Journal: Cell

Article Title: A Flat BAR Protein Promotes Actin Polymerization at the Base of Clathrin-Coated Pits

doi: 10.1016/j.cell.2018.05.020

Figure Lengend Snippet: FCHSD2 Is Not Directly Recruited to CCPs by PI3KC2α or Its Kinase Activity, Related to Figure 2 (A) Top: Transferrin uptake assay by flow cytometry comparing wild-type and FCHSD2 KO cells silenced for PI3KC2α. Uptake of Alexa488 labeled transferrin normalized by the amount of surface transferrin receptor for each condition and against uptake for the wild-type cells in each experiment. Each value represents median fluorescence from at least 5000 cells (n = 12, mean ± SD). ∗∗∗ p > 0.001, ns = non-significant. One-way ANOVA with Tukey’s post hoc analysis. Bottom: Immunoblots showing PI3KC2α knockdown in wild-type and FCHSD2 KO cells. (B) Kymographs of HeLa FCHSD2-Venus stables silenced for PI3KC2α and control cells. Cells were transfected with mCherry-ClathrinLC 24 hs before imaging. Kymographs generated from 120 s movies at 1Hz. Note the elongated CCP lifetimes in PI3KC2α knockdown cells as described in Posor et al. (2013) . (C) Autoinhibition of FCHSD2. Representative images of a center slice from cells expressing different FCHSD2 truncation constructs and co-stained with phalloidin (Actin). The bar graph (upper right) shows the quantification of cellular protrusions/μm for each construct. The non-inhibited BAR domain produces many protrusions. Numbers inside bars represent number of cells measured. The line graph (bottom right) shows the fluorescence profile of sum intensity projections for cells expressing each construct. Due to the natural thinning of cells from their centers to the edge, a gradually decaying line indicates that the fluorescent protein is primarily cytosolic while a flat line with an abrupt fall on the cell edge indicates that the fluorescent protein is primarily bound to the membrane. While the presence of SH3-1 significantly reduces the generation of cellular protrusions generated by the FCHSD2 F-BAR, a significant fraction of the protein remains bound to the membrane (green line). Only the combined presence of SH3-1 and SH3-2 is capable to avoid promiscuous binding of the BAR domain to the membrane. Data is shown as mean ± SD in bar graph and as ± SEM in fluorescence profiles. ∗∗∗ p > 0.001, ns = non-significant. One-way ANOVA with Tukey’s post hoc analysis. (D) Immunoblots for intersectin knockdown in FCHSD2-Venus HeLa cells.

Article Snippet: PI3KC2α siRNA pool , Origene , Cat# SR303516.

Techniques: Activity Assay, Flow Cytometry, Labeling, Fluorescence, Western Blot, Knockdown, Control, Transfection, Imaging, Generated, Expressing, Construct, Staining, Membrane, Binding Assay

Journal: Cell

Article Title: A Flat BAR Protein Promotes Actin Polymerization at the Base of Clathrin-Coated Pits

doi: 10.1016/j.cell.2018.05.020

Figure Lengend Snippet:

Article Snippet: PI3KC2α siRNA pool , Origene , Cat# SR303516.

Techniques: Virus, Recombinant, Labeling, Cloning, Stable Transfection, Expressing, esiRNA, Plasmid Preparation, Software

Journal: Scientific reports

Article Title: TNFα induces matrix metalloproteinase-9 expression in monocytic cells through ACSL1/JNK/ERK/NF-kB signaling pathways.

doi: 10.1038/s41598-023-41514-6

Figure Lengend Snippet: Figure 3. ACSL1 siRNA transfection reduced TNFα-mediated MMP-9 production. We transfected THP-1 monocytic cells with siRNA targeting human ACSL1 gene expression or scrambled siRNA (a control siRNA). (A,B) After 36 h, we performed real-time PCR to measure ACSL1 gene expression or western blotting for protein to test the knocking down efficiency. (C) We then incubated ACSL1-deficient cells with TNFα for 24 h. We determined mRNA expression of MMP-9 by real-time PCR. (D) We determined MMP-9 protein in culture media using ELISA. (E) The effect of siRNA transfection in combination with TNFα on cell viability was evaluated by measuring cell metabolic activity (MTT assay). The cell viability is expressed as the percentage of cells compared to the condition of vehicle control. Three independent experiments were performed with similar results. All data are expressed as mean ± SEM (n ≥ 3). t test or onene way ANOVA (Dunnett’s Test) for comparing treatments vs control) were used). **p < 0.01, ***p < 0.001.

