Journal: Molecular cancer research : MCR

Article Title: The Microtubule Network and Cell Death are Regulated by a miR-34a/Stathmin 1/βIII-tubulin Axis

doi: 10.1158/1541-7786.MCR-16-0372

Figure Lengend Snippet: (A) Top – Schematic of the alignment between the miR-34a seed sequence and the target recognition sequence of STMN1 mRNA 3′UTR, predicted by microRNA.org. Bottom - Expression of miR-34a and STMN1 mRNA in OS cell lines (n=5) and xenografts (n=9) was measured by RT-qPCR and expressed relative to normal osteoblasts. Values are mean of three measurements. (B) The correlation between miR-34a expression in OS cells and xenografts and STMN1 mRNA was analyzed by Pearson’s correlation coefficient. (C) SaOS and 143B cells were transfected with non-targeting miR-C (control) oligonucleotides or miR-34a precursors and STMN1 mRNA was measured by RT-qPCR. Results are represented as fold-change relative to miR-C. Graph depicts mean ± SE of one representation of three independent experiments. * denotes p<0.05 (D) STMN1 protein expression in untreated, miR-C and miR-34a transfected SaOS, 143B, HOS and U2OS cells was measured by immunoblot. GAPDH was loading control. Representative results of three analyses are shown.

Article Snippet: Cell culture Human OS cell lines SaOS (p53-null), 143B, HOS, U2OS (wild-type p53), MG-63 (mutant p53) and human osteoblasts (CRL-1132) were purchased from ATCC (Manassas, VA).

Techniques: Sequencing, Expressing, Quantitative RT-PCR, Transfection, Control, Western Blot

Journal: Molecular cancer research : MCR

Article Title: The Microtubule Network and Cell Death are Regulated by a miR-34a/Stathmin 1/βIII-tubulin Axis

doi: 10.1158/1541-7786.MCR-16-0372

Figure Lengend Snippet: (A) SaOS, 143B, HOS and U2OS were transfected with either miR-34a or with siRNA targeting Sp1 (bottom panels) and STMN1 and βIII-tubulin protein expression were measured by immunoblot. GAPDH was loading control. Representative results of three analyses are shown. (B) 143B cells were transfected with miR-34a or an STMN1 expression plasmid. Immunoblot for STMN1 protein (left) is indicated. Cell cycle distribution analysis for untreated, miR-34a-expressing or STMN1 overexpressing 143B cells was assessed by PI staining and flow cytometry analysis. Arrow indicates apoptotic cells. (C) Immunoblot analysis of cell cycle markers (cyclin D1 and p27) and apoptotic marker (cleaved PARP) in miR-34a-expressing and STMN1 overexpressing OS cells. GAPDH was loading control. Representative results of three immunoblot analyses are shown. (D) Confocal microscopy images for 143B cells transfected with miR-34a for 48 hours and immunostained for LC3 I/II (red) and βIII-tubulin (green). Cells were counterstained with Hoechst (blue) to visualize nuclei. Scale bars are 25 μm.

Article Snippet: Cell culture Human OS cell lines SaOS (p53-null), 143B, HOS, U2OS (wild-type p53), MG-63 (mutant p53) and human osteoblasts (CRL-1132) were purchased from ATCC (Manassas, VA).

Techniques: Transfection, Expressing, Western Blot, Control, Plasmid Preparation, Staining, Flow Cytometry, Marker, Confocal Microscopy

Journal: The Journal of Biological Chemistry

Article Title: Progesterone receptor membrane component 1 facilitates Ca 2+ signal amplification between endosomes and the endoplasmic reticulum

doi: 10.1016/j.jbc.2023.105378

Figure Lengend Snippet: Expression of PGRMC1 potentiates NAADP-evoked Ca 2+ release via TPC1. A , fluorescence traces from single U2OS cells microinjected with buffer (“mock injection,” blue ) or NAADP (100 nM pipette concentration) in the absence ( black ) or presence ( red ) of RFP-PGRMC1. B – D , similar microinjection experiments in single U2OS cells expressing ( B ) TPC1, ( C ) TPC2 or ( D ) TPC2[1-31]-TPC1[32-816] in the absence ( black ) or presence ( red ) of RFP-PGRMC1. Traces report the fluorescence of GCaMP6M over time-averaged (mean ± sd) from n ≥ 3 injected cells, with only every third data point shown for clarity. E , area under the curve from NAADP-evoked Ca 2+ mobilization responses, values represent mean area ± sd from independent fluorescence traces recorded from n ≥ 3 injected cells. Statistical significance was determined using paired two-tailed Student’s t test, p -values, ∗ p < 0.05, ∗∗∗ p < 0.001.

Article Snippet: Wild-type parental U2OS cells (RRID:CVCL_0042) were sourced from ATCC.

