Structured Review

Millipore anti sp1 antibody
Nucleolin and <t>Sp1</t> regulate G4 → duplex transition at MAPK12 promoter. A FRET probe. MAPK12-G4 motifs conjugated with 6-FAM at 5′-end of GQ-1 and internal Cy3 at T22. The complementary strand of G4-forming sequence contains Cy5 at 3′-end. G4-formation gives high FRET signal between 6-FAM and Cy3. Duplex formation gives high FRET signal between 6-FAM and Cy5. B Fluorometric titration of 90 nM FRET probe with increasing gradient of recombinant Nucleolin (0–3 µM) in 50 mM Tris–Cl, pH 7.0, 100 mM KCl at 15 °C at excitation wavelength (λex) of 497 nm. Decline of FRET signal at Cy5 emission ((λem) 650 nm) upon Nucleolin titration suggests disruption of duplex with concomitant increase of Cy3 emission ((λem) 595 nm) indicating G4 evolution. C Fluorometric titration of 90 nM FRET probe with increasing gradient of recombinant Sp1 (0–1 µM) in 50 mM Tris–Cl, pH 7.0, 100 mM KCl at 15 °C at excitation wavelength (λex) of 497 nm. Decline of FRET signal at Cy3 emission ((λem) 595 nm) upon Sp1 titration suggests G4 resolution with concomitant increase of Cy5 emission ((λem) 650 nm) indicating duplex transition. D FRET efficiency calculated following the equation: E = 1 – (IDA/ID); IDA and ID are the emission intensity of donor fluorophore in presence and absence of acceptor fluorophore respectively. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). With increasing Sp1, distance between 6-FAM and Cy5 decreases. Nucleolin titration decreases the distance between 6-FAM and Cy3. The change in distances (r) between donor and acceptor fluorophore calculated by E = R06/(R06 + r6). R0 denotes Forster distance between donor and acceptor. The distance between 6-FAM and Cy3 decreased from 6.5 ± 0.1 nm (R0 = 6.5 nm) in the free DNA to 5.2 ± 0.1 nm in the complex with Nucleolin indicating G4 formation. The distance between 6-FAM and Cy5 decreased from 6.8 ± 0.2 in free DNA to 5.3 ± 0.3 under Sp1-bound conditions indicating duplex formation (E) pGL4.72[hRlucCP] luciferase constructs having the inserts containing MAPK12 promoter sequences having adjacent G4-elements (wild-type). Dual-luciferase assays. Evaluation of MAPK12 promoter activities in MDAMB-231 cells under the following conditions: siRNA knockdown of Nucleolin at 48 h and TGS24 (50 nM) treatment for 24 h, nucleolin-knockdown + TGS24 treatment, nucleolin knockdown + TGS25 treatment, and TGS24 and TGS25 treated alone separately Error bars represent mean ± SE (N = 3). Statistical differences in the luciferase activities compared to that of the control cells use two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). Statistical differences in the luciferase activities in presence of TGS24-treated samples compared to that of cells, treated with recombinant Sp1 and Nucleolin, siRNA-knockdown of Nucleolin use two-tailed Student's t test (###P < 0.001). F RT-PCR analysis. Expression profile of MAPK12 transcripts from P1 promoter in MDAMB-231 cells under following conditions: TGS24 (50 nM) treatment for 24 h and siRNA knockdown of Nucleolin for 48 h, nucleolin-knockdown + TGS24 treatment, nucleolin knockdown + TGS25 treatment, and TGS25 treated alone separately. Quantification of the transcripts’ level relative to the control by qPCR analyses. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). Statistical differences between recombinant Nucleolin-treated and Nucleolin-knockdown cells are determined by two-tailed Student's t test (###P < 0.001). GAPDH considered as housekeeping gene
Anti Sp1 Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/anti sp1 antibody/product/Millipore
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anti sp1 antibody - by Bioz Stars, 2024-07
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1) Product Images from "G-quadruplex structural dynamics at MAPK12 promoter dictates transcriptional switch to determine stemness in breast cancer"

Article Title: G-quadruplex structural dynamics at MAPK12 promoter dictates transcriptional switch to determine stemness in breast cancer

Journal: Cellular and Molecular Life Sciences: CMLS

doi: 10.1007/s00018-023-05046-6

Nucleolin and Sp1 regulate G4 → duplex transition at MAPK12 promoter. A FRET probe. MAPK12-G4 motifs conjugated with 6-FAM at 5′-end of GQ-1 and internal Cy3 at T22. The complementary strand of G4-forming sequence contains Cy5 at 3′-end. G4-formation gives high FRET signal between 6-FAM and Cy3. Duplex formation gives high FRET signal between 6-FAM and Cy5. B Fluorometric titration of 90 nM FRET probe with increasing gradient of recombinant Nucleolin (0–3 µM) in 50 mM Tris–Cl, pH 7.0, 100 mM KCl at 15 °C at excitation wavelength (λex) of 497 nm. Decline of FRET signal at Cy5 emission ((λem) 650 nm) upon Nucleolin titration suggests disruption of duplex with concomitant increase of Cy3 emission ((λem) 595 nm) indicating G4 evolution. C Fluorometric titration of 90 nM FRET probe with increasing gradient of recombinant Sp1 (0–1 µM) in 50 mM Tris–Cl, pH 7.0, 100 mM KCl at 15 °C at excitation wavelength (λex) of 497 nm. Decline of FRET signal at Cy3 emission ((λem) 595 nm) upon Sp1 titration suggests G4 resolution with concomitant increase of Cy5 emission ((λem) 650 nm) indicating duplex transition. D FRET efficiency calculated following the equation: E = 1 – (IDA/ID); IDA and ID are the emission intensity of donor fluorophore in presence and absence of acceptor fluorophore respectively. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). With increasing Sp1, distance between 6-FAM and Cy5 decreases. Nucleolin titration decreases the distance between 6-FAM and Cy3. The change in distances (r) between donor and acceptor fluorophore calculated by E = R06/(R06 + r6). R0 denotes Forster distance between donor and acceptor. The distance between 6-FAM and Cy3 decreased from 6.5 ± 0.1 nm (R0 = 6.5 nm) in the free DNA to 5.2 ± 0.1 nm in the complex with Nucleolin indicating G4 formation. The distance between 6-FAM and Cy5 decreased from 6.8 ± 0.2 in free DNA to 5.3 ± 0.3 under Sp1-bound conditions indicating duplex formation (E) pGL4.72[hRlucCP] luciferase constructs having the inserts containing MAPK12 promoter sequences having adjacent G4-elements (wild-type). Dual-luciferase assays. Evaluation of MAPK12 promoter activities in MDAMB-231 cells under the following conditions: siRNA knockdown of Nucleolin at 48 h and TGS24 (50 nM) treatment for 24 h, nucleolin-knockdown + TGS24 treatment, nucleolin knockdown + TGS25 treatment, and TGS24 and TGS25 treated alone separately Error bars represent mean ± SE (N = 3). Statistical differences in the luciferase activities compared to that of the control cells use two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). Statistical differences in the luciferase activities in presence of TGS24-treated samples compared to that of cells, treated with recombinant Sp1 and Nucleolin, siRNA-knockdown of Nucleolin use two-tailed Student's t test (###P < 0.001). F RT-PCR analysis. Expression profile of MAPK12 transcripts from P1 promoter in MDAMB-231 cells under following conditions: TGS24 (50 nM) treatment for 24 h and siRNA knockdown of Nucleolin for 48 h, nucleolin-knockdown + TGS24 treatment, nucleolin knockdown + TGS25 treatment, and TGS25 treated alone separately. Quantification of the transcripts’ level relative to the control by qPCR analyses. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). Statistical differences between recombinant Nucleolin-treated and Nucleolin-knockdown cells are determined by two-tailed Student's t test (###P < 0.001). GAPDH considered as housekeeping gene
Figure Legend Snippet: Nucleolin and Sp1 regulate G4 → duplex transition at MAPK12 promoter. A FRET probe. MAPK12-G4 motifs conjugated with 6-FAM at 5′-end of GQ-1 and internal Cy3 at T22. The complementary strand of G4-forming sequence contains Cy5 at 3′-end. G4-formation gives high FRET signal between 6-FAM and Cy3. Duplex formation gives high FRET signal between 6-FAM and Cy5. B Fluorometric titration of 90 nM FRET probe with increasing gradient of recombinant Nucleolin (0–3 µM) in 50 mM Tris–Cl, pH 7.0, 100 mM KCl at 15 °C at excitation wavelength (λex) of 497 nm. Decline of FRET signal at Cy5 emission ((λem) 650 nm) upon Nucleolin titration suggests disruption of duplex with concomitant increase of Cy3 emission ((λem) 595 nm) indicating G4 evolution. C Fluorometric titration of 90 nM FRET probe with increasing gradient of recombinant Sp1 (0–1 µM) in 50 mM Tris–Cl, pH 7.0, 100 mM KCl at 15 °C at excitation wavelength (λex) of 497 nm. Decline of FRET signal at Cy3 emission ((λem) 595 nm) upon Sp1 titration suggests G4 resolution with concomitant increase of Cy5 emission ((λem) 650 nm) indicating duplex transition. D FRET efficiency calculated following the equation: E = 1 – (IDA/ID); IDA and ID are the emission intensity of donor fluorophore in presence and absence of acceptor fluorophore respectively. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). With increasing Sp1, distance between 6-FAM and Cy5 decreases. Nucleolin titration decreases the distance between 6-FAM and Cy3. The change in distances (r) between donor and acceptor fluorophore calculated by E = R06/(R06 + r6). R0 denotes Forster distance between donor and acceptor. The distance between 6-FAM and Cy3 decreased from 6.5 ± 0.1 nm (R0 = 6.5 nm) in the free DNA to 5.2 ± 0.1 nm in the complex with Nucleolin indicating G4 formation. The distance between 6-FAM and Cy5 decreased from 6.8 ± 0.2 in free DNA to 5.3 ± 0.3 under Sp1-bound conditions indicating duplex formation (E) pGL4.72[hRlucCP] luciferase constructs having the inserts containing MAPK12 promoter sequences having adjacent G4-elements (wild-type). Dual-luciferase assays. Evaluation of MAPK12 promoter activities in MDAMB-231 cells under the following conditions: siRNA knockdown of Nucleolin at 48 h and TGS24 (50 nM) treatment for 24 h, nucleolin-knockdown + TGS24 treatment, nucleolin knockdown + TGS25 treatment, and TGS24 and TGS25 treated alone separately Error bars represent mean ± SE (N = 3). Statistical differences in the luciferase activities compared to that of the control cells use two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). Statistical differences in the luciferase activities in presence of TGS24-treated samples compared to that of cells, treated with recombinant Sp1 and Nucleolin, siRNA-knockdown of Nucleolin use two-tailed Student's t test (###P < 0.001). F RT-PCR analysis. Expression profile of MAPK12 transcripts from P1 promoter in MDAMB-231 cells under following conditions: TGS24 (50 nM) treatment for 24 h and siRNA knockdown of Nucleolin for 48 h, nucleolin-knockdown + TGS24 treatment, nucleolin knockdown + TGS25 treatment, and TGS25 treated alone separately. Quantification of the transcripts’ level relative to the control by qPCR analyses. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). Statistical differences between recombinant Nucleolin-treated and Nucleolin-knockdown cells are determined by two-tailed Student's t test (###P < 0.001). GAPDH considered as housekeeping gene

Techniques Used: Sequencing, Titration, Recombinant, Disruption, Two Tailed Test, Luciferase, Construct, Reverse Transcription Polymerase Chain Reaction, Expressing

Downstream effects of TGS24-treatment and its selective interaction with MAPK12-G4 motifs. A RNAseq analyses of TGS24 treatment in MDAMB-231 spheres expressing high MAPK12. Heatmap of biological replicates of differentially expressed genes upon 24 h treatment of 50 nM TGS24 treatment in MDAMB-231 spheres using the log10(count) values for each replicate. The screening threshold for the differentially expressed genes is set to: |log2(Fold Change)|> 1 and P value < 0.05. Differential expression of the transcripts clustered by Euclidean correlation, with distinct upregulation, unaltered, and downregulation patterns in expression for TGS24 treatment, compared to control condition. The count values are colour coded blue to yellow to red in increasing order. Differentially expressed genes are clustered based on their involvement with RAS, apoptosis, and stemness- and metastasis-related pathways. B RT-qPCR-verification of RNAseq data for differentially expressed genes in RAS pathway upon 50 nM TGS24 treatment in MDAMB-231 spheres for 24 h: MAP4K3, RAS, MAPK12, c-FOS, c-JUN, HSP90. Quantification of the transcripts’ level relative to the control by qPCR analyses. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). GAPDH considered as housekeeping gene. C RT-qPCR-verification of RNAseq data for differentially expressed genes in metastasis and stemness-related pathway upon 50 nM TGS24 treatment in MDAMB-231 spheres for 24 h: ALDHβ1, WNT-1, β-Catenin, SOX2, MMP12, Vimentin, CD-44. Quantification of the transcripts’ level relative to the control by qPCR analyses. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). GAPDH considered as housekeeping gene. D RT-qPCR-verification of RNAseq data for differentially expressed genes in apoptotic pathway upon 50 nM TGS24 treatment in MDAMB-231 spheres for 24 h: NF-κβ, Sp1, GATA3, CASP9, PARP-1. Quantification of the transcripts’ level relative to the control by qPCR analyses. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). GAPDH considered as housekeeping gene. E Correlation of log2(fold-change) values from RNA-Seq and RT-PCR analysis. The R2 value is 0.854. F Interaction network of upregulated and downregulated genes, obtained using STRING database, with the minimum required interaction score of 0.400 and network edges representing evidence of an interaction
Figure Legend Snippet: Downstream effects of TGS24-treatment and its selective interaction with MAPK12-G4 motifs. A RNAseq analyses of TGS24 treatment in MDAMB-231 spheres expressing high MAPK12. Heatmap of biological replicates of differentially expressed genes upon 24 h treatment of 50 nM TGS24 treatment in MDAMB-231 spheres using the log10(count) values for each replicate. The screening threshold for the differentially expressed genes is set to: |log2(Fold Change)|> 1 and P value < 0.05. Differential expression of the transcripts clustered by Euclidean correlation, with distinct upregulation, unaltered, and downregulation patterns in expression for TGS24 treatment, compared to control condition. The count values are colour coded blue to yellow to red in increasing order. Differentially expressed genes are clustered based on their involvement with RAS, apoptosis, and stemness- and metastasis-related pathways. B RT-qPCR-verification of RNAseq data for differentially expressed genes in RAS pathway upon 50 nM TGS24 treatment in MDAMB-231 spheres for 24 h: MAP4K3, RAS, MAPK12, c-FOS, c-JUN, HSP90. Quantification of the transcripts’ level relative to the control by qPCR analyses. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). GAPDH considered as housekeeping gene. C RT-qPCR-verification of RNAseq data for differentially expressed genes in metastasis and stemness-related pathway upon 50 nM TGS24 treatment in MDAMB-231 spheres for 24 h: ALDHβ1, WNT-1, β-Catenin, SOX2, MMP12, Vimentin, CD-44. Quantification of the transcripts’ level relative to the control by qPCR analyses. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). GAPDH considered as housekeeping gene. D RT-qPCR-verification of RNAseq data for differentially expressed genes in apoptotic pathway upon 50 nM TGS24 treatment in MDAMB-231 spheres for 24 h: NF-κβ, Sp1, GATA3, CASP9, PARP-1. Quantification of the transcripts’ level relative to the control by qPCR analyses. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). GAPDH considered as housekeeping gene. E Correlation of log2(fold-change) values from RNA-Seq and RT-PCR analysis. The R2 value is 0.854. F Interaction network of upregulated and downregulated genes, obtained using STRING database, with the minimum required interaction score of 0.400 and network edges representing evidence of an interaction

Techniques Used: Expressing, Quantitative RT-PCR, Two Tailed Test, RNA Sequencing Assay, Reverse Transcription Polymerase Chain Reaction

Dynamics of two tandem G4-motifs at MAPK12 promoter regulated by spatiotemporal binding of Nucleolin and Sp1 regulate cancer stemness via MAPK12-HSP90-RAS-c-JUN-NANOG axis
Figure Legend Snippet: Dynamics of two tandem G4-motifs at MAPK12 promoter regulated by spatiotemporal binding of Nucleolin and Sp1 regulate cancer stemness via MAPK12-HSP90-RAS-c-JUN-NANOG axis