Article Snippet: We transfected the cells separately with siRNA against ACSL1 (30 nM; OriGene Technologies, Inc., Rockville, MD, USA), scramble siRNA (30 nM; OriGene Technologies, Inc., Rockville, MD, USA), and pmaxGFP (0.5 ug; Amaxa Nucleofector Kit V for THP-1cells, Lonza, Cologne, Germany).

Techniques: Transfection, Gene Expression, Control, Real-time Polymerase Chain Reaction, Western Blot, Incubation, Expressing, Enzyme-linked Immunosorbent Assay, Activity Assay, MTT Assay

Journal: Neurotherapeutics

Article Title: Chronic morphine-mediated upregulation of high mobility group box 1 in the spinal cord contributes to analgesic tolerance and hyperalgesia in rats

doi: 10.1007/s13311-019-00800-w

Figure Lengend Snippet: The expression of HMGB1, TLR4, and RAGE proteins and mRNA in the spinal dorsal horn. (a) Diagram of the timeline of this experiment. (b-d) Images showing HMGB1 expression in the spinal dorsal horn of naïve rats (b) and rats receiving intrathecal (i.t.) injection of saline (c) and i.t. injection of morphine (d). (e-g) Images showing TLR4 expression in the spinal dorsal horn of naïve rats (e) and rats receiving i.t. injection of saline (f) and i.t. injection of morphine (g). (gh-j) The images showing RAGE expressed in spinal dorsal horn in rats of naïve rats (h) and rats receiving i.t. injection of saline (i) and i.t. injection of morphine (j). (k) Repeated intrathecal (i.t.) injections of morphine led to a significant increase in the expression of HMGB1, TLR4, and RAGE proteins in the spinal dorsal horn. * P < 0.05, ** P < 0.01 vs. control group (i.t. injection of saline daily for 6 days). (l) Repeated i.t. injections of morphine led to a significant increase in the expression of HMGB1, TLR4, and RAGE mRNA in the spinal dorsal horn. * P < 0.05, ** P < 0.01, *** P < 0.001 vs. saline group (i.t. injection of saline daily for 6 days). (m) Repeated subcutaneous (s.c.) injections of morphine caused increased expression of HMGB1 mRNA in the spinal dorsal horn of mice. * P < 0.05; ** P < 0.01 vs. control (s.c. saline daily for 9 days). Data are presented as the mean ± SEM and were analyzed with one-way ANOVA. Images in b-j, scale bar = 200 μm. Sa: saline

Article Snippet: The HMGB1 small interfering RNA (siRNA) target rat hmgb1 was purchased from OriGene (OriGene Technologies, Rockville, SR506455).

Techniques: Expressing, Injection, Saline, Control

Journal: Neurotherapeutics

Article Title: Chronic morphine-mediated upregulation of high mobility group box 1 in the spinal cord contributes to analgesic tolerance and hyperalgesia in rats

doi: 10.1007/s13311-019-00800-w

Figure Lengend Snippet: The cell types that express HMGB1, TLR4, and RAGE in the rats spinal dorsal horn following repeated intrathecal (i.t.) injections of morphine. (a-c) Representative images showing that HMGB1 colocalized with the spinal neuronal marker NeuN (a), the astrocytic marker GFAP (b), and the microglial marker OX42 (c). (d-f) Representative images showing TLR4 colocalization with NeuN (d), GFAP (e), and OX42 (f). (g-i) Representative images showing RAGE colocalization with NeuN (g) and GFAP (h) but not with OX42 (i). Images in a-i, scale bar = 50 μm.

Article Snippet: The HMGB1 small interfering RNA (siRNA) target rat hmgb1 was purchased from OriGene (OriGene Technologies, Rockville, SR506455).

