Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Curcumin regulates cell fate and metabolism by inhibiting hedgehog signaling in hepatic stellate cells.

doi: 10.1038/labinvest.2015.59

Figure Lengend Snippet: Figure 1 Curcumin inhibits Hh signaling in rat fibrotic liver. Rats were grouped as follows: group 1, vehicle control (no CCl4, no treatment); group 2, model group (with CCl4, no treatment); group 3, colchicine-treated group (0.1 mg/kg+CCl4); group 4, curcumin-treated group (100 mg/kg+CCl4); group 5, curcumin-treated group (200 mg/kg+CCl4); and group 6, curcumin-treated group (300 mg/kg+CCl4). Liver sections were stained with immunofluo- rescence using antibodies against Patched, Smo, and Hhip. Antibody against α-SMA was used to specifically stain HSCs, and DAPI to stain the nucleus.

Article Snippet: The following primary antibodies were used in this study: Patched, Gli1, Cyclin D1, CDK4, Cyclin E1, CDK2, Bax, Pro-caspase-9, Cleaved-caspase-9, Pro-caspase-3, Cleaved-caspase-3, Pro-caspase-8, Cleaved-caspase-8, Full-length PARP-1, CleavedPARP-1, PPARγ, and β-Actin (Cell Signaling Technology, Danvers, MA, USA); Smo, Hhip, Bcl-2 and CTGF (Santa Cruz Technology, Santa Cruz, CA, USA); α-SMA, α1(I)Procollagen, and Fibronectin (Epitomics, San Francisco, CA, USA); p-PKA, PKA, Lamin A/C, C/EBPα, HK, PFK2, Glut 4, and MCT4 (Bioworld Technology, St Louis Park, MN, USA).

Techniques: Control, Staining

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Curcumin regulates cell fate and metabolism by inhibiting hedgehog signaling in hepatic stellate cells.

doi: 10.1038/labinvest.2015.59

Figure Lengend Snippet: Figure 2 Curcumin blocks Hh signaling in HSCs. HSCs were treated with DMSO (0.02%, w/v) and curcumin at indicated concentrations for 24 h. (a) Shh levels in supernatant were examined by ELISA. Significance: *Po0.05 vs DMSO. (b) Western blot analyses of protein expression of Patched, Smo, and Hhip. (c) Western blot analyses of phosphorylation of PKA. (d) Immunofluorescence using antibody against Gli. Hoechst reagent was used to stain the nucleus. (e) Western blot analyses of protein abundance of Gli in the cytoplasm and nucleus, respectively. (f) EMSA for examining the binding capacity of Gli1 to DNA sequences. Lane 1 indicates samples treated with probe alone. Lane 2 was samples treated with DMSO without curcumin. (g) Real-time PCR analyses of Hh signaling target genes cyclin D1, cyclin D2, and Bcl-2. Significance: *Po0.05 vs DMSO.

Article Snippet: The following primary antibodies were used in this study: Patched, Gli1, Cyclin D1, CDK4, Cyclin E1, CDK2, Bax, Pro-caspase-9, Cleaved-caspase-9, Pro-caspase-3, Cleaved-caspase-3, Pro-caspase-8, Cleaved-caspase-8, Full-length PARP-1, CleavedPARP-1, PPARγ, and β-Actin (Cell Signaling Technology, Danvers, MA, USA); Smo, Hhip, Bcl-2 and CTGF (Santa Cruz Technology, Santa Cruz, CA, USA); α-SMA, α1(I)Procollagen, and Fibronectin (Epitomics, San Francisco, CA, USA); p-PKA, PKA, Lamin A/C, C/EBPα, HK, PFK2, Glut 4, and MCT4 (Bioworld Technology, St Louis Park, MN, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Phospho-proteomics, Immunofluorescence, Staining, Quantitative Proteomics, Binding Assay, Real-time Polymerase Chain Reaction

Journal: Aging (Albany NY)

Article Title: lncRNA HHIP-AS1/HHIP modulates osteogenic differentiation of BM-MSCs by regulating Hedgehog signaling pathway

doi: 10.18632/aging.204381

Figure Lengend Snippet: lncRNA HHIP-AS1 and HHIP are continuously down-regulated during osteogenic differentiation of BM-MSCs. Up-regulated or down-regulated ( A ) lncRNAs and ( B ) mRNAs were analyzed by Venn diagram during osteogenic differentiation at days 0, 7 and 14 of osteogenic differentiation of BM-MSCs. Heatmap of ( C ) lncRNA and ( D ) mRNA changes at days 0, 7 and 14 of osteogenic differentiation of BM-MSCs. ( E ) FISH showing that lncRNA HHIP-AS1 was expressed in the nucleus and cytoplasm of BM-MSCs at day 7 of osteogenic differentiation of BM-MSCs. mRNA expression level of ( F ) lncRNA HHIP-AS1 and ( G ) HHIP during osteogenic differentiation. ( H ) Protein expression level of HHIP, RUNX2, and osteocalcin during osteogenic differentiation of BM-MSCs. The histogram data for each group are the average of three independent replicates; bars represent standard deviation; * P < 0.05, ** P < 0.01, *** P < 0.001; Scale bar = 10 μm.