Techniques Used: Binding Assay


Structured Review

Millipore sp1 antibody
Nucleolin and <t>Sp1</t> regulate G4 → duplex transition at MAPK12 promoter. A FRET probe. MAPK12-G4 motifs conjugated with 6-FAM at 5′-end of GQ-1 and internal Cy3 at T22. The complementary strand of G4-forming sequence contains Cy5 at 3′-end. G4-formation gives high FRET signal between 6-FAM and Cy3. Duplex formation gives high FRET signal between 6-FAM and Cy5. B Fluorometric titration of 90 nM FRET probe with increasing gradient of recombinant Nucleolin (0–3 µM) in 50 mM Tris–Cl, pH 7.0, 100 mM KCl at 15 °C at excitation wavelength (λex) of 497 nm. Decline of FRET signal at Cy5 emission ((λem) 650 nm) upon Nucleolin titration suggests disruption of duplex with concomitant increase of Cy3 emission ((λem) 595 nm) indicating G4 evolution. C Fluorometric titration of 90 nM FRET probe with increasing gradient of recombinant Sp1 (0–1 µM) in 50 mM Tris–Cl, pH 7.0, 100 mM KCl at 15 °C at excitation wavelength (λex) of 497 nm. Decline of FRET signal at Cy3 emission ((λem) 595 nm) upon Sp1 titration suggests G4 resolution with concomitant increase of Cy5 emission ((λem) 650 nm) indicating duplex transition. D FRET efficiency calculated following the equation: E = 1 – (IDA/ID); IDA and ID are the emission intensity of donor fluorophore in presence and absence of acceptor fluorophore respectively. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). With increasing Sp1, distance between 6-FAM and Cy5 decreases. Nucleolin titration decreases the distance between 6-FAM and Cy3. The change in distances (r) between donor and acceptor fluorophore calculated by E = R06/(R06 + r6). R0 denotes Forster distance between donor and acceptor. The distance between 6-FAM and Cy3 decreased from 6.5 ± 0.1 nm (R0 = 6.5 nm) in the free DNA to 5.2 ± 0.1 nm in the complex with Nucleolin indicating G4 formation. The distance between 6-FAM and Cy5 decreased from 6.8 ± 0.2 in free DNA to 5.3 ± 0.3 under Sp1-bound conditions indicating duplex formation (E) pGL4.72[hRlucCP] luciferase constructs having the inserts containing MAPK12 promoter sequences having adjacent G4-elements (wild-type). Dual-luciferase assays. Evaluation of MAPK12 promoter activities in MDAMB-231 cells under the following conditions: siRNA knockdown of Nucleolin at 48 h and TGS24 (50 nM) treatment for 24 h, nucleolin-knockdown + TGS24 treatment, nucleolin knockdown + TGS25 treatment, and TGS24 and TGS25 treated alone separately Error bars represent mean ± SE (N = 3). Statistical differences in the luciferase activities compared to that of the control cells use two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). Statistical differences in the luciferase activities in presence of TGS24-treated samples compared to that of cells, treated with recombinant Sp1 and Nucleolin, siRNA-knockdown of Nucleolin use two-tailed Student's t test (###P < 0.001). F RT-PCR analysis. Expression profile of MAPK12 transcripts from P1 promoter in MDAMB-231 cells under following conditions: TGS24 (50 nM) treatment for 24 h and siRNA knockdown of Nucleolin for 48 h, nucleolin-knockdown + TGS24 treatment, nucleolin knockdown + TGS25 treatment, and TGS25 treated alone separately. Quantification of the transcripts’ level relative to the control by qPCR analyses. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). Statistical differences between recombinant Nucleolin-treated and Nucleolin-knockdown cells are determined by two-tailed Student's t test (###P < 0.001). GAPDH considered as housekeeping gene
Sp1 Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/sp1 antibody/product/Millipore
Average 86 stars, based on 1 article reviews
Price from $9.99 to $1999.99
sp1 antibody - by Bioz Stars, 2024-07
86/100 stars

Images

1) Product Images from "G-quadruplex structural dynamics at MAPK12 promoter dictates transcriptional switch to determine stemness in breast cancer"

Article Title: G-quadruplex structural dynamics at MAPK12 promoter dictates transcriptional switch to determine stemness in breast cancer

Journal: Cellular and Molecular Life Sciences: CMLS

doi: 10.1007/s00018-023-05046-6

Nucleolin and Sp1 regulate G4 → duplex transition at MAPK12 promoter. A FRET probe. MAPK12-G4 motifs conjugated with 6-FAM at 5′-end of GQ-1 and internal Cy3 at T22. The complementary strand of G4-forming sequence contains Cy5 at 3′-end. G4-formation gives high FRET signal between 6-FAM and Cy3. Duplex formation gives high FRET signal between 6-FAM and Cy5. B Fluorometric titration of 90 nM FRET probe with increasing gradient of recombinant Nucleolin (0–3 µM) in 50 mM Tris–Cl, pH 7.0, 100 mM KCl at 15 °C at excitation wavelength (λex) of 497 nm. Decline of FRET signal at Cy5 emission ((λem) 650 nm) upon Nucleolin titration suggests disruption of duplex with concomitant increase of Cy3 emission ((λem) 595 nm) indicating G4 evolution. C Fluorometric titration of 90 nM FRET probe with increasing gradient of recombinant Sp1 (0–1 µM) in 50 mM Tris–Cl, pH 7.0, 100 mM KCl at 15 °C at excitation wavelength (λex) of 497 nm. Decline of FRET signal at Cy3 emission ((λem) 595 nm) upon Sp1 titration suggests G4 resolution with concomitant increase of Cy5 emission ((λem) 650 nm) indicating duplex transition. D FRET efficiency calculated following the equation: E = 1 – (IDA/ID); IDA and ID are the emission intensity of donor fluorophore in presence and absence of acceptor fluorophore respectively. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). With increasing Sp1, distance between 6-FAM and Cy5 decreases. Nucleolin titration decreases the distance between 6-FAM and Cy3. The change in distances (r) between donor and acceptor fluorophore calculated by E = R06/(R06 + r6). R0 denotes Forster distance between donor and acceptor. The distance between 6-FAM and Cy3 decreased from 6.5 ± 0.1 nm (R0 = 6.5 nm) in the free DNA to 5.2 ± 0.1 nm in the complex with Nucleolin indicating G4 formation. The distance between 6-FAM and Cy5 decreased from 6.8 ± 0.2 in free DNA to 5.3 ± 0.3 under Sp1-bound conditions indicating duplex formation (E) pGL4.72[hRlucCP] luciferase constructs having the inserts containing MAPK12 promoter sequences having adjacent G4-elements (wild-type). Dual-luciferase assays. Evaluation of MAPK12 promoter activities in MDAMB-231 cells under the following conditions: siRNA knockdown of Nucleolin at 48 h and TGS24 (50 nM) treatment for 24 h, nucleolin-knockdown + TGS24 treatment, nucleolin knockdown + TGS25 treatment, and TGS24 and TGS25 treated alone separately Error bars represent mean ± SE (N = 3). Statistical differences in the luciferase activities compared to that of the control cells use two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). Statistical differences in the luciferase activities in presence of TGS24-treated samples compared to that of cells, treated with recombinant Sp1 and Nucleolin, siRNA-knockdown of Nucleolin use two-tailed Student's t test (###P < 0.001). F RT-PCR analysis. Expression profile of MAPK12 transcripts from P1 promoter in MDAMB-231 cells under following conditions: TGS24 (50 nM) treatment for 24 h and siRNA knockdown of Nucleolin for 48 h, nucleolin-knockdown + TGS24 treatment, nucleolin knockdown + TGS25 treatment, and TGS25 treated alone separately. Quantification of the transcripts’ level relative to the control by qPCR analyses. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). Statistical differences between recombinant Nucleolin-treated and Nucleolin-knockdown cells are determined by two-tailed Student's t test (###P < 0.001). GAPDH considered as housekeeping gene
Figure Legend Snippet: Nucleolin and Sp1 regulate G4 → duplex transition at MAPK12 promoter. A FRET probe. MAPK12-G4 motifs conjugated with 6-FAM at 5′-end of GQ-1 and internal Cy3 at T22. The complementary strand of G4-forming sequence contains Cy5 at 3′-end. G4-formation gives high FRET signal between 6-FAM and Cy3. Duplex formation gives high FRET signal between 6-FAM and Cy5. B Fluorometric titration of 90 nM FRET probe with increasing gradient of recombinant Nucleolin (0–3 µM) in 50 mM Tris–Cl, pH 7.0, 100 mM KCl at 15 °C at excitation wavelength (λex) of 497 nm. Decline of FRET signal at Cy5 emission ((λem) 650 nm) upon Nucleolin titration suggests disruption of duplex with concomitant increase of Cy3 emission ((λem) 595 nm) indicating G4 evolution. C Fluorometric titration of 90 nM FRET probe with increasing gradient of recombinant Sp1 (0–1 µM) in 50 mM Tris–Cl, pH 7.0, 100 mM KCl at 15 °C at excitation wavelength (λex) of 497 nm. Decline of FRET signal at Cy3 emission ((λem) 595 nm) upon Sp1 titration suggests G4 resolution with concomitant increase of Cy5 emission ((λem) 650 nm) indicating duplex transition. D FRET efficiency calculated following the equation: E = 1 – (IDA/ID); IDA and ID are the emission intensity of donor fluorophore in presence and absence of acceptor fluorophore respectively. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). With increasing Sp1, distance between 6-FAM and Cy5 decreases. Nucleolin titration decreases the distance between 6-FAM and Cy3. The change in distances (r) between donor and acceptor fluorophore calculated by E = R06/(R06 + r6). R0 denotes Forster distance between donor and acceptor. The distance between 6-FAM and Cy3 decreased from 6.5 ± 0.1 nm (R0 = 6.5 nm) in the free DNA to 5.2 ± 0.1 nm in the complex with Nucleolin indicating G4 formation. The distance between 6-FAM and Cy5 decreased from 6.8 ± 0.2 in free DNA to 5.3 ± 0.3 under Sp1-bound conditions indicating duplex formation (E) pGL4.72[hRlucCP] luciferase constructs having the inserts containing MAPK12 promoter sequences having adjacent G4-elements (wild-type). Dual-luciferase assays. Evaluation of MAPK12 promoter activities in MDAMB-231 cells under the following conditions: siRNA knockdown of Nucleolin at 48 h and TGS24 (50 nM) treatment for 24 h, nucleolin-knockdown + TGS24 treatment, nucleolin knockdown + TGS25 treatment, and TGS24 and TGS25 treated alone separately Error bars represent mean ± SE (N = 3). Statistical differences in the luciferase activities compared to that of the control cells use two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). Statistical differences in the luciferase activities in presence of TGS24-treated samples compared to that of cells, treated with recombinant Sp1 and Nucleolin, siRNA-knockdown of Nucleolin use two-tailed Student's t test (###P < 0.001). F RT-PCR analysis. Expression profile of MAPK12 transcripts from P1 promoter in MDAMB-231 cells under following conditions: TGS24 (50 nM) treatment for 24 h and siRNA knockdown of Nucleolin for 48 h, nucleolin-knockdown + TGS24 treatment, nucleolin knockdown + TGS25 treatment, and TGS25 treated alone separately. Quantification of the transcripts’ level relative to the control by qPCR analyses. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). Statistical differences between recombinant Nucleolin-treated and Nucleolin-knockdown cells are determined by two-tailed Student's t test (###P < 0.001). GAPDH considered as housekeeping gene

Techniques Used: Sequencing, Titration, Recombinant, Disruption, Two Tailed Test, Luciferase, Construct, Reverse Transcription Polymerase Chain Reaction, Expressing

Downstream effects of TGS24-treatment and its selective interaction with MAPK12-G4 motifs. A RNAseq analyses of TGS24 treatment in MDAMB-231 spheres expressing high MAPK12. Heatmap of biological replicates of differentially expressed genes upon 24 h treatment of 50 nM TGS24 treatment in MDAMB-231 spheres using the log10(count) values for each replicate. The screening threshold for the differentially expressed genes is set to: |log2(Fold Change)|> 1 and P value < 0.05. Differential expression of the transcripts clustered by Euclidean correlation, with distinct upregulation, unaltered, and downregulation patterns in expression for TGS24 treatment, compared to control condition. The count values are colour coded blue to yellow to red in increasing order. Differentially expressed genes are clustered based on their involvement with RAS, apoptosis, and stemness- and metastasis-related pathways. B RT-qPCR-verification of RNAseq data for differentially expressed genes in RAS pathway upon 50 nM TGS24 treatment in MDAMB-231 spheres for 24 h: MAP4K3, RAS, MAPK12, c-FOS, c-JUN, HSP90. Quantification of the transcripts’ level relative to the control by qPCR analyses. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). GAPDH considered as housekeeping gene. C RT-qPCR-verification of RNAseq data for differentially expressed genes in metastasis and stemness-related pathway upon 50 nM TGS24 treatment in MDAMB-231 spheres for 24 h: ALDHβ1, WNT-1, β-Catenin, SOX2, MMP12, Vimentin, CD-44. Quantification of the transcripts’ level relative to the control by qPCR analyses. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). GAPDH considered as housekeeping gene. D RT-qPCR-verification of RNAseq data for differentially expressed genes in apoptotic pathway upon 50 nM TGS24 treatment in MDAMB-231 spheres for 24 h: NF-κβ, Sp1, GATA3, CASP9, PARP-1. Quantification of the transcripts’ level relative to the control by qPCR analyses. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). GAPDH considered as housekeeping gene. E Correlation of log2(fold-change) values from RNA-Seq and RT-PCR analysis. The R2 value is 0.854. F Interaction network of upregulated and downregulated genes, obtained using STRING database, with the minimum required interaction score of 0.400 and network edges representing evidence of an interaction
Figure Legend Snippet: Downstream effects of TGS24-treatment and its selective interaction with MAPK12-G4 motifs. A RNAseq analyses of TGS24 treatment in MDAMB-231 spheres expressing high MAPK12. Heatmap of biological replicates of differentially expressed genes upon 24 h treatment of 50 nM TGS24 treatment in MDAMB-231 spheres using the log10(count) values for each replicate. The screening threshold for the differentially expressed genes is set to: |log2(Fold Change)|> 1 and P value < 0.05. Differential expression of the transcripts clustered by Euclidean correlation, with distinct upregulation, unaltered, and downregulation patterns in expression for TGS24 treatment, compared to control condition. The count values are colour coded blue to yellow to red in increasing order. Differentially expressed genes are clustered based on their involvement with RAS, apoptosis, and stemness- and metastasis-related pathways. B RT-qPCR-verification of RNAseq data for differentially expressed genes in RAS pathway upon 50 nM TGS24 treatment in MDAMB-231 spheres for 24 h: MAP4K3, RAS, MAPK12, c-FOS, c-JUN, HSP90. Quantification of the transcripts’ level relative to the control by qPCR analyses. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). GAPDH considered as housekeeping gene. C RT-qPCR-verification of RNAseq data for differentially expressed genes in metastasis and stemness-related pathway upon 50 nM TGS24 treatment in MDAMB-231 spheres for 24 h: ALDHβ1, WNT-1, β-Catenin, SOX2, MMP12, Vimentin, CD-44. Quantification of the transcripts’ level relative to the control by qPCR analyses. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). GAPDH considered as housekeeping gene. D RT-qPCR-verification of RNAseq data for differentially expressed genes in apoptotic pathway upon 50 nM TGS24 treatment in MDAMB-231 spheres for 24 h: NF-κβ, Sp1, GATA3, CASP9, PARP-1. Quantification of the transcripts’ level relative to the control by qPCR analyses. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). GAPDH considered as housekeeping gene. E Correlation of log2(fold-change) values from RNA-Seq and RT-PCR analysis. The R2 value is 0.854. F Interaction network of upregulated and downregulated genes, obtained using STRING database, with the minimum required interaction score of 0.400 and network edges representing evidence of an interaction

Techniques Used: Expressing, Quantitative RT-PCR, Two Tailed Test, RNA Sequencing Assay, Reverse Transcription Polymerase Chain Reaction

Dynamics of two tandem G4-motifs at MAPK12 promoter regulated by spatiotemporal binding of Nucleolin and Sp1 regulate cancer stemness via MAPK12-HSP90-RAS-c-JUN-NANOG axis
Figure Legend Snippet: Dynamics of two tandem G4-motifs at MAPK12 promoter regulated by spatiotemporal binding of Nucleolin and Sp1 regulate cancer stemness via MAPK12-HSP90-RAS-c-JUN-NANOG axis

Techniques Used: Binding Assay


Structured Review

Millipore anti sp1 antibody
Nucleolin and <t>Sp1</t> regulate G4 → duplex transition at MAPK12 promoter. A FRET probe. MAPK12-G4 motifs conjugated with 6-FAM at 5′-end of GQ-1 and internal Cy3 at T22. The complementary strand of G4-forming sequence contains Cy5 at 3′-end. G4-formation gives high FRET signal between 6-FAM and Cy3. Duplex formation gives high FRET signal between 6-FAM and Cy5. B Fluorometric titration of 90 nM FRET probe with increasing gradient of recombinant Nucleolin (0–3 µM) in 50 mM Tris–Cl, pH 7.0, 100 mM KCl at 15 °C at excitation wavelength (λex) of 497 nm. Decline of FRET signal at Cy5 emission ((λem) 650 nm) upon Nucleolin titration suggests disruption of duplex with concomitant increase of Cy3 emission ((λem) 595 nm) indicating G4 evolution. C Fluorometric titration of 90 nM FRET probe with increasing gradient of recombinant Sp1 (0–1 µM) in 50 mM Tris–Cl, pH 7.0, 100 mM KCl at 15 °C at excitation wavelength (λex) of 497 nm. Decline of FRET signal at Cy3 emission ((λem) 595 nm) upon Sp1 titration suggests G4 resolution with concomitant increase of Cy5 emission ((λem) 650 nm) indicating duplex transition. D FRET efficiency calculated following the equation: E = 1 – (IDA/ID); IDA and ID are the emission intensity of donor fluorophore in presence and absence of acceptor fluorophore respectively. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). With increasing Sp1, distance between 6-FAM and Cy5 decreases. Nucleolin titration decreases the distance between 6-FAM and Cy3. The change in distances (r) between donor and acceptor fluorophore calculated by E = R06/(R06 + r6). R0 denotes Forster distance between donor and acceptor. The distance between 6-FAM and Cy3 decreased from 6.5 ± 0.1 nm (R0 = 6.5 nm) in the free DNA to 5.2 ± 0.1 nm in the complex with Nucleolin indicating G4 formation. The distance between 6-FAM and Cy5 decreased from 6.8 ± 0.2 in free DNA to 5.3 ± 0.3 under Sp1-bound conditions indicating duplex formation (E) pGL4.72[hRlucCP] luciferase constructs having the inserts containing MAPK12 promoter sequences having adjacent G4-elements (wild-type). Dual-luciferase assays. Evaluation of MAPK12 promoter activities in MDAMB-231 cells under the following conditions: siRNA knockdown of Nucleolin at 48 h and TGS24 (50 nM) treatment for 24 h, nucleolin-knockdown + TGS24 treatment, nucleolin knockdown + TGS25 treatment, and TGS24 and TGS25 treated alone separately Error bars represent mean ± SE (N = 3). Statistical differences in the luciferase activities compared to that of the control cells use two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). Statistical differences in the luciferase activities in presence of TGS24-treated samples compared to that of cells, treated with recombinant Sp1 and Nucleolin, siRNA-knockdown of Nucleolin use two-tailed Student's t test (###P < 0.001). F RT-PCR analysis. Expression profile of MAPK12 transcripts from P1 promoter in MDAMB-231 cells under following conditions: TGS24 (50 nM) treatment for 24 h and siRNA knockdown of Nucleolin for 48 h, nucleolin-knockdown + TGS24 treatment, nucleolin knockdown + TGS25 treatment, and TGS25 treated alone separately. Quantification of the transcripts’ level relative to the control by qPCR analyses. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). Statistical differences between recombinant Nucleolin-treated and Nucleolin-knockdown cells are determined by two-tailed Student's t test (###P < 0.001). GAPDH considered as housekeeping gene
Anti Sp1 Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/anti sp1 antibody/product/Millipore
Average 86 stars, based on 1 article reviews
Price from $9.99 to $1999.99
anti sp1 antibody - by Bioz Stars, 2024-07
86/100 stars