Techniques: Expressing, Fluorescence, Injection, Transferring, Concentration Assay, Microinjection, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Progesterone receptor membrane component 1 facilitates Ca 2+ signal amplification between endosomes and the endoplasmic reticulum

doi: 10.1016/j.jbc.2023.105378

Figure Lengend Snippet: Molecular disruption of the PGRMC1-TPC1 interaction impairs PGRMC1 potentiation of NAADP-evoked Ca 2+ release. A – C , heme binding is necessary for interaction with TPC1. A , Myc-tagged WT PGRMC1 and PGRMC1[Y113F] constructs were co-expressed in U2OS cells with GFP-tagged TPC1, with detection by Western blot. B , immunodetection of myc-tagged PGRMC1 after TPC1-GFP immunoprecipitation. C , densitometry quantification of immunodetection of myc-tagged PGRMC1 in TPC1-GFP immunoprecipitates. D – F , interaction of PGRMC1 with TPC1 is mediated by the COOH-terminal coiled-coil domain of PGRMC1. D , PGRMC1 mutants harboring point-mutations in predicted carboxyl-terminus helices co-expressed with TPC1-GFP in U2OS cells, detected by Western blot. E , immunodetection of myc-tagged PGRMC1 mutants after TPC1-GFP immunoprecipitation. F , densitometry quantification of immunodetected myc-tagged PGRMC1 mutants in TPC1-GFP immunoprecipitates, values represent mean ± sd from n = 2 independent experiments. G , traces of Ca 2+ flux in U2OS cells expressing the indicated PGRMC1 constructs microinjected with NAADP (100 nM pipette concentration) as detected by GCaMP6M fluorescence changes. Individual single-cell traces from n ≥ 3 injections are shown, with averaged responses bolded. H , peak F/F 0 values (mean ± sd) from cumulative dataset shown in ( G ). I , area under the curve (mean ± sd) of traces shown in ( G ). J , averaged traces of Ca 2+ flux in U2OS cells expressing TPC1-RFP and the indicated PGRMC1 constructs microinjected with NAADP (100 nM pipette concentration) as detected by GCaMP6M fluorescence changes. Individual single-cell traces from n ≥ 3 injections are shown, with averaged responses bolded. K , peak F/F 0 values (mean ± sd) from cumulative dataset shown in ( J ). L , area under the curve (mean ± sd) of traces shown in ( J ). Statistical significance was determined using paired two-tailed Student’s t test, p -values, ∗ p < 0.05, ∗∗∗ p < 0.001.

Article Snippet: Wild-type parental U2OS cells (RRID:CVCL_0042) were sourced from ATCC.

Techniques: Disruption, Binding Assay, Construct, Western Blot, Immunodetection, Immunoprecipitation, Expressing, Transferring, Concentration Assay, Fluorescence, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Progesterone receptor membrane component 1 facilitates Ca 2+ signal amplification between endosomes and the endoplasmic reticulum

doi: 10.1016/j.jbc.2023.105378

Figure Lengend Snippet: Pharmacological disruption of the PGRMC1-TPC1 interaction blocks PGRMC1 potentiation of NAADP-evoked Ca 2+ release. A and D , immunodetection of PGRMC-myc and TPC1-GFP in lysates from U2OS cells pretreated for 1 h with the indicated concentration of CORM3 ( A ) or AG-205 ( D ). B and E , immunodetection of PGRMC1-myc after immunoprecipitation of TPC1-GFP from lysates shown in ( A and D ). C and F , densitometric quantification of myc-tagged PGRMC1 from TPC1-GFP immunoprecipitates, values represent mean ± sd from n = 2 experiments. G – I , averaged fluorescence traces (mean ± sd from n ≥ 3 injections) showing NAADP responses in U2OS cells transfected with empty vector ( black ) or PGRMC1 ( red , pretreated with ( G ) DMSO (0.1%), ( H ) CORM3 (30 μM), or ( I ) AG-205 (30 μM) for 1 h, detected by following GCaMP6M fluorescence. J , peak F/F 0 values (mean ± sd) of independent NAADP responses summarized in ( G – I ). K , area under the curves (mean ± sd) of independent traces of NAADP-responses summarized in ( G – I ). Statistical significance was determined using paired two-tailed Student’s t test, p -values, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: Wild-type parental U2OS cells (RRID:CVCL_0042) were sourced from ATCC.

Techniques: Disruption, Immunodetection, Concentration Assay, Immunoprecipitation, Fluorescence, Transfection, Plasmid Preparation, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Progesterone receptor membrane component 1 facilitates Ca 2+ signal amplification between endosomes and the endoplasmic reticulum

doi: 10.1016/j.jbc.2023.105378

Figure Lengend Snippet: PGRMC1 localizes to the endoplasmic reticulum in U2OS cells. A , U2OS cells imaged using various live cell markers ( left ) and co-transfected with PGRMC1-RFP ( center ). The fluorescence overlay of PGRMC1-RFP and the organelle marker channel is shown on the right . Markers were TPC1-GFP, TPC2-GFP, an endoplasmic reticulum marker (CYP2C9[1-27]-GCaMP-6M), a plasma membrane marker (GAP43[1-20]-GFP) and a mitochondrial marker (COX8A[1-29]-CFP), Scale bar, 10 μm. Right , dashed line represents a line scan of fluorescence intensity from the green , blue or red fluorescence channels along the indicated axis. Gamma values were uniformly adjusted for clarity.

Article Snippet: Wild-type parental U2OS cells (RRID:CVCL_0042) were sourced from ATCC.