Article Snippet: When the cells were seeded at 80% confluence, the third-passage BM-MSCs were transfected. siRNAs targeting HHIP-AS1 (si-HHIP-AS1), HHIP (si-HHIP), ELAVL1 (si-ELAVL1) or a control non-targeting siRNA (NC) (Santa Cruz, Tokyo, Japan) were transfected into BM-MSCs using Lipofectamine RNAi MAX reagent (Thermo Fisher Scientific, Waltham, MA, USA) as per the manufacturer’s procedure.

Techniques: Expressing, Standard Deviation

Journal: Aging (Albany NY)

Article Title: lncRNA HHIP-AS1/HHIP modulates osteogenic differentiation of BM-MSCs by regulating Hedgehog signaling pathway

doi: 10.18632/aging.204381

Figure Lengend Snippet: lncRNA HHIP-AS1 up-regulates HHIP expression and inhibits osteogenic differentiation of BM-MSCs. ( A ) The complementary sequence of the first exon of HHIP-AS1 and the first exon of HHIP. ( B ) lncRNA HHIP-AS1 in si-HHIP-AS1 or HHIP-AS1 transfected BM-MSCs was detected by PCR. ( C ) Fluorescence images of lncRNA HHIP-AS1 (Red), HHIP protein (Green) and DAPI (Blue) in BM-MSCs transfected with NC, si-HHIP-AS1 and HHIP-AS1. ( D ) HHIP mRNA in si-HHIP-AS1 or HHIP-AS1 transfected BM-MSCs detected by PCR. ( E ) HHIP protein in cell or in cell fractions of cytoplasm and cell membrane was detected by western blot after overexpression or knockdown of lncRNA HHIP-AS1. ( F ) RUNX2 and OCN as well as ( G ) ALP and OPN protein levels in cells were detected by western blot after overexpression or knockdown of lncRNA HHIP-AS1. ( H ) ALP and ( I ) alizarin red staining after 14 days of osteogenic induction of BM-MSCs. The histogram data for each group are the average of three independent replicates; bars represent standard deviation; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Scale bar = 10 μm. Note: Protein levels in lncRNA HHIP-AS1 knockdown and overexpression groups on day 7 were compared to those in NC groups on day7. Similarly, protein levels in lncRNA HHIP-AS1 knockdown and overexpression groups on day 14 were compared to those in NC groups on day14.

Article Snippet: When the cells were seeded at 80% confluence, the third-passage BM-MSCs were transfected. siRNAs targeting HHIP-AS1 (si-HHIP-AS1), HHIP (si-HHIP), ELAVL1 (si-ELAVL1) or a control non-targeting siRNA (NC) (Santa Cruz, Tokyo, Japan) were transfected into BM-MSCs using Lipofectamine RNAi MAX reagent (Thermo Fisher Scientific, Waltham, MA, USA) as per the manufacturer’s procedure.

Techniques: Expressing, Sequencing, Transfection, Fluorescence, Membrane, Western Blot, Over Expression, Knockdown, Staining, Standard Deviation

Journal: Aging (Albany NY)

Article Title: lncRNA HHIP-AS1/HHIP modulates osteogenic differentiation of BM-MSCs by regulating Hedgehog signaling pathway

doi: 10.18632/aging.204381

Figure Lengend Snippet: HHIP protein inhibits osteogenic differentiation of BM-MSCs. ( A ) We constructed HHIP-silenced (si-HHIP) and HHIP-overexpressed (HHIP) BM-MSCs. RUNX2, OCN, ALP and OPN protein levels were detected after 14 days of osteogenic induction of the BM-MSCs. ( B ) ALP and ( C ) alizarin red staining after 14 days of osteogenic induction of BM-MSCs. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: When the cells were seeded at 80% confluence, the third-passage BM-MSCs were transfected. siRNAs targeting HHIP-AS1 (si-HHIP-AS1), HHIP (si-HHIP), ELAVL1 (si-ELAVL1) or a control non-targeting siRNA (NC) (Santa Cruz, Tokyo, Japan) were transfected into BM-MSCs using Lipofectamine RNAi MAX reagent (Thermo Fisher Scientific, Waltham, MA, USA) as per the manufacturer’s procedure.