Images

1) Product Images from "G-quadruplex structural dynamics at MAPK12 promoter dictates transcriptional switch to determine stemness in breast cancer"

Article Title: G-quadruplex structural dynamics at MAPK12 promoter dictates transcriptional switch to determine stemness in breast cancer

Journal: Cellular and Molecular Life Sciences: CMLS

doi: 10.1007/s00018-023-05046-6

Nucleolin and Sp1 regulate G4 → duplex transition at MAPK12 promoter. A FRET probe. MAPK12-G4 motifs conjugated with 6-FAM at 5′-end of GQ-1 and internal Cy3 at T22. The complementary strand of G4-forming sequence contains Cy5 at 3′-end. G4-formation gives high FRET signal between 6-FAM and Cy3. Duplex formation gives high FRET signal between 6-FAM and Cy5. B Fluorometric titration of 90 nM FRET probe with increasing gradient of recombinant Nucleolin (0–3 µM) in 50 mM Tris–Cl, pH 7.0, 100 mM KCl at 15 °C at excitation wavelength (λex) of 497 nm. Decline of FRET signal at Cy5 emission ((λem) 650 nm) upon Nucleolin titration suggests disruption of duplex with concomitant increase of Cy3 emission ((λem) 595 nm) indicating G4 evolution. C Fluorometric titration of 90 nM FRET probe with increasing gradient of recombinant Sp1 (0–1 µM) in 50 mM Tris–Cl, pH 7.0, 100 mM KCl at 15 °C at excitation wavelength (λex) of 497 nm. Decline of FRET signal at Cy3 emission ((λem) 595 nm) upon Sp1 titration suggests G4 resolution with concomitant increase of Cy5 emission ((λem) 650 nm) indicating duplex transition. D FRET efficiency calculated following the equation: E = 1 – (IDA/ID); IDA and ID are the emission intensity of donor fluorophore in presence and absence of acceptor fluorophore respectively. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). With increasing Sp1, distance between 6-FAM and Cy5 decreases. Nucleolin titration decreases the distance between 6-FAM and Cy3. The change in distances (r) between donor and acceptor fluorophore calculated by E = R06/(R06 + r6). R0 denotes Forster distance between donor and acceptor. The distance between 6-FAM and Cy3 decreased from 6.5 ± 0.1 nm (R0 = 6.5 nm) in the free DNA to 5.2 ± 0.1 nm in the complex with Nucleolin indicating G4 formation. The distance between 6-FAM and Cy5 decreased from 6.8 ± 0.2 in free DNA to 5.3 ± 0.3 under Sp1-bound conditions indicating duplex formation (E) pGL4.72[hRlucCP] luciferase constructs having the inserts containing MAPK12 promoter sequences having adjacent G4-elements (wild-type). Dual-luciferase assays. Evaluation of MAPK12 promoter activities in MDAMB-231 cells under the following conditions: siRNA knockdown of Nucleolin at 48 h and TGS24 (50 nM) treatment for 24 h, nucleolin-knockdown + TGS24 treatment, nucleolin knockdown + TGS25 treatment, and TGS24 and TGS25 treated alone separately Error bars represent mean ± SE (N = 3). Statistical differences in the luciferase activities compared to that of the control cells use two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). Statistical differences in the luciferase activities in presence of TGS24-treated samples compared to that of cells, treated with recombinant Sp1 and Nucleolin, siRNA-knockdown of Nucleolin use two-tailed Student's t test (###P < 0.001). F RT-PCR analysis. Expression profile of MAPK12 transcripts from P1 promoter in MDAMB-231 cells under following conditions: TGS24 (50 nM) treatment for 24 h and siRNA knockdown of Nucleolin for 48 h, nucleolin-knockdown + TGS24 treatment, nucleolin knockdown + TGS25 treatment, and TGS25 treated alone separately. Quantification of the transcripts’ level relative to the control by qPCR analyses. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). Statistical differences between recombinant Nucleolin-treated and Nucleolin-knockdown cells are determined by two-tailed Student's t test (###P < 0.001). GAPDH considered as housekeeping gene
Figure Legend Snippet: Nucleolin and Sp1 regulate G4 → duplex transition at MAPK12 promoter. A FRET probe. MAPK12-G4 motifs conjugated with 6-FAM at 5′-end of GQ-1 and internal Cy3 at T22. The complementary strand of G4-forming sequence contains Cy5 at 3′-end. G4-formation gives high FRET signal between 6-FAM and Cy3. Duplex formation gives high FRET signal between 6-FAM and Cy5. B Fluorometric titration of 90 nM FRET probe with increasing gradient of recombinant Nucleolin (0–3 µM) in 50 mM Tris–Cl, pH 7.0, 100 mM KCl at 15 °C at excitation wavelength (λex) of 497 nm. Decline of FRET signal at Cy5 emission ((λem) 650 nm) upon Nucleolin titration suggests disruption of duplex with concomitant increase of Cy3 emission ((λem) 595 nm) indicating G4 evolution. C Fluorometric titration of 90 nM FRET probe with increasing gradient of recombinant Sp1 (0–1 µM) in 50 mM Tris–Cl, pH 7.0, 100 mM KCl at 15 °C at excitation wavelength (λex) of 497 nm. Decline of FRET signal at Cy3 emission ((λem) 595 nm) upon Sp1 titration suggests G4 resolution with concomitant increase of Cy5 emission ((λem) 650 nm) indicating duplex transition. D FRET efficiency calculated following the equation: E = 1 – (IDA/ID); IDA and ID are the emission intensity of donor fluorophore in presence and absence of acceptor fluorophore respectively. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). With increasing Sp1, distance between 6-FAM and Cy5 decreases. Nucleolin titration decreases the distance between 6-FAM and Cy3. The change in distances (r) between donor and acceptor fluorophore calculated by E = R06/(R06 + r6). R0 denotes Forster distance between donor and acceptor. The distance between 6-FAM and Cy3 decreased from 6.5 ± 0.1 nm (R0 = 6.5 nm) in the free DNA to 5.2 ± 0.1 nm in the complex with Nucleolin indicating G4 formation. The distance between 6-FAM and Cy5 decreased from 6.8 ± 0.2 in free DNA to 5.3 ± 0.3 under Sp1-bound conditions indicating duplex formation (E) pGL4.72[hRlucCP] luciferase constructs having the inserts containing MAPK12 promoter sequences having adjacent G4-elements (wild-type). Dual-luciferase assays. Evaluation of MAPK12 promoter activities in MDAMB-231 cells under the following conditions: siRNA knockdown of Nucleolin at 48 h and TGS24 (50 nM) treatment for 24 h, nucleolin-knockdown + TGS24 treatment, nucleolin knockdown + TGS25 treatment, and TGS24 and TGS25 treated alone separately Error bars represent mean ± SE (N = 3). Statistical differences in the luciferase activities compared to that of the control cells use two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). Statistical differences in the luciferase activities in presence of TGS24-treated samples compared to that of cells, treated with recombinant Sp1 and Nucleolin, siRNA-knockdown of Nucleolin use two-tailed Student's t test (###P < 0.001). F RT-PCR analysis. Expression profile of MAPK12 transcripts from P1 promoter in MDAMB-231 cells under following conditions: TGS24 (50 nM) treatment for 24 h and siRNA knockdown of Nucleolin for 48 h, nucleolin-knockdown + TGS24 treatment, nucleolin knockdown + TGS25 treatment, and TGS25 treated alone separately. Quantification of the transcripts’ level relative to the control by qPCR analyses. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). Statistical differences between recombinant Nucleolin-treated and Nucleolin-knockdown cells are determined by two-tailed Student's t test (###P < 0.001). GAPDH considered as housekeeping gene

Techniques Used: Sequencing, Titration, Recombinant, Disruption, Two Tailed Test, Luciferase, Construct, Reverse Transcription Polymerase Chain Reaction, Expressing

Downstream effects of TGS24-treatment and its selective interaction with MAPK12-G4 motifs. A RNAseq analyses of TGS24 treatment in MDAMB-231 spheres expressing high MAPK12. Heatmap of biological replicates of differentially expressed genes upon 24 h treatment of 50 nM TGS24 treatment in MDAMB-231 spheres using the log10(count) values for each replicate. The screening threshold for the differentially expressed genes is set to: |log2(Fold Change)|> 1 and P value < 0.05. Differential expression of the transcripts clustered by Euclidean correlation, with distinct upregulation, unaltered, and downregulation patterns in expression for TGS24 treatment, compared to control condition. The count values are colour coded blue to yellow to red in increasing order. Differentially expressed genes are clustered based on their involvement with RAS, apoptosis, and stemness- and metastasis-related pathways. B RT-qPCR-verification of RNAseq data for differentially expressed genes in RAS pathway upon 50 nM TGS24 treatment in MDAMB-231 spheres for 24 h: MAP4K3, RAS, MAPK12, c-FOS, c-JUN, HSP90. Quantification of the transcripts’ level relative to the control by qPCR analyses. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). GAPDH considered as housekeeping gene. C RT-qPCR-verification of RNAseq data for differentially expressed genes in metastasis and stemness-related pathway upon 50 nM TGS24 treatment in MDAMB-231 spheres for 24 h: ALDHβ1, WNT-1, β-Catenin, SOX2, MMP12, Vimentin, CD-44. Quantification of the transcripts’ level relative to the control by qPCR analyses. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). GAPDH considered as housekeeping gene. D RT-qPCR-verification of RNAseq data for differentially expressed genes in apoptotic pathway upon 50 nM TGS24 treatment in MDAMB-231 spheres for 24 h: NF-κβ, Sp1, GATA3, CASP9, PARP-1. Quantification of the transcripts’ level relative to the control by qPCR analyses. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). GAPDH considered as housekeeping gene. E Correlation of log2(fold-change) values from RNA-Seq and RT-PCR analysis. The R2 value is 0.854. F Interaction network of upregulated and downregulated genes, obtained using STRING database, with the minimum required interaction score of 0.400 and network edges representing evidence of an interaction
Figure Legend Snippet: Downstream effects of TGS24-treatment and its selective interaction with MAPK12-G4 motifs. A RNAseq analyses of TGS24 treatment in MDAMB-231 spheres expressing high MAPK12. Heatmap of biological replicates of differentially expressed genes upon 24 h treatment of 50 nM TGS24 treatment in MDAMB-231 spheres using the log10(count) values for each replicate. The screening threshold for the differentially expressed genes is set to: |log2(Fold Change)|> 1 and P value < 0.05. Differential expression of the transcripts clustered by Euclidean correlation, with distinct upregulation, unaltered, and downregulation patterns in expression for TGS24 treatment, compared to control condition. The count values are colour coded blue to yellow to red in increasing order. Differentially expressed genes are clustered based on their involvement with RAS, apoptosis, and stemness- and metastasis-related pathways. B RT-qPCR-verification of RNAseq data for differentially expressed genes in RAS pathway upon 50 nM TGS24 treatment in MDAMB-231 spheres for 24 h: MAP4K3, RAS, MAPK12, c-FOS, c-JUN, HSP90. Quantification of the transcripts’ level relative to the control by qPCR analyses. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). GAPDH considered as housekeeping gene. C RT-qPCR-verification of RNAseq data for differentially expressed genes in metastasis and stemness-related pathway upon 50 nM TGS24 treatment in MDAMB-231 spheres for 24 h: ALDHβ1, WNT-1, β-Catenin, SOX2, MMP12, Vimentin, CD-44. Quantification of the transcripts’ level relative to the control by qPCR analyses. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). GAPDH considered as housekeeping gene. D RT-qPCR-verification of RNAseq data for differentially expressed genes in apoptotic pathway upon 50 nM TGS24 treatment in MDAMB-231 spheres for 24 h: NF-κβ, Sp1, GATA3, CASP9, PARP-1. Quantification of the transcripts’ level relative to the control by qPCR analyses. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). GAPDH considered as housekeeping gene. E Correlation of log2(fold-change) values from RNA-Seq and RT-PCR analysis. The R2 value is 0.854. F Interaction network of upregulated and downregulated genes, obtained using STRING database, with the minimum required interaction score of 0.400 and network edges representing evidence of an interaction

Techniques Used: Expressing, Quantitative RT-PCR, Two Tailed Test, RNA Sequencing Assay, Reverse Transcription Polymerase Chain Reaction

Dynamics of two tandem G4-motifs at MAPK12 promoter regulated by spatiotemporal binding of Nucleolin and Sp1 regulate cancer stemness via MAPK12-HSP90-RAS-c-JUN-NANOG axis
Figure Legend Snippet: Dynamics of two tandem G4-motifs at MAPK12 promoter regulated by spatiotemporal binding of Nucleolin and Sp1 regulate cancer stemness via MAPK12-HSP90-RAS-c-JUN-NANOG axis

Techniques Used: Binding Assay


Structured Review

Millipore sp1 antibody
Nucleolin and <t>Sp1</t> regulate G4 → duplex transition at MAPK12 promoter. A FRET probe. MAPK12-G4 motifs conjugated with 6-FAM at 5′-end of GQ-1 and internal Cy3 at T22. The complementary strand of G4-forming sequence contains Cy5 at 3′-end. G4-formation gives high FRET signal between 6-FAM and Cy3. Duplex formation gives high FRET signal between 6-FAM and Cy5. B Fluorometric titration of 90 nM FRET probe with increasing gradient of recombinant Nucleolin (0–3 µM) in 50 mM Tris–Cl, pH 7.0, 100 mM KCl at 15 °C at excitation wavelength (λex) of 497 nm. Decline of FRET signal at Cy5 emission ((λem) 650 nm) upon Nucleolin titration suggests disruption of duplex with concomitant increase of Cy3 emission ((λem) 595 nm) indicating G4 evolution. C Fluorometric titration of 90 nM FRET probe with increasing gradient of recombinant Sp1 (0–1 µM) in 50 mM Tris–Cl, pH 7.0, 100 mM KCl at 15 °C at excitation wavelength (λex) of 497 nm. Decline of FRET signal at Cy3 emission ((λem) 595 nm) upon Sp1 titration suggests G4 resolution with concomitant increase of Cy5 emission ((λem) 650 nm) indicating duplex transition. D FRET efficiency calculated following the equation: E = 1 – (IDA/ID); IDA and ID are the emission intensity of donor fluorophore in presence and absence of acceptor fluorophore respectively. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). With increasing Sp1, distance between 6-FAM and Cy5 decreases. Nucleolin titration decreases the distance between 6-FAM and Cy3. The change in distances (r) between donor and acceptor fluorophore calculated by E = R06/(R06 + r6). R0 denotes Forster distance between donor and acceptor. The distance between 6-FAM and Cy3 decreased from 6.5 ± 0.1 nm (R0 = 6.5 nm) in the free DNA to 5.2 ± 0.1 nm in the complex with Nucleolin indicating G4 formation. The distance between 6-FAM and Cy5 decreased from 6.8 ± 0.2 in free DNA to 5.3 ± 0.3 under Sp1-bound conditions indicating duplex formation (E) pGL4.72[hRlucCP] luciferase constructs having the inserts containing MAPK12 promoter sequences having adjacent G4-elements (wild-type). Dual-luciferase assays. Evaluation of MAPK12 promoter activities in MDAMB-231 cells under the following conditions: siRNA knockdown of Nucleolin at 48 h and TGS24 (50 nM) treatment for 24 h, nucleolin-knockdown + TGS24 treatment, nucleolin knockdown + TGS25 treatment, and TGS24 and TGS25 treated alone separately Error bars represent mean ± SE (N = 3). Statistical differences in the luciferase activities compared to that of the control cells use two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). Statistical differences in the luciferase activities in presence of TGS24-treated samples compared to that of cells, treated with recombinant Sp1 and Nucleolin, siRNA-knockdown of Nucleolin use two-tailed Student's t test (###P < 0.001). F RT-PCR analysis. Expression profile of MAPK12 transcripts from P1 promoter in MDAMB-231 cells under following conditions: TGS24 (50 nM) treatment for 24 h and siRNA knockdown of Nucleolin for 48 h, nucleolin-knockdown + TGS24 treatment, nucleolin knockdown + TGS25 treatment, and TGS25 treated alone separately. Quantification of the transcripts’ level relative to the control by qPCR analyses. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). Statistical differences between recombinant Nucleolin-treated and Nucleolin-knockdown cells are determined by two-tailed Student's t test (###P < 0.001). GAPDH considered as housekeeping gene
Sp1 Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/sp1 antibody/product/Millipore
Average 86 stars, based on 1 article reviews
Price from $9.99 to $1999.99
sp1 antibody - by Bioz Stars, 2024-07
86/100 stars