Techniques: Marker

Journal: Neurotherapeutics

Article Title: Chronic morphine-mediated upregulation of high mobility group box 1 in the spinal cord contributes to analgesic tolerance and hyperalgesia in rats

doi: 10.1007/s13311-019-00800-w

Figure Lengend Snippet: Effects of morphine exposure on the expression and release of HMGB1 in primary cultured spinal neurons. (A) Diagram of the timeline of this experiment. (b-g) The cultured neuron purity was calculated by labeling neurons with the neuronal marker MAP2 and glial cells with GFAP (an astrocytic marker) and OX42 (a microglial marker). The images were merged in (d) and (g). Scale bar = 50 μm. (h-j) Representative images showing the colocalization of the neuronal marker NeuN (h) with the TLR4 protein (i) in cultured cells (j). (k-m) Images showing the neuronal marker NeuN (k) colocalized with TLR4 mRNA (i) in cultured cells (m). Images in h-m, scale bar = 50 μm. (n, o) Morphine exposure promoted the expression of HMGB1 in primary cultured spinal neurons in a dose- (neurons were cultured with different concentrations of morphine for 12 h) (n) and time- (neurons were cultured in medium containing 20 μM morphine for different amounts of time) (o) dependent manner. * P < 0.05; ** P < 0.0; *** P < 0.001 vs. control group. (p, q) Morphine challenge resulted in the increased release of HMGB1 from cultured neurons in a dose- (p) and time- (q) dependent manner. * P < 0.05; ** P < 0.01; *** P < 0.001 vs. control group. Data are presented as the mean ± SEM and were analyzed with two-way ANOVA.

Article Snippet: The HMGB1 small interfering RNA (siRNA) target rat hmgb1 was purchased from OriGene (OriGene Technologies, Rockville, SR506455).

Techniques: Expressing, Cell Culture, Labeling, Marker, Control

Journal: Neurotherapeutics

Article Title: Chronic morphine-mediated upregulation of high mobility group box 1 in the spinal cord contributes to analgesic tolerance and hyperalgesia in rats

doi: 10.1007/s13311-019-00800-w

Figure Lengend Snippet: The role of the chronic i.t. morphine exposure-induced upregulation of HMGB1 in the spinal dorsal horn in the development of analgesic tolerance and hyperalgesia. (a) Diagram of the timeline of this experiment. (b) Repeated i.t. injections of morphine led to a significant reduction in morphine’s maximal possible analgesic effect (% MPAE). # P < 0.05; ## P < 0.01 vs. day one. Data are presented as the mean ± SEM and were analyzed by two-way ANOVA. Intrathecal coadministration of morphine (Mor) plus glycyrrhizin (GL), an inhibitor of HMGB1, dose-dependently prevented the decrease in the MPAE. * P < 0.05; ** P < 0.01; *** P < 0.001 vs. morphine plus vehicle (Veh) group (one-way ANOVA). (c, d) Chronic i.t. morphine exposure resulted in decreased paw withdrawal threshold (PWT) (c) and paw withdrawal latency (PWL) (d) in the left hind paw. ## P < 0.01 vs. baseline. Repeated intrathecal coinjections of morphine plus GL prevented the reduction in PWT and PWL after morphine withdrawal. * P < 0.05; ** P < 0.01 vs. morphine plus vehicle group (Student’s t-test). The basal tail-flick response, PWT and PWL were not changed by repeated i.t. injections of GL alone. (e) Repeated i.t. injections of morphine plus vehicle (Veh: transfection regent) or morphine plus scramble (sc) RNA resulted in significant reductions in the MPAE at day 5 and day 7. ## P < 0.01 vs. day one (two-way ANOVA). This effect was clearly prevented by the coadministration of morphine with HMGB1 siRNA intrathecally. ** P < 0.01; *** P < 0.001 vs. morphine plus vehicle or morphine plus HMGB1 scRNA (one-way ANOVA). (f, g) Repeated i.t. injections of morphine plus vehicle or morphine plus HMGB1 scRNA led to significant decreases in PWT (f) and PWL (g) in the left hind paw, but these reductions were prevented by i.t. coinjections of morphine plus HMGB1 siRNA. # P < 0.05, ## P < 0.01 vs. baseline; * P < 0.05, ** P < 0.01 vs. morphine plus vehicle or morphine plus HMGB1 scRNA (Student’s t-test). The basal tail-flick response, PWT, and PWL were not changed by repeated i.t. injections of HMGB1 siRNA or transfection regent alone. (h, i) Repeated i.t. coinjections of morphine plus vehicle or morphine plus HMGB1 scRNA led to significantly increased expression of the HMGB1 protein (h) and HMGB1 mRNA (i) in the spinal dorsal horn. These effects were inhibited by i.t. coadministration of morphine with HMGB1 siRNA. * P < 0.05, ** P < 0.01 vs. vehicle group; ### P < 0.001 vs. morphine plus vehicle or morphine plus scramble RNA (one-way ANOVA).