Techniques: Construct, Staining

Journal: Aging (Albany NY)

Article Title: lncRNA HHIP-AS1/HHIP modulates osteogenic differentiation of BM-MSCs by regulating Hedgehog signaling pathway

doi: 10.18632/aging.204381

Figure Lengend Snippet: lncRNA HHIP-AS1 increases HHIP mRNA stability by ELAVL1. ( A ) BM-MSCs transfected with overlapping sequence or non-overlapping sequence of lncRNA HHIP-AS1 and HHIP mRNA were analyzed by PCR. ( B ) qPCR validation of HHIP enrichment versus input after RNA pull down by two different specific probes (Bio-HHIP-AS1 OL and Bio-HHIP-AS1 non-OL) as compared to a non-specific one (Bio-NC) in BM-MSCs. ( C ) Measurement of the stability of HHIP mRNA by RT-qPCR in the presence of transcriptional inhibitor (actinomycin D) at the indicated time as compared to an internal control 18S rRNA. ( D, E ) Using the web ( http://service.tartaglialab.com/page/catrapid_group ), we found that ELAVL1 has high affinity to the lncRNA HHIP-AS1, especially with the region that is complementary with HHIP mRNA. ( F ) RIP assay was performed in lncRNA HHIP-AS1-overexpressed and silenced BM-MSCs and the control cells. The qPCR assay was further performed to determine the enrichments of HHIP-AS1 and HHIP mRNA in ELAVL1 protein. IgG is as a negative control. ( G ) BM-MSCs were transfected with lncRNA HHIP-AS1 overexpression vector alone or in combination with ELAVL1 siRNA. Measurement of the stability of HHIP mRNA by RT-qPCR in the presence of transcriptional inhibitor (actinomycin D) at the indicated time as compared to an internal control 18S rRNA. The histogram data for each group are the average of three independent replicates; bars represent standard deviation; * P < 0.05, ** P < 0.01 and *** P < 0.001 vs. NC or BM-MSCs without transfection; # P < 0.01 vs. BM-MSCs transfected with lncRNA HHIP-AS1 overexpression vector.

Article Snippet: When the cells were seeded at 80% confluence, the third-passage BM-MSCs were transfected. siRNAs targeting HHIP-AS1 (si-HHIP-AS1), HHIP (si-HHIP), ELAVL1 (si-ELAVL1) or a control non-targeting siRNA (NC) (Santa Cruz, Tokyo, Japan) were transfected into BM-MSCs using Lipofectamine RNAi MAX reagent (Thermo Fisher Scientific, Waltham, MA, USA) as per the manufacturer’s procedure.

Techniques: Transfection, Sequencing, Biomarker Discovery, Quantitative RT-PCR, Control, Negative Control, Over Expression, Plasmid Preparation, Standard Deviation

Journal: Aging (Albany NY)

Article Title: lncRNA HHIP-AS1/HHIP modulates osteogenic differentiation of BM-MSCs by regulating Hedgehog signaling pathway

doi: 10.18632/aging.204381

Figure Lengend Snippet: HHIP-AS1 OL inhibits osteogenic differentiation of BM-MSCs by HHIP. ( A ) HHIP, RUNX2, OCN, ALP and OPN protein expression level after overexpression lncRNA HHIP-AS1 OL or non OL by western blot. ( B ) Immunofluorescence images of RUNX2 (red), osteocalcin (Green), and DAPI (Blue) in BM-MSCs transfected with NC, HHIP-AS1 OL, or HHIP-AS1 non-OL. ( C ) ALP and ( D ) alizarin red staining after 14 days of osteogenic induction of BM-MSCs. The histogram data for each group are the average of three independent replicates; bars represent standard deviation; * P < 0.05, *** P < 0.001; Scale bar = 10 μm.

Article Snippet: When the cells were seeded at 80% confluence, the third-passage BM-MSCs were transfected. siRNAs targeting HHIP-AS1 (si-HHIP-AS1), HHIP (si-HHIP), ELAVL1 (si-ELAVL1) or a control non-targeting siRNA (NC) (Santa Cruz, Tokyo, Japan) were transfected into BM-MSCs using Lipofectamine RNAi MAX reagent (Thermo Fisher Scientific, Waltham, MA, USA) as per the manufacturer’s procedure.

