1∶5,000, 1∶5,000, 1∶5,000) were added for one hour at room temperature.

Techniques: Control, Western Blot, Cell Culture, Incubation

Journal: International Journal of Molecular Sciences

Article Title: Myogenic Potential of Extracellular Matrix Derived from Decellularized Bovine Pericardium

doi: 10.3390/ijms22179406

Figure Lengend Snippet: Expression of myogenic markers, analyzed by Western blot. C2C12 myoblasts were incubated either on uncoated (CTR) or dECM pre-coated wells and induced to differentiate in differentiation medium. At different time points the contents of α-smooth muscle actin ( A ), myogenin ( B ), and MHC ( C ) were measured by Western blots of whole cell lysates and the intensity of the bands quantified, relative to the housekeeping proteins vinculin or tubulin used to normalize. Representative Western blots out of the three for each condition are shown. Statistics were determined by t -test. ** p < 0.01, *** p < 0.001.

Article Snippet: Results were normalized with the housekeeping proteins vinculin and tubulin (anti-vinculin, anti-tubulin 1:1000; Merck Millipore, Milan, Italy).

Techniques: Expressing, Western Blot, Incubation

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