Article Snippet: The HMGB1 small interfering RNA (siRNA) target rat hmgb1 was purchased from OriGene (OriGene Technologies, Rockville, SR506455).

Techniques: Tail Flick Test, Transfection, Expressing

Journal: Neurotherapeutics

Article Title: Chronic morphine-mediated upregulation of high mobility group box 1 in the spinal cord contributes to analgesic tolerance and hyperalgesia in rats

doi: 10.1007/s13311-019-00800-w

Figure Lengend Snippet: The effects of repeated i.t. administration of glycyrrhizin (GL) and an HMGB1 neutralizing antibody on established morphine tolerance and hyperalgesia. (a) Diagram of the timeline of this experiment. (b-d) Repeated i.t. coinjections of morphine plus vehicle (saline) resulted in a significant decrease in the MPAE on day 7, day 9 and day 11. ### P < 0.001 vs. day one (two-way ANOVA) (b). This reduction was reversed by the i.t. coadministration of morphine with GL started at day 7. ** P < 0.01, *** P < 0.001 vs. morphine plus vehicle group (Student’s t-test). The morphine withdrawal-induced reductions in PWT (c) and PWL (d) in the left hind paw were also partially reversed in the morphine plus GL group. # P < 0.05, ### P < 0.001 vs. baseline. * P < 0.01, ** P < 0.01 vs. morphine plus vehicle group (Student’s t-test). The basal tail-flick response, PWT, and PWL were not changed by repeated i.t. injections of GL or saline alone. (e) The reduction in the MPAE induced by repeated i.t. injections of morphine were also partially reversed by the i.t. coinjection of morphine with an HMGB1 neutralizing antibody starting at day 7. * P < 0.05, ** P < 0.01 vs. morphine plus vehicle group (Student’s t-test). (f, g) Coadministration of morphine with an HMGB1 neutralizing antibody alleviated morphine withdrawal-induced mechanical allodynia (f) and thermal hyperalgesia (g). * P < 0.05 vs. morphine plus vehicle group (Student’s t-test). The basal tail-flick response, PWT, and PWL were not changed by repeated i.t. injections of control IgG or ACSF alone. ACSF: artificial cerebrospinal fluid; Mor: morphine; Veh: vehicle.

Article Snippet: The HMGB1 small interfering RNA (siRNA) target rat hmgb1 was purchased from OriGene (OriGene Technologies, Rockville, SR506455).

Techniques: Saline, Tail Flick Test, Control

Journal: Neurotherapeutics

Article Title: Chronic morphine-mediated upregulation of high mobility group box 1 in the spinal cord contributes to analgesic tolerance and hyperalgesia in rats