Techniques: Expressing, Over Expression, Western Blot, Immunofluorescence, Transfection, Staining, Standard Deviation

Journal: Aging (Albany NY)

Article Title: lncRNA HHIP-AS1/HHIP modulates osteogenic differentiation of BM-MSCs by regulating Hedgehog signaling pathway

doi: 10.18632/aging.204381

Figure Lengend Snippet: lncRNA HHIP-AS1 inhibits osteogenic differentiation of BM-MSCs by inhibiting Hedgehog signaling pathway. ( A ) Functional annotation of BM-MSCs lncRNAs at days 0, 7, and 14 of osteogenic differentiation by GO enrichment analysis based on the data obtained from Microarray analysis. ( B , C ) Volcano plot of the up-regulated lncRNAs (Red) and down-regulated lncRNAs (Green). ( D ) Protein expression of HHIP, Gli1, Gli2, RUNX2, and osteocalcin in BM-MSCs transfected with HHIP-AS1 or si-HHIP and treated with PF-5274857. ( E ) ALP and ( F ) alizarin red staining after 14 days of osteogenic induction of BM-MSCs. The histogram data for each group are the average of three independent replicates; bars represent standard deviation; *** P < 0.001.

Article Snippet: When the cells were seeded at 80% confluence, the third-passage BM-MSCs were transfected. siRNAs targeting HHIP-AS1 (si-HHIP-AS1), HHIP (si-HHIP), ELAVL1 (si-ELAVL1) or a control non-targeting siRNA (NC) (Santa Cruz, Tokyo, Japan) were transfected into BM-MSCs using Lipofectamine RNAi MAX reagent (Thermo Fisher Scientific, Waltham, MA, USA) as per the manufacturer’s procedure.

Techniques: Functional Assay, Microarray, Expressing, Transfection, Staining, Standard Deviation

Journal: Aging (Albany NY)

Article Title: lncRNA HHIP-AS1/HHIP modulates osteogenic differentiation of BM-MSCs by regulating Hedgehog signaling pathway

doi: 10.18632/aging.204381

Figure Lengend Snippet: The schematic diagram of lncRNA HHIP-AS1/HHIP modulating osteogenic differentiation of BM-MSCs. lncRNA HHIP-AS1 bound to HHIP mRNA through complementary base pairing. This interaction increased ELAVL1 binding to HHIP mRNA and thus improved the mRNA stability and expression. HHIP protein can binds to SHH, which suppresses the activation of SMO by SHH. As the result, osteogenic differentiation of BM-MSCs induced by Hedgehog signal is suppressed.

Article Snippet: When the cells were seeded at 80% confluence, the third-passage BM-MSCs were transfected. siRNAs targeting HHIP-AS1 (si-HHIP-AS1), HHIP (si-HHIP), ELAVL1 (si-ELAVL1) or a control non-targeting siRNA (NC) (Santa Cruz, Tokyo, Japan) were transfected into BM-MSCs using Lipofectamine RNAi MAX reagent (Thermo Fisher Scientific, Waltham, MA, USA) as per the manufacturer’s procedure.

Techniques: Binding Assay, Expressing, Activation Assay

Journal: Molecular Therapy. Nucleic Acids

Article Title: HIV-1 Tat and cocaine impact astrocytic energy reservoirs and epigenetic regulation by influencing the LINC01133-hsa-miR-4726-5p-NDUFA9 axis

doi: 10.1016/j.omtn.2022.07.001

Figure Lengend Snippet: Effects of HIV-1 Tat, cocaine, and TC on miRNA and lncRNA expression Human primary astrocytes were exposed to cocaine (1 μM) and HIV-1 Tat (50 ng/mL) either alone or in combination for 24 h. Controls were maintained in drug-free medium (without drug exposure). (A) LINC01133, (B) H19, (C) NOPA-14, and (D) HHIP lncRNA expression levels in astrocytes were determined by quantitative real-time PCR analysis using the housekeeping gene β-actin as a loading control. Two-way ANOVA was performed to compare the groups. The data are expressed as the mean ± SEM of the TAI from three independent experiments. N = 3. Moreover, (E) hsa-miR-2355-5p and (F) hsa-miR-4726-5p miRNA expression levels in astrocytes were determined by quantitative real-time PCR analysis using the housekeeping gene U6 snRNA as a loading control. The data are expressed as the mean ± SEM of the TAI from three independent experiments. n = 3. ∗∗∗∗p < 0.0001, ∗∗∗p < 0.001, ∗∗p < 0.01, ∗p < 0.05.

Article Snippet: Total RNA was screened for purity according to a 260/280 ratio of ∼2.0. cDNA was synthesized and amplified using specific primers for NDUFA9 (assay ID Hs00245308_m1, catalog no. 4448892), LIPG (assay ID Hs00195812_m1, catalog no. 4331182), NOP14-AS1 (assay ID Hs03653612_m1, catalog no. 4331182), HHIP-AS1 (assay ID Hs04232235_m1, catalog no. 4426961), LINC01133 (assay ID Hs04274447_m1, catalog no. 4331182), H19 (assay ID Hs00399294_g1, catalog no. 4453320), and β-actin (Hs99999903_m1) (Applied Biosystems, Foster City, CA, USA).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Control