Techniques: Transfection, Fluorescence, Marker, Clinical Proteomics, Membrane

Journal: The Journal of Biological Chemistry

Article Title: Progesterone receptor membrane component 1 facilitates Ca 2+ signal amplification between endosomes and the endoplasmic reticulum

doi: 10.1016/j.jbc.2023.105378

Figure Lengend Snippet: Expression of PGRMC1 increases NAADP-evoked Ca 2+ release from the endoplasmic reticulum. A and B , averaged fluorescence traces as detected by GCaMP6M fluorescence changes reporting NAADP-evoked Ca 2+ release in U2OS cells transfected with empty vector or in cells expressing PGRMC1. Microinjections were performed with NAADP (100 nM pipette concentration) in the presence of ( A ) thapsigargin (1 μM) or ( B ) bafilomycin A1 (100 nM). C , quantification of peak ΔF/F 0 values from ( A and B ). D , quantification of the area under the curves from ( A and B ). E , left , Images of U2OS cell co-expressing GCaMP6M ( top ) and RCEPIA1er ( bottom ). Middle , pseudocolored images of GCaMP6M and RCEPIA1er fluorescence intensity before (middle image) and after ( right image) microinjection of NAADP. Scale bar,10 μm. F , representative traces of GCaMP6M fluorescence ( top ) and RCEPIA1er fluorescence ( bottom ) in response to microinjection of NAADP (100 nM pipette concentration) in U2OS cells transfected with empty vector ( grey ), or from cells expressing PGRMC1 ( red ). G , quantification of ΔF/F 0 values from the cumulative dataset for GCaMP6M ( top ) and RCEPIA1er fluorescence ( bottom ). H , quantification of area under the curves from the cumulative dataset for GCaMP6M ( top ) and RCEPIA1er fluorescence ( bottom ). Statistical significance was determined using paired two-tailed Student’s t test, p -values, ∗ p < 0.05, ∗∗ p < 0.01.

Article Snippet: Wild-type parental U2OS cells (RRID:CVCL_0042) were sourced from ATCC.

Techniques: Expressing, Fluorescence, Transfection, Plasmid Preparation, Transferring, Concentration Assay, Microinjection, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Progesterone receptor membrane component 1 facilitates Ca 2+ signal amplification between endosomes and the endoplasmic reticulum

doi: 10.1016/j.jbc.2023.105378

Figure Lengend Snippet: Expression of PGRMC1 inhibits EGF-evoked Ca 2+ release. A , U2OS cells were transfected with vector encoding PGRMC1-RFP ( top right ) and loaded with fluo-4 dye ( top left ). Pseudocolored images of fluo-4 fluorescence intensity after addition of buffer ( bottom , left ) or EGF (32 PM, bottom , right ). Kinetics of Ca 2+ signals (fluo-4 fluorescence) from U2OS cells expressing PGRMC1-RFP ( red boxes ) and untransfected cells ( white boxes ) were compared. Scale bar, 20 μm. B , single-cell fluorescence traces from EGF responses in U2OS cells in the absence ( black , top ) or presence of PGRMC1-RFP ( red , bottom ). C and D , quantification of ( C ) peak F/F 0 or ( D ) area under the curve (AUC) in response to EGF (32 PM) or acetylcholine (ACh, 100 μM) in control U2OS cells ( black ) or cells overexpressing PGRMC1 ( red ). Data are shown as average±sem. Statistical significance was determined using paired two-tailed Student’s t test, p -values, ∗ p < 0.05; ns, not significant.

Article Snippet: Wild-type parental U2OS cells (RRID:CVCL_0042) were sourced from ATCC.

Techniques: Expressing, Transfection, Plasmid Preparation, Fluorescence, Control, Two Tailed Test

Journal: Current Research in Structural Biology

Article Title: Comparative computational and experimental analyses of some natural small molecules to restore transcriptional activation function of p53 in cancer cells harbouring wild type and p53 Ser46 mutant

doi: 10.1016/j.crstbi.2022.09.002

Figure Lengend Snippet: Summary of the structures downloaded from PDB and PubChem along with their identification number.

Article Snippet: Human osteosarcoma (U2OS; wild type p53) and oral squamous carcinoma (HSC3; p53 Ser46mutant ) cells were obtained from the Japanese Collection of Research Bioresources (JCRB, Tokyo, Japan).

Techniques:

Journal: Current Research in Structural Biology

Article Title: Comparative computational and experimental analyses of some natural small molecules to restore transcriptional activation function of p53 in cancer cells harbouring wild type and p53 Ser46 mutant

doi: 10.1016/j.crstbi.2022.09.002

Figure Lengend Snippet: Docking scores of the ligands which bound with mutated p53/p62 complexes.

Article Snippet: Human osteosarcoma (U2OS; wild type p53) and oral squamous carcinoma (HSC3; p53 Ser46mutant ) cells were obtained from the Japanese Collection of Research Bioresources (JCRB, Tokyo, Japan).