doi: 10.1007/s13311-019-00800-w

Figure Lengend Snippet: The role of spinal HMGB1 in morphine tolerance and morphine withdrawal-induced hyperalgesia in female rats. (a) Repeated intrathecal (i.t.) injections of morphine led to a significant increase in the expression of HMGB1, TLR4, and RAGE proteins in the spinal dorsal horn. * P < 0.05, ** P < 0.01 vs. control group (i.t. injection of saline daily for 6 days). (b) Intrathecal coadministration of morphine (Mor) plus glycyrrhizin (GL), an inhibitor of HMGB1, prevented the decrease in the MPAE. * P < 0.05; ** P < 0.01; *** P < 0.001 vs. morphine plus vehicle (Veh) group (one-way ANOVA). (c, d) Repeated intrathecal coinjections of morphine plus GL prevented the reduction in PWT and PWL after morphine withdrawal. * P < 0.05; ** P < 0.01 vs. morphine plus vehicle group (Student’s t-test). (e) The reduction in the MPAE induced by repeated i.t. injections of morphine were also partially reversed by the i.t. coinjection of morphine with an HMGB1 neutralizing antibody starting at day 7. * P < 0.05, ** P < 0.01 vs. morphine plus vehicle group (Student’s t-test). (f, g) Coadministration of morphine with an HMGB1 neutralizing antibody alleviated morphine withdrawal-induced mechanical allodynia (f) and thermal hyperalgesia (g). * P < 0.05 vs. morphine plus vehicle group (Student’s t-test). (h, i) All rats, male (h) and female (i) showed equivalent time in the pairing chambers prior to conditioning day. After rats received vehicle, clonidine, and GL i.t. injection, the times that spent in chambers between vehicle-, clonidine-, and GL-paired group were no reached statistical difference at testing day in male(h) and female rats (i).

Article Snippet: The HMGB1 small interfering RNA (siRNA) target rat hmgb1 was purchased from OriGene (OriGene Technologies, Rockville, SR506455).

Techniques: Expressing, Control, Injection, Saline

Journal: Neurotherapeutics

Article Title: Chronic morphine-mediated upregulation of high mobility group box 1 in the spinal cord contributes to analgesic tolerance and hyperalgesia in rats

doi: 10.1007/s13311-019-00800-w

Figure Lengend Snippet: The signaling pathway involved in the morphine-mediated increase in the expression and release of HMGB1 in primary cultured spinal neurons. (a) Diagram of the timeline of this experiment. (b-e) The morphine plus vehicle-induced increased expression of HMGB1 in cultured spinal neurons was not blocked by the treatment of morphine plus CTOP (b) but was blocked by morphine plus TAK-242 (c), morphine plus naloxone (d), and morphine plus TLR4 siRNA (e). The concentration of morphine in the media was 20 μM, and the neurons were cultured for 12 hours. (f-i) The treatment of morphine plus CTOP in cultured spinal neurons did not reduce the morphine-stimulated increase in the release of HMGB1 (f). However, morphine plus TAK-242 (g), morphine plus naloxone (h), and morphine plus TLR4 siRNA (i) treatments resulted in significant reductions in the release of HMGB1 from cultured neurons. Compared to the normal control neurons (in which nothing was added to the media), the neurons treated with vehicle alone did not exhibit a change in the levels of HMGB1 released. * P < 0.05, ** P < 0.01 vs. control (Vehicle). # P < 0.05, ## P < 0.01 vs. morphine plus vehicle (Veh: b, c, d, f, g, and h: normal saline containing 10% DMSO; e and i: transfection regent). Data are presented as the mean ± SEM and were analyzed by one-way ANOVA. (j-m) Images showing the results of the cell death assay for cultured spinal neurons. (j) and (k) are the normal cultured neurons under different magnifications. (l) and (m) are the neurons treated with morphine and costained with Hoechst/propidium iodide (PI). Images in j scale bar = 100 μm; k-m scale bar = 50 μm.

Article Snippet: The HMGB1 small interfering RNA (siRNA) target rat hmgb1 was purchased from OriGene (OriGene Technologies, Rockville, SR506455).

Techniques: Expressing, Cell Culture, Concentration Assay, Control, Saline, Transfection

Journal: Neurotherapeutics

Article Title: Chronic morphine-mediated upregulation of high mobility group box 1 in the spinal cord contributes to analgesic tolerance and hyperalgesia in rats