Images

1) Product Images from "G-quadruplex structural dynamics at MAPK12 promoter dictates transcriptional switch to determine stemness in breast cancer"

Article Title: G-quadruplex structural dynamics at MAPK12 promoter dictates transcriptional switch to determine stemness in breast cancer

Journal: Cellular and Molecular Life Sciences: CMLS

doi: 10.1007/s00018-023-05046-6

Nucleolin and Sp1 regulate G4 → duplex transition at MAPK12 promoter. A FRET probe. MAPK12-G4 motifs conjugated with 6-FAM at 5′-end of GQ-1 and internal Cy3 at T22. The complementary strand of G4-forming sequence contains Cy5 at 3′-end. G4-formation gives high FRET signal between 6-FAM and Cy3. Duplex formation gives high FRET signal between 6-FAM and Cy5. B Fluorometric titration of 90 nM FRET probe with increasing gradient of recombinant Nucleolin (0–3 µM) in 50 mM Tris–Cl, pH 7.0, 100 mM KCl at 15 °C at excitation wavelength (λex) of 497 nm. Decline of FRET signal at Cy5 emission ((λem) 650 nm) upon Nucleolin titration suggests disruption of duplex with concomitant increase of Cy3 emission ((λem) 595 nm) indicating G4 evolution. C Fluorometric titration of 90 nM FRET probe with increasing gradient of recombinant Sp1 (0–1 µM) in 50 mM Tris–Cl, pH 7.0, 100 mM KCl at 15 °C at excitation wavelength (λex) of 497 nm. Decline of FRET signal at Cy3 emission ((λem) 595 nm) upon Sp1 titration suggests G4 resolution with concomitant increase of Cy5 emission ((λem) 650 nm) indicating duplex transition. D FRET efficiency calculated following the equation: E = 1 – (IDA/ID); IDA and ID are the emission intensity of donor fluorophore in presence and absence of acceptor fluorophore respectively. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). With increasing Sp1, distance between 6-FAM and Cy5 decreases. Nucleolin titration decreases the distance between 6-FAM and Cy3. The change in distances (r) between donor and acceptor fluorophore calculated by E = R06/(R06 + r6). R0 denotes Forster distance between donor and acceptor. The distance between 6-FAM and Cy3 decreased from 6.5 ± 0.1 nm (R0 = 6.5 nm) in the free DNA to 5.2 ± 0.1 nm in the complex with Nucleolin indicating G4 formation. The distance between 6-FAM and Cy5 decreased from 6.8 ± 0.2 in free DNA to 5.3 ± 0.3 under Sp1-bound conditions indicating duplex formation (E) pGL4.72[hRlucCP] luciferase constructs having the inserts containing MAPK12 promoter sequences having adjacent G4-elements (wild-type). Dual-luciferase assays. Evaluation of MAPK12 promoter activities in MDAMB-231 cells under the following conditions: siRNA knockdown of Nucleolin at 48 h and TGS24 (50 nM) treatment for 24 h, nucleolin-knockdown + TGS24 treatment, nucleolin knockdown + TGS25 treatment, and TGS24 and TGS25 treated alone separately Error bars represent mean ± SE (N = 3). Statistical differences in the luciferase activities compared to that of the control cells use two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). Statistical differences in the luciferase activities in presence of TGS24-treated samples compared to that of cells, treated with recombinant Sp1 and Nucleolin, siRNA-knockdown of Nucleolin use two-tailed Student's t test (###P < 0.001). F RT-PCR analysis. Expression profile of MAPK12 transcripts from P1 promoter in MDAMB-231 cells under following conditions: TGS24 (50 nM) treatment for 24 h and siRNA knockdown of Nucleolin for 48 h, nucleolin-knockdown + TGS24 treatment, nucleolin knockdown + TGS25 treatment, and TGS25 treated alone separately. Quantification of the transcripts’ level relative to the control by qPCR analyses. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). Statistical differences between recombinant Nucleolin-treated and Nucleolin-knockdown cells are determined by two-tailed Student's t test (###P < 0.001). GAPDH considered as housekeeping gene
Figure Legend Snippet: Nucleolin and Sp1 regulate G4 → duplex transition at MAPK12 promoter. A FRET probe. MAPK12-G4 motifs conjugated with 6-FAM at 5′-end of GQ-1 and internal Cy3 at T22. The complementary strand of G4-forming sequence contains Cy5 at 3′-end. G4-formation gives high FRET signal between 6-FAM and Cy3. Duplex formation gives high FRET signal between 6-FAM and Cy5. B Fluorometric titration of 90 nM FRET probe with increasing gradient of recombinant Nucleolin (0–3 µM) in 50 mM Tris–Cl, pH 7.0, 100 mM KCl at 15 °C at excitation wavelength (λex) of 497 nm. Decline of FRET signal at Cy5 emission ((λem) 650 nm) upon Nucleolin titration suggests disruption of duplex with concomitant increase of Cy3 emission ((λem) 595 nm) indicating G4 evolution. C Fluorometric titration of 90 nM FRET probe with increasing gradient of recombinant Sp1 (0–1 µM) in 50 mM Tris–Cl, pH 7.0, 100 mM KCl at 15 °C at excitation wavelength (λex) of 497 nm. Decline of FRET signal at Cy3 emission ((λem) 595 nm) upon Sp1 titration suggests G4 resolution with concomitant increase of Cy5 emission ((λem) 650 nm) indicating duplex transition. D FRET efficiency calculated following the equation: E = 1 – (IDA/ID); IDA and ID are the emission intensity of donor fluorophore in presence and absence of acceptor fluorophore respectively. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). With increasing Sp1, distance between 6-FAM and Cy5 decreases. Nucleolin titration decreases the distance between 6-FAM and Cy3. The change in distances (r) between donor and acceptor fluorophore calculated by E = R06/(R06 + r6). R0 denotes Forster distance between donor and acceptor. The distance between 6-FAM and Cy3 decreased from 6.5 ± 0.1 nm (R0 = 6.5 nm) in the free DNA to 5.2 ± 0.1 nm in the complex with Nucleolin indicating G4 formation. The distance between 6-FAM and Cy5 decreased from 6.8 ± 0.2 in free DNA to 5.3 ± 0.3 under Sp1-bound conditions indicating duplex formation (E) pGL4.72[hRlucCP] luciferase constructs having the inserts containing MAPK12 promoter sequences having adjacent G4-elements (wild-type). Dual-luciferase assays. Evaluation of MAPK12 promoter activities in MDAMB-231 cells under the following conditions: siRNA knockdown of Nucleolin at 48 h and TGS24 (50 nM) treatment for 24 h, nucleolin-knockdown + TGS24 treatment, nucleolin knockdown + TGS25 treatment, and TGS24 and TGS25 treated alone separately Error bars represent mean ± SE (N = 3). Statistical differences in the luciferase activities compared to that of the control cells use two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). Statistical differences in the luciferase activities in presence of TGS24-treated samples compared to that of cells, treated with recombinant Sp1 and Nucleolin, siRNA-knockdown of Nucleolin use two-tailed Student's t test (###P < 0.001). F RT-PCR analysis. Expression profile of MAPK12 transcripts from P1 promoter in MDAMB-231 cells under following conditions: TGS24 (50 nM) treatment for 24 h and siRNA knockdown of Nucleolin for 48 h, nucleolin-knockdown + TGS24 treatment, nucleolin knockdown + TGS25 treatment, and TGS25 treated alone separately. Quantification of the transcripts’ level relative to the control by qPCR analyses. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). Statistical differences between recombinant Nucleolin-treated and Nucleolin-knockdown cells are determined by two-tailed Student's t test (###P < 0.001). GAPDH considered as housekeeping gene

Techniques Used: Sequencing, Titration, Recombinant, Disruption, Two Tailed Test, Luciferase, Construct, Reverse Transcription Polymerase Chain Reaction, Expressing

Downstream effects of TGS24-treatment and its selective interaction with MAPK12-G4 motifs. A RNAseq analyses of TGS24 treatment in MDAMB-231 spheres expressing high MAPK12. Heatmap of biological replicates of differentially expressed genes upon 24 h treatment of 50 nM TGS24 treatment in MDAMB-231 spheres using the log10(count) values for each replicate. The screening threshold for the differentially expressed genes is set to: |log2(Fold Change)|> 1 and P value < 0.05. Differential expression of the transcripts clustered by Euclidean correlation, with distinct upregulation, unaltered, and downregulation patterns in expression for TGS24 treatment, compared to control condition. The count values are colour coded blue to yellow to red in increasing order. Differentially expressed genes are clustered based on their involvement with RAS, apoptosis, and stemness- and metastasis-related pathways. B RT-qPCR-verification of RNAseq data for differentially expressed genes in RAS pathway upon 50 nM TGS24 treatment in MDAMB-231 spheres for 24 h: MAP4K3, RAS, MAPK12, c-FOS, c-JUN, HSP90. Quantification of the transcripts’ level relative to the control by qPCR analyses. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). GAPDH considered as housekeeping gene. C RT-qPCR-verification of RNAseq data for differentially expressed genes in metastasis and stemness-related pathway upon 50 nM TGS24 treatment in MDAMB-231 spheres for 24 h: ALDHβ1, WNT-1, β-Catenin, SOX2, MMP12, Vimentin, CD-44. Quantification of the transcripts’ level relative to the control by qPCR analyses. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). GAPDH considered as housekeeping gene. D RT-qPCR-verification of RNAseq data for differentially expressed genes in apoptotic pathway upon 50 nM TGS24 treatment in MDAMB-231 spheres for 24 h: NF-κβ, Sp1, GATA3, CASP9, PARP-1. Quantification of the transcripts’ level relative to the control by qPCR analyses. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). GAPDH considered as housekeeping gene. E Correlation of log2(fold-change) values from RNA-Seq and RT-PCR analysis. The R2 value is 0.854. F Interaction network of upregulated and downregulated genes, obtained using STRING database, with the minimum required interaction score of 0.400 and network edges representing evidence of an interaction
Figure Legend Snippet: Downstream effects of TGS24-treatment and its selective interaction with MAPK12-G4 motifs. A RNAseq analyses of TGS24 treatment in MDAMB-231 spheres expressing high MAPK12. Heatmap of biological replicates of differentially expressed genes upon 24 h treatment of 50 nM TGS24 treatment in MDAMB-231 spheres using the log10(count) values for each replicate. The screening threshold for the differentially expressed genes is set to: |log2(Fold Change)|> 1 and P value < 0.05. Differential expression of the transcripts clustered by Euclidean correlation, with distinct upregulation, unaltered, and downregulation patterns in expression for TGS24 treatment, compared to control condition. The count values are colour coded blue to yellow to red in increasing order. Differentially expressed genes are clustered based on their involvement with RAS, apoptosis, and stemness- and metastasis-related pathways. B RT-qPCR-verification of RNAseq data for differentially expressed genes in RAS pathway upon 50 nM TGS24 treatment in MDAMB-231 spheres for 24 h: MAP4K3, RAS, MAPK12, c-FOS, c-JUN, HSP90. Quantification of the transcripts’ level relative to the control by qPCR analyses. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). GAPDH considered as housekeeping gene. C RT-qPCR-verification of RNAseq data for differentially expressed genes in metastasis and stemness-related pathway upon 50 nM TGS24 treatment in MDAMB-231 spheres for 24 h: ALDHβ1, WNT-1, β-Catenin, SOX2, MMP12, Vimentin, CD-44. Quantification of the transcripts’ level relative to the control by qPCR analyses. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). GAPDH considered as housekeeping gene. D RT-qPCR-verification of RNAseq data for differentially expressed genes in apoptotic pathway upon 50 nM TGS24 treatment in MDAMB-231 spheres for 24 h: NF-κβ, Sp1, GATA3, CASP9, PARP-1. Quantification of the transcripts’ level relative to the control by qPCR analyses. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). GAPDH considered as housekeeping gene. E Correlation of log2(fold-change) values from RNA-Seq and RT-PCR analysis. The R2 value is 0.854. F Interaction network of upregulated and downregulated genes, obtained using STRING database, with the minimum required interaction score of 0.400 and network edges representing evidence of an interaction

Techniques Used: Expressing, Quantitative RT-PCR, Two Tailed Test, RNA Sequencing Assay, Reverse Transcription Polymerase Chain Reaction

Dynamics of two tandem G4-motifs at MAPK12 promoter regulated by spatiotemporal binding of Nucleolin and Sp1 regulate cancer stemness via MAPK12-HSP90-RAS-c-JUN-NANOG axis
Figure Legend Snippet: Dynamics of two tandem G4-motifs at MAPK12 promoter regulated by spatiotemporal binding of Nucleolin and Sp1 regulate cancer stemness via MAPK12-HSP90-RAS-c-JUN-NANOG axis

Techniques Used: Binding Assay

pcdna3 1 sp1 overexpression vector  (Thermo Fisher)


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    Thermo Fisher pcdna3 1 sp1 overexpression vector
    Primer sequences used for RT-qPCR.
    Pcdna3 1 Sp1 Overexpression Vector, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "SP1‑mediated ADAMTS5 transcription promotes IL‑1β‑induced chondrocyte injury via Wnt/β‑catenin pathway in osteoarthritis"

    Article Title: SP1‑mediated ADAMTS5 transcription promotes IL‑1β‑induced chondrocyte injury via Wnt/β‑catenin pathway in osteoarthritis

    Journal: Molecular Medicine Reports

    doi: 10.3892/mmr.2024.13273

    Primer sequences used for RT-qPCR.
    Figure Legend Snippet: Primer sequences used for RT-qPCR.

    Techniques Used: Sequencing

    SP1 activated the transcription of ADAMTS5. (A) Jaspar software identified the binding sites of SP1 on the promoter region of ADAMTS5. (B) ChIP was used to confirm the binding ability of SP1 with the ADAMTS5 promoter. (C) SP1 protein expression was detected by western blotting in 293T cells transfected with pcDNA/SP1. (D) Dual-luciferase reporter assay was used to verify the binding of SP1 with the WT of the ADAMTS5 promoter. (E) Transfection efficiency of si-SP1 was confirmed by western blotting. (F) ADAMTS5 protein expression was examined by western blotting in CHON-001 cells transfected with si-NC/si-SP1. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. SP1, specific protein 1; WT, wild-type; MUT, mutant.
    Figure Legend Snippet: SP1 activated the transcription of ADAMTS5. (A) Jaspar software identified the binding sites of SP1 on the promoter region of ADAMTS5. (B) ChIP was used to confirm the binding ability of SP1 with the ADAMTS5 promoter. (C) SP1 protein expression was detected by western blotting in 293T cells transfected with pcDNA/SP1. (D) Dual-luciferase reporter assay was used to verify the binding of SP1 with the WT of the ADAMTS5 promoter. (E) Transfection efficiency of si-SP1 was confirmed by western blotting. (F) ADAMTS5 protein expression was examined by western blotting in CHON-001 cells transfected with si-NC/si-SP1. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. SP1, specific protein 1; WT, wild-type; MUT, mutant.

    Techniques Used: Software, Binding Assay, Expressing, Western Blot, Transfection, Luciferase, Reporter Assay, Mutagenesis

    Effects of si-SP1 and ADAMTS5 on IL-1β-induced chondrocyte injury. CHON-001 cells were transfected with si-NC/si-SP1/pcDNA/ADAMTS5 followed by treatment with IL-1β. (A) ADAMTS5 protein expression was assessed using western blotting. Cell proliferation and apoptosis were determined by (B) MTT assay, (C) EdU assay and (D) flow cytometry. (E) Protein expression of aggrecan and collagen II was evaluated using western blotting. (F) Concentrations of IL-6 and TNF-α were assessed by ELISA. Corresponding kits were used to test (G) SOD activity and (H) MDA levels. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. MDA, malondialdehyde; SOD, superoxide dismutase; NC, negative control; SP1, specific protein 1.
    Figure Legend Snippet: Effects of si-SP1 and ADAMTS5 on IL-1β-induced chondrocyte injury. CHON-001 cells were transfected with si-NC/si-SP1/pcDNA/ADAMTS5 followed by treatment with IL-1β. (A) ADAMTS5 protein expression was assessed using western blotting. Cell proliferation and apoptosis were determined by (B) MTT assay, (C) EdU assay and (D) flow cytometry. (E) Protein expression of aggrecan and collagen II was evaluated using western blotting. (F) Concentrations of IL-6 and TNF-α were assessed by ELISA. Corresponding kits were used to test (G) SOD activity and (H) MDA levels. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. MDA, malondialdehyde; SOD, superoxide dismutase; NC, negative control; SP1, specific protein 1.

    Techniques Used: Transfection, Expressing, Western Blot, MTT Assay, EdU Assay, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Activity Assay, Negative Control

    Effects of the SP1/ADAMTS5 axis on the activity of the Wnt/β-catenin pathway. (A) western blotting was used to measure the protein expression of β-catenin and Wnt3a in IL-1β-induced CHON-001 cells transfected with si-NC/si-ADAMTS5. (B) Protein expression of β-catenin and Wnt3a was detected by western blotting in IL-1β-induced CHON-001 cells transfected with si-NC/si-SP1/pcDNA/ADAMTS5. **P<0.01, ***P<0.001 and ****P<0.0001. NC, negative control; SP1, specific protein 1.
    Figure Legend Snippet: Effects of the SP1/ADAMTS5 axis on the activity of the Wnt/β-catenin pathway. (A) western blotting was used to measure the protein expression of β-catenin and Wnt3a in IL-1β-induced CHON-001 cells transfected with si-NC/si-ADAMTS5. (B) Protein expression of β-catenin and Wnt3a was detected by western blotting in IL-1β-induced CHON-001 cells transfected with si-NC/si-SP1/pcDNA/ADAMTS5. **P<0.01, ***P<0.001 and ****P<0.0001. NC, negative control; SP1, specific protein 1.