Techniques:

Journal: Current Research in Structural Biology

Article Title: Comparative computational and experimental analyses of some natural small molecules to restore transcriptional activation function of p53 in cancer cells harbouring wild type and p53 Ser46 mutant

doi: 10.1016/j.crstbi.2022.09.002

Figure Lengend Snippet: Molecular dynamic simulation study of natural metabolites on phosphor mutant p53-p62 complex. A) Conformational changes observed in the structure of mutant type p53 compared with wild-type p53. B) Conformational changes observed in the structure of mutant type p53 after Cuc-B interaction. C) Conformational changes observed in the structure of mutant type p53 after Wi-N interaction. D) Conformational changes observed in the structure of mutant type p53 after Wi-A interaction. E) Root mean square deviation plot of wild-type as well as mutant p53 complex with p62 before and after Cuc-B interaction simulated for 200ns. F) Root mean square deviation plot of mutant p53 complex with p62 before and after Wi-N, Withaferin-A, CAPE and ARC interaction simulated for 200ns. G) Interaction fraction diagram of wild type p53 with amino acids of p62. H) Interaction fraction diagram of phospho-mutant p53 with amino acids of p62.

Article Snippet: Human osteosarcoma (U2OS; wild type p53) and oral squamous carcinoma (HSC3; p53 Ser46mutant ) cells were obtained from the Japanese Collection of Research Bioresources (JCRB, Tokyo, Japan).

Techniques: Mutagenesis

Journal: Current Research in Structural Biology

Article Title: Comparative computational and experimental analyses of some natural small molecules to restore transcriptional activation function of p53 in cancer cells harbouring wild type and p53 Ser46 mutant

doi: 10.1016/j.crstbi.2022.09.002

Figure Lengend Snippet: Molecular dynamics simulation analysis of natural metabolites in restoring wild type p53 activity. A) Interaction fraction diagram of phospho-mutant p53 with amino acids of p62 after Cuc-B intervention. B) Interaction fraction diagram of phospho-mutant p53 with amino acids of p62 after Wi-N intervention. C) Interaction fraction diagram of phospho-mutant p53 with amino acids of p62 after Wi-A intervention. D) Change in MMMGBSA binding energy before and after Cuc-B interaction. E) Change in MMMGBSA binding energy before and after the Wi-N interaction. F) Change in MMMGBSA binding energy before and after the Wi-A interaction. G) Change in Vander Wal, electrostatic and hydrogen bonding energy of interacting molecules. H) Hydrogen bonding plot observed between p53 and p62 before and after Cuc-B intervention.

Article Snippet: Human osteosarcoma (U2OS; wild type p53) and oral squamous carcinoma (HSC3; p53 Ser46mutant ) cells were obtained from the Japanese Collection of Research Bioresources (JCRB, Tokyo, Japan).

Techniques: Activity Assay, Mutagenesis, Binding Assay

Journal: Current Research in Structural Biology

Article Title: Comparative computational and experimental analyses of some natural small molecules to restore transcriptional activation function of p53 in cancer cells harbouring wild type and p53 Ser46 mutant

doi: 10.1016/j.crstbi.2022.09.002

Figure Lengend Snippet: Molecular dynamics simulation study of Mortalin p53 abrogation by natural metabolites. A) Three-dimensional visualization of interaction between Mortalin and p53 (docked using HADDOCK server and simulated for 200ns). B) Three-dimensional visualization of Interaction between Cuc-B and p53 binding domain of Mortalin. C) Three-dimensional visualization of Interaction between Wi-N and p53 binding domain of Mortalin. D) Three-dimensional visualization of interaction between Wi-A and p53 binding domain of Mortalin. E) Three-dimensional visualization of interaction between CAPE and p53 binding domain of Mortalin. F) Three-dimensional visualization of Interaction between Cuc-B and Mortalin binding domain of p53. G) Root mean square deviation plot of natural ligands bound Mortalin complex showing stable interactions. H) Root mean square deviation plot of Cuc-B bound p53 complex showing stable interaction.

Article Snippet: Human osteosarcoma (U2OS; wild type p53) and oral squamous carcinoma (HSC3; p53 Ser46mutant ) cells were obtained from the Japanese Collection of Research Bioresources (JCRB, Tokyo, Japan).

Techniques: Binding Assay

Journal: Current Research in Structural Biology

Article Title: Comparative computational and experimental analyses of some natural small molecules to restore transcriptional activation function of p53 in cancer cells harbouring wild type and p53 Ser46 mutant

doi: 10.1016/j.crstbi.2022.09.002

Figure Lengend Snippet: Dock scores and MMGBSA binding energy for the interaction of the ligands with p53 and Mortalin.

Article Snippet: Human osteosarcoma (U2OS; wild type p53) and oral squamous carcinoma (HSC3; p53 Ser46mutant ) cells were obtained from the Japanese Collection of Research Bioresources (JCRB, Tokyo, Japan).

Techniques: Binding Assay

Journal: Current Research in Structural Biology

Article Title: Comparative computational and experimental analyses of some natural small molecules to restore transcriptional activation function of p53 in cancer cells harbouring wild type and p53 Ser46 mutant

doi: 10.1016/j.crstbi.2022.09.002

Figure Lengend Snippet: Interacting residues of p53 and Mortalin with the ligands after simulation for 200ns.

Article Snippet: Human osteosarcoma (U2OS; wild type p53) and oral squamous carcinoma (HSC3; p53 Ser46mutant ) cells were obtained from the Japanese Collection of Research Bioresources (JCRB, Tokyo, Japan).