doi: 10.1007/s13311-019-00800-w

Figure Lengend Snippet: Effects of TLR4/NF-κB signaling activation on the expression of HMGB1 in the spinal cord and the development of morphine tolerance and hyperalgesia. (a) Repeated i.t. coinjections of morphine with TAK-242 inhibited the phosphorylation of NF-κB p65 and reduced HMGB1 expression. (b) Repeated i.t. coinjections of morphine with PDTC reduced HMGB1 expression. Data shown in (a) and (b) are presented as the mean ± SEM and were analyzed by one-way ANOVA. * P < 0.05, ** P < 0.01 vs. control (i.t. saline containing 10% DMSO); # P < 0.05, ## P < 0.01 vs. morphine plus vehicle group. (c) Repeated i.t. coinjections of morphine plus TAK-242 dose-dependently prevented the decrease in morphine’s MPAE. ### P < 0.001 vs. day one (two-way ANOVA); * P < 0.05, ** P < 0.01, *** P < 0.001 vs. morphine plus vehicle group (one-way ANOVA). Repeated i.t. injections of saline or TAK-242 alone did not change the basal MPAE value. (d, e) Chronic i.t. morphine exposure resulted in decreased paw withdrawal threshold (PWT) (d) and paw withdrawal latency (PWL) (e) in the left hind paw. ## P < 0.01 vs. baseline. Repeated i.t. coinjections of morphine plus TAK-242 prevented the reduction in PWT and PWL after morphine withdrawal. * P < 0.05; ** P < 0.01 vs. morphine plus vehicle group (Student’s t-test). The basal tail-flick response, PWT and PWL were not changed by repeated i.t. injections of TAK-242 alone.

Article Snippet: The HMGB1 small interfering RNA (siRNA) target rat hmgb1 was purchased from OriGene (OriGene Technologies, Rockville, SR506455).

Techniques: Activation Assay, Expressing, Phospho-proteomics, Control, Saline, Tail Flick Test

Journal: Neurotherapeutics

Article Title: Chronic morphine-mediated upregulation of high mobility group box 1 in the spinal cord contributes to analgesic tolerance and hyperalgesia in rats

doi: 10.1007/s13311-019-00800-w

Figure Lengend Snippet: Signaling pathway of spinal HMGB1-mediated morphine tolerance and hyperalgesia. (a) Compared to morphine plus vehicle treatment, intrathecal administration of morphine with HMGB1 siRNA inhibited the increase in NF-κB p-p65 levels and the decrease in cytoplasmic IκB-α levels. However, p-p38 and p-JNK levels were not changed by this treatment. Data are presented as the mean ± SEM and were analyzed by one-way ANOVA. * P < 0.05, ** P < 0.01 vs. vehicle (Veh: transfection regent); # P < 0.05, ## P < 0.01 vs. morphine plus vehicle or morphine plus scramble (sc) RNA group. (b, c) A single i.t. injection of recombinant HMGB1 resulted in the increased expression of p-p65, TNF-α and IL-1β in the dorsal horn at 3 hours, which persisted to 6 hours after the injection (b). These effects were partially blocked by the coinjection of HMGB1 with TAK-242 (c). * P < 0.05, ** P < 0.01 vs. control (i.t. ACSF); # P < 0.05 vs. HMGB1 plus vehicle (Veh: ACSF). (d) A bolus i.t. coinjection of morphine with HMGB1 reduced the analgesic efficacy of morphine. * P < 0.05 vs. morphine plus ACSF group. (e-f) Bolus i.t. injection of HMGB1 led to a reduction in the paw withdrawal threshold (e) and paw withdrawal latency (f) in the left hind paw, which occurred at 3 hours and persisted to 6 hours after the injection. * P < 0.05 vs. baseline; # P < 0.05 vs. ACSF group. ACSF: artificial cerebrospinal fluid. Data of (d), (e), and (f) were analyzed by Student’s t-test.

Article Snippet: The HMGB1 small interfering RNA (siRNA) target rat hmgb1 was purchased from OriGene (OriGene Technologies, Rockville, SR506455).

Techniques: Transfection, Injection, Recombinant, Expressing, Control

Journal: JCI insight

Article Title: Sclerostin inhibition alleviates breast cancer-induced bone metastases and muscle weakness.