    Techniques Used: Activity Assay, Western Blot, Expressing, Transfection, Negative Control

    Summary diagram of the present study. In IL-1β-induced chondrocytes, the SP1/ADAMTS5/Wnt/β-catenin pathway suppressed proliferation and accelerated apoptosis, ECM degradation, inflammation and oxidative stress to promote OA progression. OA, osteoarthritis; ECM, extracellular matrix; SP1, specific protein 1; MDA, malondialdehyde; SOD, superoxide dismutase.
    Figure Legend Snippet: Summary diagram of the present study. In IL-1β-induced chondrocytes, the SP1/ADAMTS5/Wnt/β-catenin pathway suppressed proliferation and accelerated apoptosis, ECM degradation, inflammation and oxidative stress to promote OA progression. OA, osteoarthritis; ECM, extracellular matrix; SP1, specific protein 1; MDA, malondialdehyde; SOD, superoxide dismutase.

    Techniques Used:

    pcdna3 1 sp1 overexpression vector  (ATCC)


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    Structured Review

    ATCC pcdna3 1 sp1 overexpression vector
    Primer sequences used for RT-qPCR.
    Pcdna3 1 Sp1 Overexpression Vector, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcdna3 1 sp1 overexpression vector/product/ATCC
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pcdna3 1 sp1 overexpression vector - by Bioz Stars, 2024-07
    86/100 stars

    Images

    1) Product Images from "SP1‑mediated ADAMTS5 transcription promotes IL‑1β‑induced chondrocyte injury via Wnt/β‑catenin pathway in osteoarthritis"

    Article Title: SP1‑mediated ADAMTS5 transcription promotes IL‑1β‑induced chondrocyte injury via Wnt/β‑catenin pathway in osteoarthritis

    Journal: Molecular Medicine Reports

    doi: 10.3892/mmr.2024.13273

    Primer sequences used for RT-qPCR.
    Figure Legend Snippet: Primer sequences used for RT-qPCR.

    Techniques Used: Sequencing

    SP1 activated the transcription of ADAMTS5. (A) Jaspar software identified the binding sites of SP1 on the promoter region of ADAMTS5. (B) ChIP was used to confirm the binding ability of SP1 with the ADAMTS5 promoter. (C) SP1 protein expression was detected by western blotting in 293T cells transfected with pcDNA/SP1. (D) Dual-luciferase reporter assay was used to verify the binding of SP1 with the WT of the ADAMTS5 promoter. (E) Transfection efficiency of si-SP1 was confirmed by western blotting. (F) ADAMTS5 protein expression was examined by western blotting in CHON-001 cells transfected with si-NC/si-SP1. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. SP1, specific protein 1; WT, wild-type; MUT, mutant.
    Figure Legend Snippet: SP1 activated the transcription of ADAMTS5. (A) Jaspar software identified the binding sites of SP1 on the promoter region of ADAMTS5. (B) ChIP was used to confirm the binding ability of SP1 with the ADAMTS5 promoter. (C) SP1 protein expression was detected by western blotting in 293T cells transfected with pcDNA/SP1. (D) Dual-luciferase reporter assay was used to verify the binding of SP1 with the WT of the ADAMTS5 promoter. (E) Transfection efficiency of si-SP1 was confirmed by western blotting. (F) ADAMTS5 protein expression was examined by western blotting in CHON-001 cells transfected with si-NC/si-SP1. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. SP1, specific protein 1; WT, wild-type; MUT, mutant.

    Techniques Used: Software, Binding Assay, Expressing, Western Blot, Transfection, Luciferase, Reporter Assay, Mutagenesis

    Effects of si-SP1 and ADAMTS5 on IL-1β-induced chondrocyte injury. CHON-001 cells were transfected with si-NC/si-SP1/pcDNA/ADAMTS5 followed by treatment with IL-1β. (A) ADAMTS5 protein expression was assessed using western blotting. Cell proliferation and apoptosis were determined by (B) MTT assay, (C) EdU assay and (D) flow cytometry. (E) Protein expression of aggrecan and collagen II was evaluated using western blotting. (F) Concentrations of IL-6 and TNF-α were assessed by ELISA. Corresponding kits were used to test (G) SOD activity and (H) MDA levels. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. MDA, malondialdehyde; SOD, superoxide dismutase; NC, negative control; SP1, specific protein 1.
    Figure Legend Snippet: Effects of si-SP1 and ADAMTS5 on IL-1β-induced chondrocyte injury. CHON-001 cells were transfected with si-NC/si-SP1/pcDNA/ADAMTS5 followed by treatment with IL-1β. (A) ADAMTS5 protein expression was assessed using western blotting. Cell proliferation and apoptosis were determined by (B) MTT assay, (C) EdU assay and (D) flow cytometry. (E) Protein expression of aggrecan and collagen II was evaluated using western blotting. (F) Concentrations of IL-6 and TNF-α were assessed by ELISA. Corresponding kits were used to test (G) SOD activity and (H) MDA levels. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. MDA, malondialdehyde; SOD, superoxide dismutase; NC, negative control; SP1, specific protein 1.

    Techniques Used: Transfection, Expressing, Western Blot, MTT Assay, EdU Assay, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Activity Assay, Negative Control

    Effects of the SP1/ADAMTS5 axis on the activity of the Wnt/β-catenin pathway. (A) western blotting was used to measure the protein expression of β-catenin and Wnt3a in IL-1β-induced CHON-001 cells transfected with si-NC/si-ADAMTS5. (B) Protein expression of β-catenin and Wnt3a was detected by western blotting in IL-1β-induced CHON-001 cells transfected with si-NC/si-SP1/pcDNA/ADAMTS5. **P<0.01, ***P<0.001 and ****P<0.0001. NC, negative control; SP1, specific protein 1.
    Figure Legend Snippet: Effects of the SP1/ADAMTS5 axis on the activity of the Wnt/β-catenin pathway. (A) western blotting was used to measure the protein expression of β-catenin and Wnt3a in IL-1β-induced CHON-001 cells transfected with si-NC/si-ADAMTS5. (B) Protein expression of β-catenin and Wnt3a was detected by western blotting in IL-1β-induced CHON-001 cells transfected with si-NC/si-SP1/pcDNA/ADAMTS5. **P<0.01, ***P<0.001 and ****P<0.0001. NC, negative control; SP1, specific protein 1.

    Techniques Used: Activity Assay, Western Blot, Expressing, Transfection, Negative Control

    Summary diagram of the present study. In IL-1β-induced chondrocytes, the SP1/ADAMTS5/Wnt/β-catenin pathway suppressed proliferation and accelerated apoptosis, ECM degradation, inflammation and oxidative stress to promote OA progression. OA, osteoarthritis; ECM, extracellular matrix; SP1, specific protein 1; MDA, malondialdehyde; SOD, superoxide dismutase.
    Figure Legend Snippet: Summary diagram of the present study. In IL-1β-induced chondrocytes, the SP1/ADAMTS5/Wnt/β-catenin pathway suppressed proliferation and accelerated apoptosis, ECM degradation, inflammation and oxidative stress to promote OA progression. OA, osteoarthritis; ECM, extracellular matrix; SP1, specific protein 1; MDA, malondialdehyde; SOD, superoxide dismutase.

    Techniques Used:

    pcdna3 1 sp1 overexpression vector  (Thermo Fisher)


    Bioz Verified Symbol Thermo Fisher is a verified supplier
    Bioz Manufacturer Symbol Thermo Fisher manufactures this product  
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    Structured Review

    Thermo Fisher pcdna3 1 sp1 overexpression vector
    Primer sequences used for RT-qPCR.
    Pcdna3 1 Sp1 Overexpression Vector, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcdna3 1 sp1 overexpression vector/product/Thermo Fisher
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pcdna3 1 sp1 overexpression vector - by Bioz Stars, 2024-07
    86/100 stars

    Images

    1) Product Images from "SP1‑mediated ADAMTS5 transcription promotes IL‑1β‑induced chondrocyte injury via Wnt/β‑catenin pathway in osteoarthritis"

    Article Title: SP1‑mediated ADAMTS5 transcription promotes IL‑1β‑induced chondrocyte injury via Wnt/β‑catenin pathway in osteoarthritis

    Journal: Molecular Medicine Reports

    doi: 10.3892/mmr.2024.13273

    Primer sequences used for RT-qPCR.
    Figure Legend Snippet: Primer sequences used for RT-qPCR.

    Techniques Used: Sequencing

    SP1 activated the transcription of ADAMTS5. (A) Jaspar software identified the binding sites of SP1 on the promoter region of ADAMTS5. (B) ChIP was used to confirm the binding ability of SP1 with the ADAMTS5 promoter. (C) SP1 protein expression was detected by western blotting in 293T cells transfected with pcDNA/SP1. (D) Dual-luciferase reporter assay was used to verify the binding of SP1 with the WT of the ADAMTS5 promoter. (E) Transfection efficiency of si-SP1 was confirmed by western blotting. (F) ADAMTS5 protein expression was examined by western blotting in CHON-001 cells transfected with si-NC/si-SP1. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. SP1, specific protein 1; WT, wild-type; MUT, mutant.
    Figure Legend Snippet: SP1 activated the transcription of ADAMTS5. (A) Jaspar software identified the binding sites of SP1 on the promoter region of ADAMTS5. (B) ChIP was used to confirm the binding ability of SP1 with the ADAMTS5 promoter. (C) SP1 protein expression was detected by western blotting in 293T cells transfected with pcDNA/SP1. (D) Dual-luciferase reporter assay was used to verify the binding of SP1 with the WT of the ADAMTS5 promoter. (E) Transfection efficiency of si-SP1 was confirmed by western blotting. (F) ADAMTS5 protein expression was examined by western blotting in CHON-001 cells transfected with si-NC/si-SP1. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. SP1, specific protein 1; WT, wild-type; MUT, mutant.

    Techniques Used: Software, Binding Assay, Expressing, Western Blot, Transfection, Luciferase, Reporter Assay, Mutagenesis

    Effects of si-SP1 and ADAMTS5 on IL-1β-induced chondrocyte injury. CHON-001 cells were transfected with si-NC/si-SP1/pcDNA/ADAMTS5 followed by treatment with IL-1β. (A) ADAMTS5 protein expression was assessed using western blotting. Cell proliferation and apoptosis were determined by (B) MTT assay, (C) EdU assay and (D) flow cytometry. (E) Protein expression of aggrecan and collagen II was evaluated using western blotting. (F) Concentrations of IL-6 and TNF-α were assessed by ELISA. Corresponding kits were used to test (G) SOD activity and (H) MDA levels. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. MDA, malondialdehyde; SOD, superoxide dismutase; NC, negative control; SP1, specific protein 1.
    Figure Legend Snippet: Effects of si-SP1 and ADAMTS5 on IL-1β-induced chondrocyte injury. CHON-001 cells were transfected with si-NC/si-SP1/pcDNA/ADAMTS5 followed by treatment with IL-1β. (A) ADAMTS5 protein expression was assessed using western blotting. Cell proliferation and apoptosis were determined by (B) MTT assay, (C) EdU assay and (D) flow cytometry. (E) Protein expression of aggrecan and collagen II was evaluated using western blotting. (F) Concentrations of IL-6 and TNF-α were assessed by ELISA. Corresponding kits were used to test (G) SOD activity and (H) MDA levels. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. MDA, malondialdehyde; SOD, superoxide dismutase; NC, negative control; SP1, specific protein 1.

    Techniques Used: Transfection, Expressing, Western Blot, MTT Assay, EdU Assay, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Activity Assay, Negative Control

    Effects of the SP1/ADAMTS5 axis on the activity of the Wnt/β-catenin pathway. (A) western blotting was used to measure the protein expression of β-catenin and Wnt3a in IL-1β-induced CHON-001 cells transfected with si-NC/si-ADAMTS5. (B) Protein expression of β-catenin and Wnt3a was detected by western blotting in IL-1β-induced CHON-001 cells transfected with si-NC/si-SP1/pcDNA/ADAMTS5. **P<0.01, ***P<0.001 and ****P<0.0001. NC, negative control; SP1, specific protein 1.
    Figure Legend Snippet: Effects of the SP1/ADAMTS5 axis on the activity of the Wnt/β-catenin pathway. (A) western blotting was used to measure the protein expression of β-catenin and Wnt3a in IL-1β-induced CHON-001 cells transfected with si-NC/si-ADAMTS5. (B) Protein expression of β-catenin and Wnt3a was detected by western blotting in IL-1β-induced CHON-001 cells transfected with si-NC/si-SP1/pcDNA/ADAMTS5. **P<0.01, ***P<0.001 and ****P<0.0001. NC, negative control; SP1, specific protein 1.

    Techniques Used: Activity Assay, Western Blot, Expressing, Transfection, Negative Control

    Summary diagram of the present study. In IL-1β-induced chondrocytes, the SP1/ADAMTS5/Wnt/β-catenin pathway suppressed proliferation and accelerated apoptosis, ECM degradation, inflammation and oxidative stress to promote OA progression. OA, osteoarthritis; ECM, extracellular matrix; SP1, specific protein 1; MDA, malondialdehyde; SOD, superoxide dismutase.
    Figure Legend Snippet: Summary diagram of the present study. In IL-1β-induced chondrocytes, the SP1/ADAMTS5/Wnt/β-catenin pathway suppressed proliferation and accelerated apoptosis, ECM degradation, inflammation and oxidative stress to promote OA progression. OA, osteoarthritis; ECM, extracellular matrix; SP1, specific protein 1; MDA, malondialdehyde; SOD, superoxide dismutase.

    Techniques Used:

    anti sp1  (Danaher Inc)


    Bioz Verified Symbol Danaher Inc is a verified supplier
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    Structured Review

    Danaher Inc anti sp1
    Primer sequences used for RT-qPCR.
    Anti Sp1, supplied by Danaher Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti sp1/product/Danaher Inc
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti sp1 - by Bioz Stars, 2024-07
    86/100 stars

    Images

    1) Product Images from "SP1‑mediated ADAMTS5 transcription promotes IL‑1β‑induced chondrocyte injury via Wnt/β‑catenin pathway in osteoarthritis"

    Article Title: SP1‑mediated ADAMTS5 transcription promotes IL‑1β‑induced chondrocyte injury via Wnt/β‑catenin pathway in osteoarthritis

    Journal: Molecular Medicine Reports

    doi: 10.3892/mmr.2024.13273

    Primer sequences used for RT-qPCR.
    Figure Legend Snippet: Primer sequences used for RT-qPCR.

    Techniques Used: Sequencing

    SP1 activated the transcription of ADAMTS5. (A) Jaspar software identified the binding sites of SP1 on the promoter region of ADAMTS5. (B) ChIP was used to confirm the binding ability of SP1 with the ADAMTS5 promoter. (C) SP1 protein expression was detected by western blotting in 293T cells transfected with pcDNA/SP1. (D) Dual-luciferase reporter assay was used to verify the binding of SP1 with the WT of the ADAMTS5 promoter. (E) Transfection efficiency of si-SP1 was confirmed by western blotting. (F) ADAMTS5 protein expression was examined by western blotting in CHON-001 cells transfected with si-NC/si-SP1. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. SP1, specific protein 1; WT, wild-type; MUT, mutant.
    Figure Legend Snippet: SP1 activated the transcription of ADAMTS5. (A) Jaspar software identified the binding sites of SP1 on the promoter region of ADAMTS5. (B) ChIP was used to confirm the binding ability of SP1 with the ADAMTS5 promoter. (C) SP1 protein expression was detected by western blotting in 293T cells transfected with pcDNA/SP1. (D) Dual-luciferase reporter assay was used to verify the binding of SP1 with the WT of the ADAMTS5 promoter. (E) Transfection efficiency of si-SP1 was confirmed by western blotting. (F) ADAMTS5 protein expression was examined by western blotting in CHON-001 cells transfected with si-NC/si-SP1. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. SP1, specific protein 1; WT, wild-type; MUT, mutant.

    Techniques Used: Software, Binding Assay, Expressing, Western Blot, Transfection, Luciferase, Reporter Assay, Mutagenesis

    Effects of si-SP1 and ADAMTS5 on IL-1β-induced chondrocyte injury. CHON-001 cells were transfected with si-NC/si-SP1/pcDNA/ADAMTS5 followed by treatment with IL-1β. (A) ADAMTS5 protein expression was assessed using western blotting. Cell proliferation and apoptosis were determined by (B) MTT assay, (C) EdU assay and (D) flow cytometry. (E) Protein expression of aggrecan and collagen II was evaluated using western blotting. (F) Concentrations of IL-6 and TNF-α were assessed by ELISA. Corresponding kits were used to test (G) SOD activity and (H) MDA levels. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. MDA, malondialdehyde; SOD, superoxide dismutase; NC, negative control; SP1, specific protein 1.
    Figure Legend Snippet: Effects of si-SP1 and ADAMTS5 on IL-1β-induced chondrocyte injury. CHON-001 cells were transfected with si-NC/si-SP1/pcDNA/ADAMTS5 followed by treatment with IL-1β. (A) ADAMTS5 protein expression was assessed using western blotting. Cell proliferation and apoptosis were determined by (B) MTT assay, (C) EdU assay and (D) flow cytometry. (E) Protein expression of aggrecan and collagen II was evaluated using western blotting. (F) Concentrations of IL-6 and TNF-α were assessed by ELISA. Corresponding kits were used to test (G) SOD activity and (H) MDA levels. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. MDA, malondialdehyde; SOD, superoxide dismutase; NC, negative control; SP1, specific protein 1.