Techniques:

Journal: Current Research in Structural Biology

Article Title: Comparative computational and experimental analyses of some natural small molecules to restore transcriptional activation function of p53 in cancer cells harbouring wild type and p53 Ser46 mutant

doi: 10.1016/j.crstbi.2022.09.002

Figure Lengend Snippet: In vitro analysis of the comparative wild type p53 activation function of five natural compounds. A) Western blotting of control and treated U2OS cells for p53 and p21 proteins showed increase in the treated cells. Highest increase was observed in cells treated with Withaferin-A followed by Withanone, Cucurbitacin B, CAPE and Artepillin C. B) Immunocytostaining of control and treated U2OS cells showed increase in expression of p53 and p21 in the later. C) p53-dependent luciferase reporter assay in control and treated U2OS cells showed increase in wild type p53 activity on treated cells and was in accordance to the expression analysis. D) Western blotting of control and treated HSC3 cells (harboring p53 Ser46mutant ) for p21 showed its increase in the later. Blots (A and D) were probed with β-Actin as an internal loading positive control. The results from three independent experiments are shown with statistical analysis indicated as p values. p ​> ​0.05 (non-significant), p ​≤ ​0.05 (∗significant), p ​≤ ​0.01 (∗∗ very significant), p ≤ 0.001 (∗∗∗highly significant), and p ​≤ ​0.0001 (∗∗∗∗ extremely significant).

Article Snippet: Human osteosarcoma (U2OS; wild type p53) and oral squamous carcinoma (HSC3; p53 Ser46mutant ) cells were obtained from the Japanese Collection of Research Bioresources (JCRB, Tokyo, Japan).

Techniques: In Vitro, Activation Assay, Western Blot, Control, Expressing, Luciferase, Reporter Assay, Activity Assay, Positive Control

Journal: Scientific Reports

Article Title: DBC1/CCAR2 is involved in the stabilization of androgen receptor and the progression of osteosarcoma

doi: 10.1038/srep13144

Figure Lengend Snippet: ( a , b ) The knock-down of DBC1 ( a ) or AR ( b ) with two sets of siRNAs for DBC1 or AR (siDBC1 #1, siDBC1 #2, siAR #1, and siAR #2) inhibit the proliferation of both U2OS and SaOS2 osteosarcoma cells as indicated by an MTT and colony forming assay. ( c ) The knock-down of DBC1 and AR significantly reduced the migration activity of both U2OS and SaOS2 cells. ( d ) The invasion capacity of U2OS and SaOS2 cells are significantly decreased with the knock-down of DBC1 and AR in a matrigel invasion assay. ( e ) Cell cycle analysis with flow cytometry shows that the knock-down of DBC1 significantly increases the subG1 and G0/G1 populations in the both U2OS and SaOS2 cells. The statistical analysis for the cell cycle analysis was performed from the flow-cytometry analysis six times. The MTT assay was performed by seeding 1 × 10 3 U2OS cells and 2 × 10 3 SaOS2 cells, and the absorbance measured at 560 nm. For the colony forming assays, 1 × 10 3 U2OS cells and 2 × 10 3 SaOS2 cells were seeded per well of a 24-well culture plate for seven days. The trans-chamber migration and invasion assays were performed after seeding 4 × 10 4 U2OS cells and 1 × 10 5 SaOS2 cells. * p < 0.05, ** p < 0.001.

Article Snippet: The human osteosarcoma cell lines, U2OS (wild-type P53) and SaOS2 (P53-null) were purchased from the Korean Cell Line Bank (KCLB, Seoul, Korea) and cultured in DMEM medium supplemented with 10% fetal bovine serum (Gibco BRL, Gaithersburg, MD), penicillin and streptomycin (100 U/ml), and fungizone (0.25 μg/ml, Gibco BRL, Gaithersburg, MD) at 37 °C in humidified incubator with 5% CO 2 .

Techniques: Knockdown, Migration, Activity Assay, Invasion Assay, Cell Cycle Assay, Flow Cytometry, MTT Assay

Journal: Scientific Reports

Article Title: DBC1/CCAR2 is involved in the stabilization of androgen receptor and the progression of osteosarcoma

doi: 10.1038/srep13144

Figure Lengend Snippet: ( a , b ) Western blotting indicates that the protein levels of BCL-2, TGFβ, RhoA, NFκB, and PCNA were significantly decreased and the expression of P21 and P27 were significantly increased with the knock-down of DBC1 by siRNA in the both U2OS (wild-type P52) and SaOS2 (P53-null) cells. In U2OS cells, acetylation of P53 was significantly increased with the knock-down of DBC1. Especially, the protein level of AR was significantly decreased with the knock-down of DBC1. ( c ) However, the mRNA level of AR did not decrease with the knock-down of DBC1 as demonstrated by quantitative reverse-transcription PCR. ( d , e ) The knock-down of AR increased the protein levels of P21, P27, and BAX, and decreased the protein levels of TGFβ, RhoA, and NFκB in the both U2OS and SaOS2 cells. But the protein level of DBC1 ( e ) and mRNA level of DBC1 ( f ) were not significantly changed with the knock-down of AR with siRNA for AR. The statistical analysis for the quantification of protein expression was performed from three western blots. * p < 0.05, ** p < 0.001.