doi: 10.1172/jci.insight.125543

Figure Lengend Snippet: Figure 1. Breast cancer–derived sclerostin inhibits Wnt signaling in osteoblasts. (A and B) Calvarial cells were differentiated into osteoblasts in the presence of control medium or cancer-conditioned medium (CM) collected from MDA-MB-231 metastatic breast cancer cells. Osteoblast differen- tiation was determined by quantification of Runx2 and osteocalcin (Ocn) gene expression (A) and by alizarin red S staining (B) (n = 4 independent experiments). (C) Calvarial osteoblasts were cultured in the presence of the indicated amount of MDA-MB-231–derived CM. Wnt signaling activity was determined by TOPflash reporter assay (n = 4 independent experiments). (D) Sclerostin mRNA expression was quantified in nonmetastatic MCF-7 and metastatic MDA-MB-231 breast cancer cells by qRT-PCR (n = 6 independent experiments). (E) SOST mRNA expression was analyzed in breast cancer tissue from 48 patients. Proportion of sclerostin-positive and sclerostin-negative tissue samples is shown for all patients and for triple-negative (ER–, PR–, HER–) and in hormone receptor–negative (ER–, PR–, HER+) patients. All, n = 48; ER–, PR–, HER-, n = 9; ER–, HER–, HER+, n = 7. (F) Wnt signaling activity in calvarial osteoblasts cultured with control medium or with CM from MDA-MB-231 cells transfected with scrambled control siRNA (si-Ctrl CM) or siRNA against sclerostin (si-Sclerostin CM) (n = 6 independent experiments). (G) Wnt signaling activity in calvarial osteoblasts isolated from mice heterozygous for the Lrp5 mutation G171V (Lrp5-G171V+/T) and from control littermates (Lrp5-G171V+/+) stimulated with control medium or cancer CM (n = 4 independent experiments). Data are presented as mean ± SEM. Two-tailed Student’s t test was used to compare 2 groups (A and D), and ANOVA followed by Tukey’s post hoc analysis was used to compare 3 or more groups (C, F, and G); *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: MDA-MB-231 cells were transfected with scrambled control siRNA or siRNA against sclerostin (Origene) using Lipofectamine 3000 (Thermo Fisher Scientific) according to the manufacturer’s instructions.

Techniques: Derivative Assay, Control, Gene Expression, Staining, Cell Culture, Activity Assay, Reporter Assay, Expressing, Quantitative RT-PCR, Transfection, Isolation, Mutagenesis, Two Tailed Test

Journal: JCI insight

Article Title: Sclerostin inhibition alleviates breast cancer-induced bone metastases and muscle weakness.

doi: 10.1172/jci.insight.125543

Figure Lengend Snippet: Figure 2. Pharmacological inhibition of sclerostin reduces bone metastatic burden in mice. MDA-MB-231 breast cancer cells stably expressing the lucifer- ase gene were injected into the left ventricle of 8-week-old female immune-compromised SCID mice. Micrometastases were detected 2 weeks after breast cancer cell injection by bioluminescence imaging (BLI). Mice were randomized and received either vehicle (n = 8) or anti-sclerostin antibody (Scl-Ab; n = 8) once a week for 4 weeks. (A and B) Tumor growth in bone was visualized after 4 weeks of treatment (A) and quantified (B) by BLI. Values are represented in a log10 scale. (C and D) Quantification of the metastasis area (C) in the tibia (D) of cancer-bearing mice treated with vehicle (n = 16 tibiae) or Scl-Ab (n = 16 tibiae) using histological sections. Scale bar: 1 mm. (E and F) Quantification of human leukocyte antigen (HLA) mRNA expression in the lung (E) and brain (F) by qRT-PCR (n = 8). (G) Kaplan-Meier survival curve of cancer-bearing mice treated with vehicle (n = 6) or Scl-Ab (n = 6). Data are presented as mean ± SEM. Two groups were compared using 2-tailed Student’s t test; *P < 0.05.

Article Snippet: MDA-MB-231 cells were transfected with scrambled control siRNA or siRNA against sclerostin (Origene) using Lipofectamine 3000 (Thermo Fisher Scientific) according to the manufacturer’s instructions.