    Techniques Used: Transfection, Expressing, Western Blot, MTT Assay, EdU Assay, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Activity Assay, Negative Control

    Effects of the SP1/ADAMTS5 axis on the activity of the Wnt/β-catenin pathway. (A) western blotting was used to measure the protein expression of β-catenin and Wnt3a in IL-1β-induced CHON-001 cells transfected with si-NC/si-ADAMTS5. (B) Protein expression of β-catenin and Wnt3a was detected by western blotting in IL-1β-induced CHON-001 cells transfected with si-NC/si-SP1/pcDNA/ADAMTS5. **P<0.01, ***P<0.001 and ****P<0.0001. NC, negative control; SP1, specific protein 1.
    Figure Legend Snippet: Effects of the SP1/ADAMTS5 axis on the activity of the Wnt/β-catenin pathway. (A) western blotting was used to measure the protein expression of β-catenin and Wnt3a in IL-1β-induced CHON-001 cells transfected with si-NC/si-ADAMTS5. (B) Protein expression of β-catenin and Wnt3a was detected by western blotting in IL-1β-induced CHON-001 cells transfected with si-NC/si-SP1/pcDNA/ADAMTS5. **P<0.01, ***P<0.001 and ****P<0.0001. NC, negative control; SP1, specific protein 1.

    Techniques Used: Activity Assay, Western Blot, Expressing, Transfection, Negative Control

    Summary diagram of the present study. In IL-1β-induced chondrocytes, the SP1/ADAMTS5/Wnt/β-catenin pathway suppressed proliferation and accelerated apoptosis, ECM degradation, inflammation and oxidative stress to promote OA progression. OA, osteoarthritis; ECM, extracellular matrix; SP1, specific protein 1; MDA, malondialdehyde; SOD, superoxide dismutase.
    Figure Legend Snippet: Summary diagram of the present study. In IL-1β-induced chondrocytes, the SP1/ADAMTS5/Wnt/β-catenin pathway suppressed proliferation and accelerated apoptosis, ECM degradation, inflammation and oxidative stress to promote OA progression. OA, osteoarthritis; ECM, extracellular matrix; SP1, specific protein 1; MDA, malondialdehyde; SOD, superoxide dismutase.

    Techniques Used:

    anti sp1  (Danaher Inc)


    Bioz Verified Symbol Danaher Inc is a verified supplier
    Bioz Manufacturer Symbol Danaher Inc manufactures this product  
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  • 86

    Structured Review

    Danaher Inc anti sp1
    Primer sequences used for RT-qPCR.
    Anti Sp1, supplied by Danaher Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti sp1/product/Danaher Inc
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti sp1 - by Bioz Stars, 2024-07
    86/100 stars

    Images

    1) Product Images from "SP1‑mediated ADAMTS5 transcription promotes IL‑1β‑induced chondrocyte injury via Wnt/β‑catenin pathway in osteoarthritis"

    Article Title: SP1‑mediated ADAMTS5 transcription promotes IL‑1β‑induced chondrocyte injury via Wnt/β‑catenin pathway in osteoarthritis

    Journal: Molecular Medicine Reports

    doi: 10.3892/mmr.2024.13273

    Primer sequences used for RT-qPCR.
    Figure Legend Snippet: Primer sequences used for RT-qPCR.

    Techniques Used: Sequencing

    SP1 activated the transcription of ADAMTS5. (A) Jaspar software identified the binding sites of SP1 on the promoter region of ADAMTS5. (B) ChIP was used to confirm the binding ability of SP1 with the ADAMTS5 promoter. (C) SP1 protein expression was detected by western blotting in 293T cells transfected with pcDNA/SP1. (D) Dual-luciferase reporter assay was used to verify the binding of SP1 with the WT of the ADAMTS5 promoter. (E) Transfection efficiency of si-SP1 was confirmed by western blotting. (F) ADAMTS5 protein expression was examined by western blotting in CHON-001 cells transfected with si-NC/si-SP1. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. SP1, specific protein 1; WT, wild-type; MUT, mutant.
    Figure Legend Snippet: SP1 activated the transcription of ADAMTS5. (A) Jaspar software identified the binding sites of SP1 on the promoter region of ADAMTS5. (B) ChIP was used to confirm the binding ability of SP1 with the ADAMTS5 promoter. (C) SP1 protein expression was detected by western blotting in 293T cells transfected with pcDNA/SP1. (D) Dual-luciferase reporter assay was used to verify the binding of SP1 with the WT of the ADAMTS5 promoter. (E) Transfection efficiency of si-SP1 was confirmed by western blotting. (F) ADAMTS5 protein expression was examined by western blotting in CHON-001 cells transfected with si-NC/si-SP1. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. SP1, specific protein 1; WT, wild-type; MUT, mutant.

    Techniques Used: Software, Binding Assay, Expressing, Western Blot, Transfection, Luciferase, Reporter Assay, Mutagenesis

    Effects of si-SP1 and ADAMTS5 on IL-1β-induced chondrocyte injury. CHON-001 cells were transfected with si-NC/si-SP1/pcDNA/ADAMTS5 followed by treatment with IL-1β. (A) ADAMTS5 protein expression was assessed using western blotting. Cell proliferation and apoptosis were determined by (B) MTT assay, (C) EdU assay and (D) flow cytometry. (E) Protein expression of aggrecan and collagen II was evaluated using western blotting. (F) Concentrations of IL-6 and TNF-α were assessed by ELISA. Corresponding kits were used to test (G) SOD activity and (H) MDA levels. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. MDA, malondialdehyde; SOD, superoxide dismutase; NC, negative control; SP1, specific protein 1.
    Figure Legend Snippet: Effects of si-SP1 and ADAMTS5 on IL-1β-induced chondrocyte injury. CHON-001 cells were transfected with si-NC/si-SP1/pcDNA/ADAMTS5 followed by treatment with IL-1β. (A) ADAMTS5 protein expression was assessed using western blotting. Cell proliferation and apoptosis were determined by (B) MTT assay, (C) EdU assay and (D) flow cytometry. (E) Protein expression of aggrecan and collagen II was evaluated using western blotting. (F) Concentrations of IL-6 and TNF-α were assessed by ELISA. Corresponding kits were used to test (G) SOD activity and (H) MDA levels. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. MDA, malondialdehyde; SOD, superoxide dismutase; NC, negative control; SP1, specific protein 1.

    Techniques Used: Transfection, Expressing, Western Blot, MTT Assay, EdU Assay, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Activity Assay, Negative Control

    Effects of the SP1/ADAMTS5 axis on the activity of the Wnt/β-catenin pathway. (A) western blotting was used to measure the protein expression of β-catenin and Wnt3a in IL-1β-induced CHON-001 cells transfected with si-NC/si-ADAMTS5. (B) Protein expression of β-catenin and Wnt3a was detected by western blotting in IL-1β-induced CHON-001 cells transfected with si-NC/si-SP1/pcDNA/ADAMTS5. **P<0.01, ***P<0.001 and ****P<0.0001. NC, negative control; SP1, specific protein 1.
    Figure Legend Snippet: Effects of the SP1/ADAMTS5 axis on the activity of the Wnt/β-catenin pathway. (A) western blotting was used to measure the protein expression of β-catenin and Wnt3a in IL-1β-induced CHON-001 cells transfected with si-NC/si-ADAMTS5. (B) Protein expression of β-catenin and Wnt3a was detected by western blotting in IL-1β-induced CHON-001 cells transfected with si-NC/si-SP1/pcDNA/ADAMTS5. **P<0.01, ***P<0.001 and ****P<0.0001. NC, negative control; SP1, specific protein 1.

    Techniques Used: Activity Assay, Western Blot, Expressing, Transfection, Negative Control

    Summary diagram of the present study. In IL-1β-induced chondrocytes, the SP1/ADAMTS5/Wnt/β-catenin pathway suppressed proliferation and accelerated apoptosis, ECM degradation, inflammation and oxidative stress to promote OA progression. OA, osteoarthritis; ECM, extracellular matrix; SP1, specific protein 1; MDA, malondialdehyde; SOD, superoxide dismutase.
    Figure Legend Snippet: Summary diagram of the present study. In IL-1β-induced chondrocytes, the SP1/ADAMTS5/Wnt/β-catenin pathway suppressed proliferation and accelerated apoptosis, ECM degradation, inflammation and oxidative stress to promote OA progression. OA, osteoarthritis; ECM, extracellular matrix; SP1, specific protein 1; MDA, malondialdehyde; SOD, superoxide dismutase.

    Techniques Used:


    Structured Review

    Proteintech anti sp1
    Anti Sp1, supplied by Proteintech, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti sp1/product/Proteintech
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti sp1 - by Bioz Stars, 2024-07
    86/100 stars

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  • 86
    Millipore anti sp1 antibody
    Nucleolin and <t>Sp1</t> regulate G4 → duplex transition at MAPK12 promoter. A FRET probe. MAPK12-G4 motifs conjugated with 6-FAM at 5′-end of GQ-1 and internal Cy3 at T22. The complementary strand of G4-forming sequence contains Cy5 at 3′-end. G4-formation gives high FRET signal between 6-FAM and Cy3. Duplex formation gives high FRET signal between 6-FAM and Cy5. B Fluorometric titration of 90 nM FRET probe with increasing gradient of recombinant Nucleolin (0–3 µM) in 50 mM Tris–Cl, pH 7.0, 100 mM KCl at 15 °C at excitation wavelength (λex) of 497 nm. Decline of FRET signal at Cy5 emission ((λem) 650 nm) upon Nucleolin titration suggests disruption of duplex with concomitant increase of Cy3 emission ((λem) 595 nm) indicating G4 evolution. C Fluorometric titration of 90 nM FRET probe with increasing gradient of recombinant Sp1 (0–1 µM) in 50 mM Tris–Cl, pH 7.0, 100 mM KCl at 15 °C at excitation wavelength (λex) of 497 nm. Decline of FRET signal at Cy3 emission ((λem) 595 nm) upon Sp1 titration suggests G4 resolution with concomitant increase of Cy5 emission ((λem) 650 nm) indicating duplex transition. D FRET efficiency calculated following the equation: E = 1 – (IDA/ID); IDA and ID are the emission intensity of donor fluorophore in presence and absence of acceptor fluorophore respectively. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). With increasing Sp1, distance between 6-FAM and Cy5 decreases. Nucleolin titration decreases the distance between 6-FAM and Cy3. The change in distances (r) between donor and acceptor fluorophore calculated by E = R06/(R06 + r6). R0 denotes Forster distance between donor and acceptor. The distance between 6-FAM and Cy3 decreased from 6.5 ± 0.1 nm (R0 = 6.5 nm) in the free DNA to 5.2 ± 0.1 nm in the complex with Nucleolin indicating G4 formation. The distance between 6-FAM and Cy5 decreased from 6.8 ± 0.2 in free DNA to 5.3 ± 0.3 under Sp1-bound conditions indicating duplex formation (E) pGL4.72[hRlucCP] luciferase constructs having the inserts containing MAPK12 promoter sequences having adjacent G4-elements (wild-type). Dual-luciferase assays. Evaluation of MAPK12 promoter activities in MDAMB-231 cells under the following conditions: siRNA knockdown of Nucleolin at 48 h and TGS24 (50 nM) treatment for 24 h, nucleolin-knockdown + TGS24 treatment, nucleolin knockdown + TGS25 treatment, and TGS24 and TGS25 treated alone separately Error bars represent mean ± SE (N = 3). Statistical differences in the luciferase activities compared to that of the control cells use two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). Statistical differences in the luciferase activities in presence of TGS24-treated samples compared to that of cells, treated with recombinant Sp1 and Nucleolin, siRNA-knockdown of Nucleolin use two-tailed Student's t test (###P < 0.001). F RT-PCR analysis. Expression profile of MAPK12 transcripts from P1 promoter in MDAMB-231 cells under following conditions: TGS24 (50 nM) treatment for 24 h and siRNA knockdown of Nucleolin for 48 h, nucleolin-knockdown + TGS24 treatment, nucleolin knockdown + TGS25 treatment, and TGS25 treated alone separately. Quantification of the transcripts’ level relative to the control by qPCR analyses. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). Statistical differences between recombinant Nucleolin-treated and Nucleolin-knockdown cells are determined by two-tailed Student's t test (###P < 0.001). GAPDH considered as housekeeping gene
    Anti Sp1 Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti sp1 antibody/product/Millipore
    Average 86 stars, based on 1 article reviews
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    anti sp1 antibody - by Bioz Stars, 2024-07
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    Millipore sp1 antibody
    Nucleolin and <t>Sp1</t> regulate G4 → duplex transition at MAPK12 promoter. A FRET probe. MAPK12-G4 motifs conjugated with 6-FAM at 5′-end of GQ-1 and internal Cy3 at T22. The complementary strand of G4-forming sequence contains Cy5 at 3′-end. G4-formation gives high FRET signal between 6-FAM and Cy3. Duplex formation gives high FRET signal between 6-FAM and Cy5. B Fluorometric titration of 90 nM FRET probe with increasing gradient of recombinant Nucleolin (0–3 µM) in 50 mM Tris–Cl, pH 7.0, 100 mM KCl at 15 °C at excitation wavelength (λex) of 497 nm. Decline of FRET signal at Cy5 emission ((λem) 650 nm) upon Nucleolin titration suggests disruption of duplex with concomitant increase of Cy3 emission ((λem) 595 nm) indicating G4 evolution. C Fluorometric titration of 90 nM FRET probe with increasing gradient of recombinant Sp1 (0–1 µM) in 50 mM Tris–Cl, pH 7.0, 100 mM KCl at 15 °C at excitation wavelength (λex) of 497 nm. Decline of FRET signal at Cy3 emission ((λem) 595 nm) upon Sp1 titration suggests G4 resolution with concomitant increase of Cy5 emission ((λem) 650 nm) indicating duplex transition. D FRET efficiency calculated following the equation: E = 1 – (IDA/ID); IDA and ID are the emission intensity of donor fluorophore in presence and absence of acceptor fluorophore respectively. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). With increasing Sp1, distance between 6-FAM and Cy5 decreases. Nucleolin titration decreases the distance between 6-FAM and Cy3. The change in distances (r) between donor and acceptor fluorophore calculated by E = R06/(R06 + r6). R0 denotes Forster distance between donor and acceptor. The distance between 6-FAM and Cy3 decreased from 6.5 ± 0.1 nm (R0 = 6.5 nm) in the free DNA to 5.2 ± 0.1 nm in the complex with Nucleolin indicating G4 formation. The distance between 6-FAM and Cy5 decreased from 6.8 ± 0.2 in free DNA to 5.3 ± 0.3 under Sp1-bound conditions indicating duplex formation (E) pGL4.72[hRlucCP] luciferase constructs having the inserts containing MAPK12 promoter sequences having adjacent G4-elements (wild-type). Dual-luciferase assays. Evaluation of MAPK12 promoter activities in MDAMB-231 cells under the following conditions: siRNA knockdown of Nucleolin at 48 h and TGS24 (50 nM) treatment for 24 h, nucleolin-knockdown + TGS24 treatment, nucleolin knockdown + TGS25 treatment, and TGS24 and TGS25 treated alone separately Error bars represent mean ± SE (N = 3). Statistical differences in the luciferase activities compared to that of the control cells use two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). Statistical differences in the luciferase activities in presence of TGS24-treated samples compared to that of cells, treated with recombinant Sp1 and Nucleolin, siRNA-knockdown of Nucleolin use two-tailed Student's t test (###P < 0.001). F RT-PCR analysis. Expression profile of MAPK12 transcripts from P1 promoter in MDAMB-231 cells under following conditions: TGS24 (50 nM) treatment for 24 h and siRNA knockdown of Nucleolin for 48 h, nucleolin-knockdown + TGS24 treatment, nucleolin knockdown + TGS25 treatment, and TGS25 treated alone separately. Quantification of the transcripts’ level relative to the control by qPCR analyses. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). Statistical differences between recombinant Nucleolin-treated and Nucleolin-knockdown cells are determined by two-tailed Student's t test (###P < 0.001). GAPDH considered as housekeeping gene
    Sp1 Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sp1 antibody/product/Millipore
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    Thermo Fisher pcdna3 1 sp1 overexpression vector
    Primer sequences used for RT-qPCR.
    Pcdna3 1 Sp1 Overexpression Vector, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC pcdna3 1 sp1 overexpression vector
    Primer sequences used for RT-qPCR.
    Pcdna3 1 Sp1 Overexpression Vector, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Danaher Inc anti sp1
    Primer sequences used for RT-qPCR.
    Anti Sp1, supplied by Danaher Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti sp1/product/Danaher Inc
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    Proteintech anti sp1
    Primer sequences used for RT-qPCR.
    Anti Sp1, supplied by Proteintech, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti sp1/product/Proteintech
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    anti sp1 - by Bioz Stars, 2024-07
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    Nucleolin and Sp1 regulate G4 → duplex transition at MAPK12 promoter. A FRET probe. MAPK12-G4 motifs conjugated with 6-FAM at 5′-end of GQ-1 and internal Cy3 at T22. The complementary strand of G4-forming sequence contains Cy5 at 3′-end. G4-formation gives high FRET signal between 6-FAM and Cy3. Duplex formation gives high FRET signal between 6-FAM and Cy5. B Fluorometric titration of 90 nM FRET probe with increasing gradient of recombinant Nucleolin (0–3 µM) in 50 mM Tris–Cl, pH 7.0, 100 mM KCl at 15 °C at excitation wavelength (λex) of 497 nm. Decline of FRET signal at Cy5 emission ((λem) 650 nm) upon Nucleolin titration suggests disruption of duplex with concomitant increase of Cy3 emission ((λem) 595 nm) indicating G4 evolution. C Fluorometric titration of 90 nM FRET probe with increasing gradient of recombinant Sp1 (0–1 µM) in 50 mM Tris–Cl, pH 7.0, 100 mM KCl at 15 °C at excitation wavelength (λex) of 497 nm. Decline of FRET signal at Cy3 emission ((λem) 595 nm) upon Sp1 titration suggests G4 resolution with concomitant increase of Cy5 emission ((λem) 650 nm) indicating duplex transition. D FRET efficiency calculated following the equation: E = 1 – (IDA/ID); IDA and ID are the emission intensity of donor fluorophore in presence and absence of acceptor fluorophore respectively. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). With increasing Sp1, distance between 6-FAM and Cy5 decreases. Nucleolin titration decreases the distance between 6-FAM and Cy3. The change in distances (r) between donor and acceptor fluorophore calculated by E = R06/(R06 + r6). R0 denotes Forster distance between donor and acceptor. The distance between 6-FAM and Cy3 decreased from 6.5 ± 0.1 nm (R0 = 6.5 nm) in the free DNA to 5.2 ± 0.1 nm in the complex with Nucleolin indicating G4 formation. The distance between 6-FAM and Cy5 decreased from 6.8 ± 0.2 in free DNA to 5.3 ± 0.3 under Sp1-bound conditions indicating duplex formation (E) pGL4.72[hRlucCP] luciferase constructs having the inserts containing MAPK12 promoter sequences having adjacent G4-elements (wild-type). Dual-luciferase assays. Evaluation of MAPK12 promoter activities in MDAMB-231 cells under the following conditions: siRNA knockdown of Nucleolin at 48 h and TGS24 (50 nM) treatment for 24 h, nucleolin-knockdown + TGS24 treatment, nucleolin knockdown + TGS25 treatment, and TGS24 and TGS25 treated alone separately Error bars represent mean ± SE (N = 3). Statistical differences in the luciferase activities compared to that of the control cells use two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). Statistical differences in the luciferase activities in presence of TGS24-treated samples compared to that of cells, treated with recombinant Sp1 and Nucleolin, siRNA-knockdown of Nucleolin use two-tailed Student's t test (###P < 0.001). F RT-PCR analysis. Expression profile of MAPK12 transcripts from P1 promoter in MDAMB-231 cells under following conditions: TGS24 (50 nM) treatment for 24 h and siRNA knockdown of Nucleolin for 48 h, nucleolin-knockdown + TGS24 treatment, nucleolin knockdown + TGS25 treatment, and TGS25 treated alone separately. Quantification of the transcripts’ level relative to the control by qPCR analyses. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). Statistical differences between recombinant Nucleolin-treated and Nucleolin-knockdown cells are determined by two-tailed Student's t test (###P < 0.001). GAPDH considered as housekeeping gene