Article Snippet: The human osteosarcoma cell lines, U2OS (wild-type P53) and SaOS2 (P53-null) were purchased from the Korean Cell Line Bank (KCLB, Seoul, Korea) and cultured in DMEM medium supplemented with 10% fetal bovine serum (Gibco BRL, Gaithersburg, MD), penicillin and streptomycin (100 U/ml), and fungizone (0.25 μg/ml, Gibco BRL, Gaithersburg, MD) at 37 °C in humidified incubator with 5% CO 2 .

Techniques: Western Blot, Expressing, Knockdown, Reverse Transcription

Journal: Scientific Reports

Article Title: DBC1/CCAR2 is involved in the stabilization of androgen receptor and the progression of osteosarcoma

doi: 10.1038/srep13144

Figure Lengend Snippet: ( a ) DBC1 (green) and AR (red) are co-localized mainly in the nuclei of U2OS and SaOS2 osteosarcoma cells by the confocal microscopic image with immunofluorescence staining. ( b ) Immunoprecipitation indicates direct binding of DBC1 and AR. AR is detected in samples taken with immunoprecipitation for DBC1 and vice versa . ( c ) Transfection of DBC1 siRNA into U2OS cells decreases AR stability via a proteasome-mediated pathway. The protein level of AR was decreased more in cells transfected with DBC1 siRNA than in cells transfected with control siRNA. U2OS cells transfected with control or DBC1 siRNA for 24 hours and treated with DMSO (control vehicle), cycloheximide (CHX, 20 μg/ml), or MG-132 (20 μM) for the indicated time. ( d ) Transfection of DBC1 siRNA into U2OS cells caused ubiquitination of AR. U2OS cells were transfected with control or DBC1 siRNA for 24 hours using Lipofectamine. Subsequently, the transfected cells were treated with MG-132 (20 μM) for 4 h and total lysates of cells were immuno-precipitated with anti-androgen receptor antibodies and blotted with anti-ubiquitin antibodies.

Article Snippet: The human osteosarcoma cell lines, U2OS (wild-type P53) and SaOS2 (P53-null) were purchased from the Korean Cell Line Bank (KCLB, Seoul, Korea) and cultured in DMEM medium supplemented with 10% fetal bovine serum (Gibco BRL, Gaithersburg, MD), penicillin and streptomycin (100 U/ml), and fungizone (0.25 μg/ml, Gibco BRL, Gaithersburg, MD) at 37 °C in humidified incubator with 5% CO 2 .

Techniques: Immunofluorescence, Staining, Immunoprecipitation, Binding Assay, Transfection, Control, Ubiquitin Proteomics

Journal: bioRxiv

Article Title: Regulation of FXR1 by alternative splicing is required for muscle development and controls liquid-like condensates in muscle cells

doi: 10.1101/818476

Figure Lengend Snippet: A. Whole cell extracts (WCE) from wild type U2OS cells or U2OSΔFFF cells transiently transfected with either an empty vector (pAGB1139) or P CMV 2xTetO 2 -mNeonGreen-FXR1 isoform E (pAGB1162) were prepared. Extracts were analyzed by Western blot. Right panel shows quantification of protein levels normalized to a loading control. B. U2OΔFFF cells transiently transfected with an empty vector (pAGB1139), P CMV 2xTetO 2 -mNeonGreen-FXR1 isoform A (pAGB1189), or isoform E (pAGB1162) were lysed. The cell lysate and pellet were analyzed by Western blot as above. The percent of protein in the pellet was quantified as the amount in the pellet out of the amount in the WCE. C. Representative images of cells used in panel B at increasing tetracycline (Tet) concentrations. Scale bar 5 µm. D. Quantification of droplet formation for individual cells. n >720 cells from three independent experiments. E. Zoomed in images showing examples of irregular droplets, small spheres, and large spheres. Scale bar 5 µm. F. Quantification of droplet morphology. n >185 cells from three experiments. Results: mean ± s.e.m.

Article Snippet: C2C12 cells and wild type U2OS cells were purchased from ATCC.

Techniques: Transfection, Plasmid Preparation, Western Blot, Control

Journal: bioRxiv

Article Title: Regulation of FXR1 by alternative splicing is required for muscle development and controls liquid-like condensates in muscle cells

doi: 10.1101/818476

Figure Lengend Snippet: A. Location of mutations in the domains of isoform E. B-C U2OSΔFFF cells were transfected with wild type P CMV 2xTetO 2 -mNeonGreen-FXR1 isoform E (pAGB1162), plasmids with mutated KH1 (pAGB1171), KH2 (pAGB1172), KH1/2 (pAGB1173), RGG (pAGB1174), RGG and KH1/2 (pAGB1175), complete IDD (pAGB1176), or partial IDD (pAGB1177). All constructs were induced with 25 ng/mL tetracycline (Tet) to endogenous levels. B. Quantification of droplet formation. n >350 cells from three experiments. C. Representative images of cells expressing the different mutants. Scale bar 5 µm D. Whole cell extracts (WCE), lysate, and pellets were analyzed by Western blotting using an antibody against FXR1. The right panel shows the percent protein in the pellet quantified as in . E. Quantification of droplet morphology utilizing the same three categories that were used in . Results: mean ± s.e.m.

Article Snippet: C2C12 cells and wild type U2OS cells were purchased from ATCC.