Techniques: Inhibition, Stable Transfection, Expressing, Injection, Imaging, Quantitative RT-PCR

Journal: JCI insight

Article Title: Sclerostin inhibition alleviates breast cancer-induced bone metastases and muscle weakness.

doi: 10.1172/jci.insight.125543

Figure Lengend Snippet: Figure 4. Inhibition of sclerostin prevents breast cancer–induced loss of muscle function. (A) Specific force of the extensor digitorum longus (EDL) muscle from healthy mice without treatment (n = 5), or treated with vehicle (n = 10) or anti-sclerostin antibody (Scl-Ab, n = 10); and from cancer-bearing mice treated with vehicle (n = 8) or Scl-Ab (n = 8). N, newtons. (B) Endurance of the EDL muscle of mice with bone metastases treated with vehicle (n = 8) or Scl-Ab (n = 8). max, maximum. (C) Succinate dehydroxygenase–stained (SDH-stained) tibialis anterior muscle sections from healthy mice without treatment or with vehicle or Scl-Ab treatment, as well as from mice with bone metastases treated with vehicle or Scl-Ab stained. Two representative muscles are shown per group. Scale bar: 50 μm. (D) Quantification of the cross-sectional area (CSA) of all muscle fibers, oxidative fibers, and nonoxida- tive fibers using SDH-stained muscle sections from healthy mice without treatment (n = 5), or with vehicle (n = 10) or Scl-Ab treatment (n = 10); and from cancer-bearing mice treated with vehicle (n = 8) or Scl-Ab (n = 8). Data are presented as mean ± SEM. Three or more groups were compared using ANOVA followed by Tukey’s post hoc analysis; *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: MDA-MB-231 cells were transfected with scrambled control siRNA or siRNA against sclerostin (Origene) using Lipofectamine 3000 (Thermo Fisher Scientific) according to the manufacturer’s instructions.

Techniques: Inhibition, Staining, Muscles

Journal: JCI insight

Article Title: Sclerostin inhibition alleviates breast cancer-induced bone metastases and muscle weakness.

doi: 10.1172/jci.insight.125543

Figure Lengend Snippet: Figure 5. Treatment with an anti-sclerostin antibody reverses breast cancer–induced activation of NF-κB signaling and increased number of Pax7-positive cells. (A) Immunoblot analysis of phosphorylated IKKα and IKKβ (p-IKKα and p-IKKβ), phosphorylated NF-κBp65 (p-NF-κBp65), phos- phorylated p38 (p-p38), and total p38 in the gastrocnemius (GAS) muscle of healthy nontreated mice (n = 5) and cancer-bearing mice treated with vehicle (n = 8) or Scl-Ab (n = 8). Actin was used as loading control. Representative samples are shown. (B) Immunoblot analysis of phosphorylated NF-κBp65 (p-NF-κBp65), phosphorylated p38 (p-p38) and total p38 in C2C12 myoblasts stimulated with vehicle (veh) or TGF-β1. Actin was used as loading control. Representative image of 6 independent experiments is shown. (C) Immunoblot analysis of phosphorylated NF-κBp65, p-p38, and total p38 in C2C12 cells treated with a control peptide or an NF-κB blocking peptide (NPD) and stimulated with vehicle or TGF-β1. Actin was used as loading control. Representative image of 4 independent experiments is shown. (D) Myogenin and MyoD mRNA expression was quantified by qRT-PCR in C2C12 cells after 10 days of myogenic differentiation (n = 4). (E) Pai1 mRNA expression was quantified in the GAS muscle from healthy nontreat- ed mice (n = 5) and from cancer-bearing mice treated with vehicle (n = 8) or Scl-Ab (n = 8). (F) Immunohistochemical staining of Pax7 in the tibialis anterior (TA) muscle from healthy nontreated mice and from mice with bone metastases treated with vehicle or Scl-Ab. Scale bar: 50 μm (top row) and 100 μm (bottom row). (G) Quantification of Pax7-positive cells in the TA muscle from healthy nontreated mice (n = 5) and from mice with bone metastases treated with vehicle (n = 8) or Scl-Ab (n = 8). Data are presented as mean ± SEM. Two-tailed Student’s t test was used to compare 2 groups (D), and ANOVA followed by Tukey’s post hoc analysis was used to compare 3 or more groups (E and G); *P < 0.05, ***P < 0.001.

Article Snippet: MDA-MB-231 cells were transfected with scrambled control siRNA or siRNA against sclerostin (Origene) using Lipofectamine 3000 (Thermo Fisher Scientific) according to the manufacturer’s instructions.

Techniques: Activation Assay, Western Blot, Control, Blocking Assay, Expressing, Quantitative RT-PCR, Immunohistochemical staining, Staining, Two Tailed Test