    Journal: Cellular and Molecular Life Sciences: CMLS

    Article Title: G-quadruplex structural dynamics at MAPK12 promoter dictates transcriptional switch to determine stemness in breast cancer

    doi: 10.1007/s00018-023-05046-6

    Figure Lengend Snippet: Nucleolin and Sp1 regulate G4 → duplex transition at MAPK12 promoter. A FRET probe. MAPK12-G4 motifs conjugated with 6-FAM at 5′-end of GQ-1 and internal Cy3 at T22. The complementary strand of G4-forming sequence contains Cy5 at 3′-end. G4-formation gives high FRET signal between 6-FAM and Cy3. Duplex formation gives high FRET signal between 6-FAM and Cy5. B Fluorometric titration of 90 nM FRET probe with increasing gradient of recombinant Nucleolin (0–3 µM) in 50 mM Tris–Cl, pH 7.0, 100 mM KCl at 15 °C at excitation wavelength (λex) of 497 nm. Decline of FRET signal at Cy5 emission ((λem) 650 nm) upon Nucleolin titration suggests disruption of duplex with concomitant increase of Cy3 emission ((λem) 595 nm) indicating G4 evolution. C Fluorometric titration of 90 nM FRET probe with increasing gradient of recombinant Sp1 (0–1 µM) in 50 mM Tris–Cl, pH 7.0, 100 mM KCl at 15 °C at excitation wavelength (λex) of 497 nm. Decline of FRET signal at Cy3 emission ((λem) 595 nm) upon Sp1 titration suggests G4 resolution with concomitant increase of Cy5 emission ((λem) 650 nm) indicating duplex transition. D FRET efficiency calculated following the equation: E = 1 – (IDA/ID); IDA and ID are the emission intensity of donor fluorophore in presence and absence of acceptor fluorophore respectively. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). With increasing Sp1, distance between 6-FAM and Cy5 decreases. Nucleolin titration decreases the distance between 6-FAM and Cy3. The change in distances (r) between donor and acceptor fluorophore calculated by E = R06/(R06 + r6). R0 denotes Forster distance between donor and acceptor. The distance between 6-FAM and Cy3 decreased from 6.5 ± 0.1 nm (R0 = 6.5 nm) in the free DNA to 5.2 ± 0.1 nm in the complex with Nucleolin indicating G4 formation. The distance between 6-FAM and Cy5 decreased from 6.8 ± 0.2 in free DNA to 5.3 ± 0.3 under Sp1-bound conditions indicating duplex formation (E) pGL4.72[hRlucCP] luciferase constructs having the inserts containing MAPK12 promoter sequences having adjacent G4-elements (wild-type). Dual-luciferase assays. Evaluation of MAPK12 promoter activities in MDAMB-231 cells under the following conditions: siRNA knockdown of Nucleolin at 48 h and TGS24 (50 nM) treatment for 24 h, nucleolin-knockdown + TGS24 treatment, nucleolin knockdown + TGS25 treatment, and TGS24 and TGS25 treated alone separately Error bars represent mean ± SE (N = 3). Statistical differences in the luciferase activities compared to that of the control cells use two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). Statistical differences in the luciferase activities in presence of TGS24-treated samples compared to that of cells, treated with recombinant Sp1 and Nucleolin, siRNA-knockdown of Nucleolin use two-tailed Student's t test (###P < 0.001). F RT-PCR analysis. Expression profile of MAPK12 transcripts from P1 promoter in MDAMB-231 cells under following conditions: TGS24 (50 nM) treatment for 24 h and siRNA knockdown of Nucleolin for 48 h, nucleolin-knockdown + TGS24 treatment, nucleolin knockdown + TGS25 treatment, and TGS25 treated alone separately. Quantification of the transcripts’ level relative to the control by qPCR analyses. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). Statistical differences between recombinant Nucleolin-treated and Nucleolin-knockdown cells are determined by two-tailed Student's t test (###P < 0.001). GAPDH considered as housekeeping gene

    Article Snippet: Elution fractions were run on 15% SDS-PAGE and proteins were detected by Western blot using anti-Nucleolin antibody (Abcam) and anti-Sp1 antibody (Sigma).

    Techniques: Sequencing, Titration, Recombinant, Disruption, Two Tailed Test, Luciferase, Construct, Reverse Transcription Polymerase Chain Reaction, Expressing

    Downstream effects of TGS24-treatment and its selective interaction with MAPK12-G4 motifs. A RNAseq analyses of TGS24 treatment in MDAMB-231 spheres expressing high MAPK12. Heatmap of biological replicates of differentially expressed genes upon 24 h treatment of 50 nM TGS24 treatment in MDAMB-231 spheres using the log10(count) values for each replicate. The screening threshold for the differentially expressed genes is set to: |log2(Fold Change)|> 1 and P value < 0.05. Differential expression of the transcripts clustered by Euclidean correlation, with distinct upregulation, unaltered, and downregulation patterns in expression for TGS24 treatment, compared to control condition. The count values are colour coded blue to yellow to red in increasing order. Differentially expressed genes are clustered based on their involvement with RAS, apoptosis, and stemness- and metastasis-related pathways. B RT-qPCR-verification of RNAseq data for differentially expressed genes in RAS pathway upon 50 nM TGS24 treatment in MDAMB-231 spheres for 24 h: MAP4K3, RAS, MAPK12, c-FOS, c-JUN, HSP90. Quantification of the transcripts’ level relative to the control by qPCR analyses. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). GAPDH considered as housekeeping gene. C RT-qPCR-verification of RNAseq data for differentially expressed genes in metastasis and stemness-related pathway upon 50 nM TGS24 treatment in MDAMB-231 spheres for 24 h: ALDHβ1, WNT-1, β-Catenin, SOX2, MMP12, Vimentin, CD-44. Quantification of the transcripts’ level relative to the control by qPCR analyses. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). GAPDH considered as housekeeping gene. D RT-qPCR-verification of RNAseq data for differentially expressed genes in apoptotic pathway upon 50 nM TGS24 treatment in MDAMB-231 spheres for 24 h: NF-κβ, Sp1, GATA3, CASP9, PARP-1. Quantification of the transcripts’ level relative to the control by qPCR analyses. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). GAPDH considered as housekeeping gene. E Correlation of log2(fold-change) values from RNA-Seq and RT-PCR analysis. The R2 value is 0.854. F Interaction network of upregulated and downregulated genes, obtained using STRING database, with the minimum required interaction score of 0.400 and network edges representing evidence of an interaction

    Journal: Cellular and Molecular Life Sciences: CMLS

    Article Title: G-quadruplex structural dynamics at MAPK12 promoter dictates transcriptional switch to determine stemness in breast cancer

    doi: 10.1007/s00018-023-05046-6

    Figure Lengend Snippet: Downstream effects of TGS24-treatment and its selective interaction with MAPK12-G4 motifs. A RNAseq analyses of TGS24 treatment in MDAMB-231 spheres expressing high MAPK12. Heatmap of biological replicates of differentially expressed genes upon 24 h treatment of 50 nM TGS24 treatment in MDAMB-231 spheres using the log10(count) values for each replicate. The screening threshold for the differentially expressed genes is set to: |log2(Fold Change)|> 1 and P value < 0.05. Differential expression of the transcripts clustered by Euclidean correlation, with distinct upregulation, unaltered, and downregulation patterns in expression for TGS24 treatment, compared to control condition. The count values are colour coded blue to yellow to red in increasing order. Differentially expressed genes are clustered based on their involvement with RAS, apoptosis, and stemness- and metastasis-related pathways. B RT-qPCR-verification of RNAseq data for differentially expressed genes in RAS pathway upon 50 nM TGS24 treatment in MDAMB-231 spheres for 24 h: MAP4K3, RAS, MAPK12, c-FOS, c-JUN, HSP90. Quantification of the transcripts’ level relative to the control by qPCR analyses. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). GAPDH considered as housekeeping gene. C RT-qPCR-verification of RNAseq data for differentially expressed genes in metastasis and stemness-related pathway upon 50 nM TGS24 treatment in MDAMB-231 spheres for 24 h: ALDHβ1, WNT-1, β-Catenin, SOX2, MMP12, Vimentin, CD-44. Quantification of the transcripts’ level relative to the control by qPCR analyses. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). GAPDH considered as housekeeping gene. D RT-qPCR-verification of RNAseq data for differentially expressed genes in apoptotic pathway upon 50 nM TGS24 treatment in MDAMB-231 spheres for 24 h: NF-κβ, Sp1, GATA3, CASP9, PARP-1. Quantification of the transcripts’ level relative to the control by qPCR analyses. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). GAPDH considered as housekeeping gene. E Correlation of log2(fold-change) values from RNA-Seq and RT-PCR analysis. The R2 value is 0.854. F Interaction network of upregulated and downregulated genes, obtained using STRING database, with the minimum required interaction score of 0.400 and network edges representing evidence of an interaction

    Article Snippet: Elution fractions were run on 15% SDS-PAGE and proteins were detected by Western blot using anti-Nucleolin antibody (Abcam) and anti-Sp1 antibody (Sigma).

    Techniques: Expressing, Quantitative RT-PCR, Two Tailed Test, RNA Sequencing Assay, Reverse Transcription Polymerase Chain Reaction

    Dynamics of two tandem G4-motifs at MAPK12 promoter regulated by spatiotemporal binding of Nucleolin and Sp1 regulate cancer stemness via MAPK12-HSP90-RAS-c-JUN-NANOG axis

    Journal: Cellular and Molecular Life Sciences: CMLS

    Article Title: G-quadruplex structural dynamics at MAPK12 promoter dictates transcriptional switch to determine stemness in breast cancer

    doi: 10.1007/s00018-023-05046-6

    Figure Lengend Snippet: Dynamics of two tandem G4-motifs at MAPK12 promoter regulated by spatiotemporal binding of Nucleolin and Sp1 regulate cancer stemness via MAPK12-HSP90-RAS-c-JUN-NANOG axis

    Article Snippet: Elution fractions were run on 15% SDS-PAGE and proteins were detected by Western blot using anti-Nucleolin antibody (Abcam) and anti-Sp1 antibody (Sigma).

    Techniques: Binding Assay

    Nucleolin and Sp1 regulate G4 → duplex transition at MAPK12 promoter. A FRET probe. MAPK12-G4 motifs conjugated with 6-FAM at 5′-end of GQ-1 and internal Cy3 at T22. The complementary strand of G4-forming sequence contains Cy5 at 3′-end. G4-formation gives high FRET signal between 6-FAM and Cy3. Duplex formation gives high FRET signal between 6-FAM and Cy5. B Fluorometric titration of 90 nM FRET probe with increasing gradient of recombinant Nucleolin (0–3 µM) in 50 mM Tris–Cl, pH 7.0, 100 mM KCl at 15 °C at excitation wavelength (λex) of 497 nm. Decline of FRET signal at Cy5 emission ((λem) 650 nm) upon Nucleolin titration suggests disruption of duplex with concomitant increase of Cy3 emission ((λem) 595 nm) indicating G4 evolution. C Fluorometric titration of 90 nM FRET probe with increasing gradient of recombinant Sp1 (0–1 µM) in 50 mM Tris–Cl, pH 7.0, 100 mM KCl at 15 °C at excitation wavelength (λex) of 497 nm. Decline of FRET signal at Cy3 emission ((λem) 595 nm) upon Sp1 titration suggests G4 resolution with concomitant increase of Cy5 emission ((λem) 650 nm) indicating duplex transition. D FRET efficiency calculated following the equation: E = 1 – (IDA/ID); IDA and ID are the emission intensity of donor fluorophore in presence and absence of acceptor fluorophore respectively. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). With increasing Sp1, distance between 6-FAM and Cy5 decreases. Nucleolin titration decreases the distance between 6-FAM and Cy3. The change in distances (r) between donor and acceptor fluorophore calculated by E = R06/(R06 + r6). R0 denotes Forster distance between donor and acceptor. The distance between 6-FAM and Cy3 decreased from 6.5 ± 0.1 nm (R0 = 6.5 nm) in the free DNA to 5.2 ± 0.1 nm in the complex with Nucleolin indicating G4 formation. The distance between 6-FAM and Cy5 decreased from 6.8 ± 0.2 in free DNA to 5.3 ± 0.3 under Sp1-bound conditions indicating duplex formation (E) pGL4.72[hRlucCP] luciferase constructs having the inserts containing MAPK12 promoter sequences having adjacent G4-elements (wild-type). Dual-luciferase assays. Evaluation of MAPK12 promoter activities in MDAMB-231 cells under the following conditions: siRNA knockdown of Nucleolin at 48 h and TGS24 (50 nM) treatment for 24 h, nucleolin-knockdown + TGS24 treatment, nucleolin knockdown + TGS25 treatment, and TGS24 and TGS25 treated alone separately Error bars represent mean ± SE (N = 3). Statistical differences in the luciferase activities compared to that of the control cells use two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). Statistical differences in the luciferase activities in presence of TGS24-treated samples compared to that of cells, treated with recombinant Sp1 and Nucleolin, siRNA-knockdown of Nucleolin use two-tailed Student's t test (###P < 0.001). F RT-PCR analysis. Expression profile of MAPK12 transcripts from P1 promoter in MDAMB-231 cells under following conditions: TGS24 (50 nM) treatment for 24 h and siRNA knockdown of Nucleolin for 48 h, nucleolin-knockdown + TGS24 treatment, nucleolin knockdown + TGS25 treatment, and TGS25 treated alone separately. Quantification of the transcripts’ level relative to the control by qPCR analyses. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). Statistical differences between recombinant Nucleolin-treated and Nucleolin-knockdown cells are determined by two-tailed Student's t test (###P < 0.001). GAPDH considered as housekeeping gene