Techniques: Transfection, Construct, Expressing, Western Blot

Journal: bioRxiv

Article Title: SNX10 regulates the clearance of mitochondrial proteins and mitochondrial bioenergetics

doi: 10.1101/2024.05.15.594320

Figure Lengend Snippet: A) SNX10 graphical view. The PX domain is represented in green, and the arrows indicate the position of the natural variants (SNPs) linked to ARO (R16L, Y32S, R51P, R51Q). B) Confocal imaging of U2OS cell lines stably expressing doxycycline inducible SNX10-EGFP WT or the indicated ARO-linked mutants. Nuclei were stained with Hoechst. Scale bar = 20µm. C) Representative immunoblot showing the expression levels of SNX10-EGFP and the indicated ARO mutants. The membrane was blotted using and antibody anti GFP and using Actin as loading control. D and E) Representative immunofluorescence images of U2OS cells stably expressing SNX10-EGFP WT or the Y32S mutant (green) immuno-stained with anti-EEA1 (D) or anti-LAMP1 antibodies (E) (magenta) after treating cells with 5µM VPS34-IN1 for 2h. Nuclei were stained with Hoechst. Scale bar = 10µm F) U2OS cells with stable inducible expression of SNX10-EGFP were infected with lentiviral particles to express mScarlet-RAB5/RAB7/RAB9 vectors. Nuclei were stained with Hoechst. Scale bars, 10 μm. G) U2OS SNX10-EGFP cells fixed for CLEM analysis. The area analyzed and showed in (ii) is indicated with a square in the confocal image (i). (iii) shows the transmission EM and (iv) the z-slide from the tomogram. The white arrows indicate clathrin coated vesicles. (v) green: endosomes; red: lipid droplets; yellow: vesicles; and pink: clathrin coated vesicles Scale bars, 10 μm (i and ii), 1 μm (iii and iv).

Article Snippet: U2OS cells expressing SNX10-EGFP wild type or the Y32S mutant were subjected to GFP pulldown using the ChromoTek GFP-Trap®, following the vendor’s specifications.

Techniques: Imaging, Stable Transfection, Expressing, Staining, Western Blot, Membrane, Control, Immunofluorescence, Mutagenesis, Infection, Transmission Assay

Journal: bioRxiv

Article Title: SNX10 regulates the clearance of mitochondrial proteins and mitochondrial bioenergetics

doi: 10.1101/2024.05.15.594320

Figure Lengend Snippet: A) Graphical description of the plasma membrane EGFR staining. Live cells are put on ice, and (1) incubated for 20 min with the primary anti-EGFR antibody, then washed and (2) incubated with a secondary antibody for 20 min, followed by (3) incubation with EGF for 15 or 50 min at 37°C before fixation and imaging. B) U2OS SNX10-EGFP cells were incubated with anti EGFR antibody as described in A), then stimulated with EGF and fixed. Cells were stain with an anti-EFG antibody after fixation. Scale bar: 10µm. C) After 72 h of siRNA transfection, U2OS SNX10-EGFP cells were serum starved for 2 h and then incubated with 50 ng/ml EGF + 10 µg/ml Cycloheximide (CHX) for the indicated times. The cells were lysed followed by western blotting for the indicated proteins. D) Quantification of EGFR and pEGFRTyr1068 protein levels normalized to GAPDH in n= 3 independent experiments. Significance was determined by two-way ANOVA followed by Šídák’s multiple comparisons test. E) Cells were reverse transfected with 20nM siRNA: siCtrl (control) and two different siSNX10 oligoes (siSNX10#1 and siSNX10#2) prior to fixation and staining for endogenous EEA1. Images were taken with Zeiss Axio Observer widefield microscope (Zen Blue 2.3, Zeiss) and a 20x objective was used. Scale bar: 10 µm. F) quantification of the data shown in E) was performed using CellProfiler software. The values were obtained from analyzing >1000 cells per condition and they were normalized to control siRNA (siCtrl). The graphs display the mean values ±SEM from n = 3 independent experiments. The significance was assessed by ordinary one-way ANOVA followed by Bonferroni’s post hoc test. (* = p < 0.05). G) Representative electron microscopy images of U2OS cells (control and siSNX10) showing immunogold-labelled EGFR containing endosomes. One experiment was performed. The number of endosomes without EGFR-gold and with EGFR-gold of which diameter was measured were 8 (siCtrl), 45 (siSNX10), and 23 (siCtrl), 10 (siSNX10) respectively. H) The graph displays the mean values ± SEM from the endosomal diameters. Significance was determined by unpaired t-test with Welch’s correction. * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001 and n.s = not significant in all graphs.

Article Snippet: U2OS cells expressing SNX10-EGFP wild type or the Y32S mutant were subjected to GFP pulldown using the ChromoTek GFP-Trap®, following the vendor’s specifications.

Techniques: Clinical Proteomics, Membrane, Staining, Incubation, Imaging, Transfection, Western Blot, Control, Microscopy, Software, Electron Microscopy

Journal: bioRxiv

Article Title: SNX10 regulates the clearance of mitochondrial proteins and mitochondrial bioenergetics

doi: 10.1101/2024.05.15.594320

Figure Lengend Snippet: A) U2OS cells expressing either WT SNX10-EGFP or the Y32S mutant underwent GFP pulldown for the subsequent analysis of their interactome using mass spectrometry assays. The resulting data were depicted in a Venn diagram, illustrating the distinct and shared interactors between the WT and mutant forms of SNX10. Significant identified proteins representing hits that interacted with SNX10 WT are listed below B) The interacting proteins of SNX10 WT were enriched for GO term analysis. The enrichment of the Cellular Component they belong to (pie chart above) and Biological Processes they are involved in (pie chart below) is expressed in percentage towards the significant hits. Graphs were plotted using plotly (python package). C) U2OS with stable inducible expression of SNX10-EGFP were treated with doxycycline for 24 hrs before incubation with MitoTracker Red for 30 min, followed by live imaging using a Dragonfly 505 (Andor) microscope, with an acquisition speed of 1 frame every 500 ms. Scale bar: 10 µm. D) U2OS cells stably expressing mScarlet-RAB5 and with inducible expression of SNX10-EGFP and were stained with MitoTracker Deep Red FM in the presence or absence of DFP. Scale bar 10 µm. E) U2OS SNX10-EGFP were treated with or without DFP (1 µM) for 24 hrs and with or without MRT68921 (1 µM) for 1 h. MitoTracker (100 nM) was added for 1 h, followed by immunofluorescence staining with antibodies against LC3B and COX-IV. Scale bars: 10 µm. F and G) Quantification of the percentage of SNX10 localizing on LC3B (F) and LC3 + MitoTracker positive structures localizing on SNX10 (G), normalized to control sample (-MRT/-DFP) (the graph shows mean ± SEM [error bars] from n=3 independent experiments. The statistical significance was calculated with a two-way ANOVA followed by a Tukey’s post analysis test. * = p < 0.05)

Article Snippet: U2OS cells expressing SNX10-EGFP wild type or the Y32S mutant were subjected to GFP pulldown using the ChromoTek GFP-Trap®, following the vendor’s specifications.

Techniques: Expressing, Mutagenesis, Mass Spectrometry, Incubation, Imaging, Microscopy, Stable Transfection, Staining, Immunofluorescence, Control

Journal: bioRxiv

Article Title: SNX10 regulates the clearance of mitochondrial proteins and mitochondrial bioenergetics

doi: 10.1101/2024.05.15.594320

Figure Lengend Snippet: A) U2OS cells with stable inducible expression of SNX10-EGFP were pre-treated with doxycycline for 16 hrs before the addition of DFP for 24 hrs. The cells were fixed and stained with antibodies against mitochondrial proteins for subsequent analysis. Scale bars: 10 µm. B) U2OS cells were reverse transfected with the indicated siRNA (20 nM) for 48 hrs, then treated or not with DFP for 24 hrs with BafA1 treatment or not the last 2 hrs, followed by western blotting for the indicated proteins. C-E) Quantification of the data in B) from n=5, 3 and 6 independent experiments. Bars show mean values of the protein levels normalized to b-Actin relative to Control conditions (siCtrl control). Significance is assessed by two-way ANOVA followed by Tukey’s post hoc test. F) U2OS cells were subjected to reverse transfection with siSNX10 (20nM) for 48 hours, followed by treatment with either IN1 or MRT for 24 hours before fixation. Post-fixation, cells were stained with a COX-IV antibody, and images were captured using an ImageXpress Micro Confocal (Molecular Devices) at 20X magnification. * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001 and n.s = not significant in all graphs

Article Snippet: U2OS cells expressing SNX10-EGFP wild type or the Y32S mutant were subjected to GFP pulldown using the ChromoTek GFP-Trap®, following the vendor’s specifications.

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

Journal: bioRxiv

Article Title: SNX10 regulates the clearance of mitochondrial proteins and mitochondrial bioenergetics

doi: 10.1101/2024.05.15.594320

Figure Lengend Snippet: A) Citrate Synthase activity was determined by spectrophotometry from lysates of U2OS cells transfected with siRNA for 72 hrs, in the presence or absence of DFP for the last 24 hrs. The graph displays mean values normalized to siCtrl. Significance was determined from n = 3 independent experiments, by two-way ANOVA followed by Tukey’s multiple comparison test. B) Mitochondrial oxygen consumption rate (OCR) was assessed in control and SNX10 knocked down cells using the Seahorse XFe24 Analyzer. OCR was measured following sequential addition of Oligomycin, carbonyl cyanide m-chlorophenyl hydrazone (CCCP), and Rotenone/Antimycin A (Rot/AntiA). C) The four basal OCR measurements per well were averaged to determine the basal OCR value, and non-mitochondrial respiration was subtracted to ascertain the basal respiration associated with each condition. D) ATP production was calculated by subtracting the proton leak from the maximal respiratory capacity. Error bars represent mean ± SEM from n=5. Statistical significance was determined using one-way ANOVA followed by Dunnett’s multiple comparison test. * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001 and n.s = not significant in all graphs

Article Snippet: U2OS cells expressing SNX10-EGFP wild type or the Y32S mutant were subjected to GFP pulldown using the ChromoTek GFP-Trap®, following the vendor’s specifications.

Techniques: Activity Assay, Spectrophotometry, Transfection, Comparison, Control