    Journal: Cellular and Molecular Life Sciences: CMLS

    Article Title: G-quadruplex structural dynamics at MAPK12 promoter dictates transcriptional switch to determine stemness in breast cancer

    doi: 10.1007/s00018-023-05046-6

    Figure Lengend Snippet: Nucleolin and Sp1 regulate G4 → duplex transition at MAPK12 promoter. A FRET probe. MAPK12-G4 motifs conjugated with 6-FAM at 5′-end of GQ-1 and internal Cy3 at T22. The complementary strand of G4-forming sequence contains Cy5 at 3′-end. G4-formation gives high FRET signal between 6-FAM and Cy3. Duplex formation gives high FRET signal between 6-FAM and Cy5. B Fluorometric titration of 90 nM FRET probe with increasing gradient of recombinant Nucleolin (0–3 µM) in 50 mM Tris–Cl, pH 7.0, 100 mM KCl at 15 °C at excitation wavelength (λex) of 497 nm. Decline of FRET signal at Cy5 emission ((λem) 650 nm) upon Nucleolin titration suggests disruption of duplex with concomitant increase of Cy3 emission ((λem) 595 nm) indicating G4 evolution. C Fluorometric titration of 90 nM FRET probe with increasing gradient of recombinant Sp1 (0–1 µM) in 50 mM Tris–Cl, pH 7.0, 100 mM KCl at 15 °C at excitation wavelength (λex) of 497 nm. Decline of FRET signal at Cy3 emission ((λem) 595 nm) upon Sp1 titration suggests G4 resolution with concomitant increase of Cy5 emission ((λem) 650 nm) indicating duplex transition. D FRET efficiency calculated following the equation: E = 1 – (IDA/ID); IDA and ID are the emission intensity of donor fluorophore in presence and absence of acceptor fluorophore respectively. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). With increasing Sp1, distance between 6-FAM and Cy5 decreases. Nucleolin titration decreases the distance between 6-FAM and Cy3. The change in distances (r) between donor and acceptor fluorophore calculated by E = R06/(R06 + r6). R0 denotes Forster distance between donor and acceptor. The distance between 6-FAM and Cy3 decreased from 6.5 ± 0.1 nm (R0 = 6.5 nm) in the free DNA to 5.2 ± 0.1 nm in the complex with Nucleolin indicating G4 formation. The distance between 6-FAM and Cy5 decreased from 6.8 ± 0.2 in free DNA to 5.3 ± 0.3 under Sp1-bound conditions indicating duplex formation (E) pGL4.72[hRlucCP] luciferase constructs having the inserts containing MAPK12 promoter sequences having adjacent G4-elements (wild-type). Dual-luciferase assays. Evaluation of MAPK12 promoter activities in MDAMB-231 cells under the following conditions: siRNA knockdown of Nucleolin at 48 h and TGS24 (50 nM) treatment for 24 h, nucleolin-knockdown + TGS24 treatment, nucleolin knockdown + TGS25 treatment, and TGS24 and TGS25 treated alone separately Error bars represent mean ± SE (N = 3). Statistical differences in the luciferase activities compared to that of the control cells use two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). Statistical differences in the luciferase activities in presence of TGS24-treated samples compared to that of cells, treated with recombinant Sp1 and Nucleolin, siRNA-knockdown of Nucleolin use two-tailed Student's t test (###P < 0.001). F RT-PCR analysis. Expression profile of MAPK12 transcripts from P1 promoter in MDAMB-231 cells under following conditions: TGS24 (50 nM) treatment for 24 h and siRNA knockdown of Nucleolin for 48 h, nucleolin-knockdown + TGS24 treatment, nucleolin knockdown + TGS25 treatment, and TGS25 treated alone separately. Quantification of the transcripts’ level relative to the control by qPCR analyses. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). Statistical differences between recombinant Nucleolin-treated and Nucleolin-knockdown cells are determined by two-tailed Student's t test (###P < 0.001). GAPDH considered as housekeeping gene

    Article Snippet: ChIP-grade antibodies (Anti-Nucleolin antibody [4E2]-ChIP Grade (Abcam)), Anti Sp1 antibody (Sigma), Anti c-JUN Rabbit polyclonal antibody (Abcam), and anti-Hsp90β polyclonal antibody (Abcam)) were employed for the experiments.

    Techniques: Sequencing, Titration, Recombinant, Disruption, Two Tailed Test, Luciferase, Construct, Reverse Transcription Polymerase Chain Reaction, Expressing

    Downstream effects of TGS24-treatment and its selective interaction with MAPK12-G4 motifs. A RNAseq analyses of TGS24 treatment in MDAMB-231 spheres expressing high MAPK12. Heatmap of biological replicates of differentially expressed genes upon 24 h treatment of 50 nM TGS24 treatment in MDAMB-231 spheres using the log10(count) values for each replicate. The screening threshold for the differentially expressed genes is set to: |log2(Fold Change)|> 1 and P value < 0.05. Differential expression of the transcripts clustered by Euclidean correlation, with distinct upregulation, unaltered, and downregulation patterns in expression for TGS24 treatment, compared to control condition. The count values are colour coded blue to yellow to red in increasing order. Differentially expressed genes are clustered based on their involvement with RAS, apoptosis, and stemness- and metastasis-related pathways. B RT-qPCR-verification of RNAseq data for differentially expressed genes in RAS pathway upon 50 nM TGS24 treatment in MDAMB-231 spheres for 24 h: MAP4K3, RAS, MAPK12, c-FOS, c-JUN, HSP90. Quantification of the transcripts’ level relative to the control by qPCR analyses. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). GAPDH considered as housekeeping gene. C RT-qPCR-verification of RNAseq data for differentially expressed genes in metastasis and stemness-related pathway upon 50 nM TGS24 treatment in MDAMB-231 spheres for 24 h: ALDHβ1, WNT-1, β-Catenin, SOX2, MMP12, Vimentin, CD-44. Quantification of the transcripts’ level relative to the control by qPCR analyses. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). GAPDH considered as housekeeping gene. D RT-qPCR-verification of RNAseq data for differentially expressed genes in apoptotic pathway upon 50 nM TGS24 treatment in MDAMB-231 spheres for 24 h: NF-κβ, Sp1, GATA3, CASP9, PARP-1. Quantification of the transcripts’ level relative to the control by qPCR analyses. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). GAPDH considered as housekeeping gene. E Correlation of log2(fold-change) values from RNA-Seq and RT-PCR analysis. The R2 value is 0.854. F Interaction network of upregulated and downregulated genes, obtained using STRING database, with the minimum required interaction score of 0.400 and network edges representing evidence of an interaction

    Journal: Cellular and Molecular Life Sciences: CMLS

    Article Title: G-quadruplex structural dynamics at MAPK12 promoter dictates transcriptional switch to determine stemness in breast cancer

    doi: 10.1007/s00018-023-05046-6

    Figure Lengend Snippet: Downstream effects of TGS24-treatment and its selective interaction with MAPK12-G4 motifs. A RNAseq analyses of TGS24 treatment in MDAMB-231 spheres expressing high MAPK12. Heatmap of biological replicates of differentially expressed genes upon 24 h treatment of 50 nM TGS24 treatment in MDAMB-231 spheres using the log10(count) values for each replicate. The screening threshold for the differentially expressed genes is set to: |log2(Fold Change)|> 1 and P value < 0.05. Differential expression of the transcripts clustered by Euclidean correlation, with distinct upregulation, unaltered, and downregulation patterns in expression for TGS24 treatment, compared to control condition. The count values are colour coded blue to yellow to red in increasing order. Differentially expressed genes are clustered based on their involvement with RAS, apoptosis, and stemness- and metastasis-related pathways. B RT-qPCR-verification of RNAseq data for differentially expressed genes in RAS pathway upon 50 nM TGS24 treatment in MDAMB-231 spheres for 24 h: MAP4K3, RAS, MAPK12, c-FOS, c-JUN, HSP90. Quantification of the transcripts’ level relative to the control by qPCR analyses. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). GAPDH considered as housekeeping gene. C RT-qPCR-verification of RNAseq data for differentially expressed genes in metastasis and stemness-related pathway upon 50 nM TGS24 treatment in MDAMB-231 spheres for 24 h: ALDHβ1, WNT-1, β-Catenin, SOX2, MMP12, Vimentin, CD-44. Quantification of the transcripts’ level relative to the control by qPCR analyses. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). GAPDH considered as housekeeping gene. D RT-qPCR-verification of RNAseq data for differentially expressed genes in apoptotic pathway upon 50 nM TGS24 treatment in MDAMB-231 spheres for 24 h: NF-κβ, Sp1, GATA3, CASP9, PARP-1. Quantification of the transcripts’ level relative to the control by qPCR analyses. Error bars represent mean ± SE (N = 3). Statistical differences are determined compared to the control by two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). GAPDH considered as housekeeping gene. E Correlation of log2(fold-change) values from RNA-Seq and RT-PCR analysis. The R2 value is 0.854. F Interaction network of upregulated and downregulated genes, obtained using STRING database, with the minimum required interaction score of 0.400 and network edges representing evidence of an interaction

    Article Snippet: ChIP-grade antibodies (Anti-Nucleolin antibody [4E2]-ChIP Grade (Abcam)), Anti Sp1 antibody (Sigma), Anti c-JUN Rabbit polyclonal antibody (Abcam), and anti-Hsp90β polyclonal antibody (Abcam)) were employed for the experiments.

    Techniques: Expressing, Quantitative RT-PCR, Two Tailed Test, RNA Sequencing Assay, Reverse Transcription Polymerase Chain Reaction

    Dynamics of two tandem G4-motifs at MAPK12 promoter regulated by spatiotemporal binding of Nucleolin and Sp1 regulate cancer stemness via MAPK12-HSP90-RAS-c-JUN-NANOG axis

    Journal: Cellular and Molecular Life Sciences: CMLS

    Article Title: G-quadruplex structural dynamics at MAPK12 promoter dictates transcriptional switch to determine stemness in breast cancer

    doi: 10.1007/s00018-023-05046-6

    Figure Lengend Snippet: Dynamics of two tandem G4-motifs at MAPK12 promoter regulated by spatiotemporal binding of Nucleolin and Sp1 regulate cancer stemness via MAPK12-HSP90-RAS-c-JUN-NANOG axis

    Article Snippet: ChIP-grade antibodies (Anti-Nucleolin antibody [4E2]-ChIP Grade (Abcam)), Anti Sp1 antibody (Sigma), Anti c-JUN Rabbit polyclonal antibody (Abcam), and anti-Hsp90β polyclonal antibody (Abcam)) were employed for the experiments.

    Techniques: Binding Assay

    Primer sequences used for RT-qPCR.

    Journal: Molecular Medicine Reports

    Article Title: SP1‑mediated ADAMTS5 transcription promotes IL‑1β‑induced chondrocyte injury via Wnt/β‑catenin pathway in osteoarthritis

    doi: 10.3892/mmr.2024.13273

    Figure Lengend Snippet: Primer sequences used for RT-qPCR.

    Article Snippet: CHON-001 cells were stimulated with different concentrations (0, 5, 10 and 15 ng/ml) of IL-1β at 37°C for 24 h to establish the OA cell model. For transfection, cells were transfected with 50 nM ADAMTS5 siRNA [si-ADAMTS5, forward (F) 5′-AAAAUGUUUGGAUUCGUGCUC-3′; reverse (R) 5′-GCACGAAUCCAAACAUUUUCC-3′], 50 nM SP1 siRNA (si-SP1, F 5′-ACUUGAUACUGAAUAUUAGGC-3′; R 5′-CUAAUAUUCAGUAUCAAGUAA-3′), 4.0 µg pcDNA3.1 ADAMTS5 overexpression vector (F 5′-AAAGGGGAGAATCTGCCTGC-3′; R 5′-CCAAGATCCCCAGTTGCCAT-3′), 4.0 µg pcDNA3.1 SP1 overexpression vector (F 5′-GTCCGCCCTCTGACCAAG-3′; R 5′-AAGGCACCACCACCATTACC-3′) or negative controls (si-NC, F 5′-GGAGUAGGGAGCAAACCUAUAGGAA-3′, R 5′-UUCCUAUAGGUUUGCUCCCUACUCC-3′; pcDNA3.1, F 5′-CTAGAGAACCCACTGCTTAC-3′, R 5′-TAGAAGGCACAGTCGAGG-3′) using Lipofectamine ® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C for 24 h. All siRNAs and pcDNAs were purchased from Guangzhou RiboBio Co., Ltd. After 24 h post-transfection, cells were treated with 10 ng/ml IL-1β for 24 h at 37°C.

    Techniques: Sequencing

    SP1 activated the transcription of ADAMTS5. (A) Jaspar software identified the binding sites of SP1 on the promoter region of ADAMTS5. (B) ChIP was used to confirm the binding ability of SP1 with the ADAMTS5 promoter. (C) SP1 protein expression was detected by western blotting in 293T cells transfected with pcDNA/SP1. (D) Dual-luciferase reporter assay was used to verify the binding of SP1 with the WT of the ADAMTS5 promoter. (E) Transfection efficiency of si-SP1 was confirmed by western blotting. (F) ADAMTS5 protein expression was examined by western blotting in CHON-001 cells transfected with si-NC/si-SP1. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. SP1, specific protein 1; WT, wild-type; MUT, mutant.

    Journal: Molecular Medicine Reports

    Article Title: SP1‑mediated ADAMTS5 transcription promotes IL‑1β‑induced chondrocyte injury via Wnt/β‑catenin pathway in osteoarthritis

    doi: 10.3892/mmr.2024.13273

    Figure Lengend Snippet: SP1 activated the transcription of ADAMTS5. (A) Jaspar software identified the binding sites of SP1 on the promoter region of ADAMTS5. (B) ChIP was used to confirm the binding ability of SP1 with the ADAMTS5 promoter. (C) SP1 protein expression was detected by western blotting in 293T cells transfected with pcDNA/SP1. (D) Dual-luciferase reporter assay was used to verify the binding of SP1 with the WT of the ADAMTS5 promoter. (E) Transfection efficiency of si-SP1 was confirmed by western blotting. (F) ADAMTS5 protein expression was examined by western blotting in CHON-001 cells transfected with si-NC/si-SP1. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. SP1, specific protein 1; WT, wild-type; MUT, mutant.

    Article Snippet: CHON-001 cells were stimulated with different concentrations (0, 5, 10 and 15 ng/ml) of IL-1β at 37°C for 24 h to establish the OA cell model. For transfection, cells were transfected with 50 nM ADAMTS5 siRNA [si-ADAMTS5, forward (F) 5′-AAAAUGUUUGGAUUCGUGCUC-3′; reverse (R) 5′-GCACGAAUCCAAACAUUUUCC-3′], 50 nM SP1 siRNA (si-SP1, F 5′-ACUUGAUACUGAAUAUUAGGC-3′; R 5′-CUAAUAUUCAGUAUCAAGUAA-3′), 4.0 µg pcDNA3.1 ADAMTS5 overexpression vector (F 5′-AAAGGGGAGAATCTGCCTGC-3′; R 5′-CCAAGATCCCCAGTTGCCAT-3′), 4.0 µg pcDNA3.1 SP1 overexpression vector (F 5′-GTCCGCCCTCTGACCAAG-3′; R 5′-AAGGCACCACCACCATTACC-3′) or negative controls (si-NC, F 5′-GGAGUAGGGAGCAAACCUAUAGGAA-3′, R 5′-UUCCUAUAGGUUUGCUCCCUACUCC-3′; pcDNA3.1, F 5′-CTAGAGAACCCACTGCTTAC-3′, R 5′-TAGAAGGCACAGTCGAGG-3′) using Lipofectamine ® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C for 24 h. All siRNAs and pcDNAs were purchased from Guangzhou RiboBio Co., Ltd. After 24 h post-transfection, cells were treated with 10 ng/ml IL-1β for 24 h at 37°C.

    Techniques: Software, Binding Assay, Expressing, Western Blot, Transfection, Luciferase, Reporter Assay, Mutagenesis

    Effects of si-SP1 and ADAMTS5 on IL-1β-induced chondrocyte injury. CHON-001 cells were transfected with si-NC/si-SP1/pcDNA/ADAMTS5 followed by treatment with IL-1β. (A) ADAMTS5 protein expression was assessed using western blotting. Cell proliferation and apoptosis were determined by (B) MTT assay, (C) EdU assay and (D) flow cytometry. (E) Protein expression of aggrecan and collagen II was evaluated using western blotting. (F) Concentrations of IL-6 and TNF-α were assessed by ELISA. Corresponding kits were used to test (G) SOD activity and (H) MDA levels. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. MDA, malondialdehyde; SOD, superoxide dismutase; NC, negative control; SP1, specific protein 1.

    Journal: Molecular Medicine Reports

    Article Title: SP1‑mediated ADAMTS5 transcription promotes IL‑1β‑induced chondrocyte injury via Wnt/β‑catenin pathway in osteoarthritis

    doi: 10.3892/mmr.2024.13273

    Figure Lengend Snippet: Effects of si-SP1 and ADAMTS5 on IL-1β-induced chondrocyte injury. CHON-001 cells were transfected with si-NC/si-SP1/pcDNA/ADAMTS5 followed by treatment with IL-1β. (A) ADAMTS5 protein expression was assessed using western blotting. Cell proliferation and apoptosis were determined by (B) MTT assay, (C) EdU assay and (D) flow cytometry. (E) Protein expression of aggrecan and collagen II was evaluated using western blotting. (F) Concentrations of IL-6 and TNF-α were assessed by ELISA. Corresponding kits were used to test (G) SOD activity and (H) MDA levels. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. MDA, malondialdehyde; SOD, superoxide dismutase; NC, negative control; SP1, specific protein 1.

    Article Snippet: CHON-001 cells were stimulated with different concentrations (0, 5, 10 and 15 ng/ml) of IL-1β at 37°C for 24 h to establish the OA cell model. For transfection, cells were transfected with 50 nM ADAMTS5 siRNA [si-ADAMTS5, forward (F) 5′-AAAAUGUUUGGAUUCGUGCUC-3′; reverse (R) 5′-GCACGAAUCCAAACAUUUUCC-3′], 50 nM SP1 siRNA (si-SP1, F 5′-ACUUGAUACUGAAUAUUAGGC-3′; R 5′-CUAAUAUUCAGUAUCAAGUAA-3′), 4.0 µg pcDNA3.1 ADAMTS5 overexpression vector (F 5′-AAAGGGGAGAATCTGCCTGC-3′; R 5′-CCAAGATCCCCAGTTGCCAT-3′), 4.0 µg pcDNA3.1 SP1 overexpression vector (F 5′-GTCCGCCCTCTGACCAAG-3′; R 5′-AAGGCACCACCACCATTACC-3′) or negative controls (si-NC, F 5′-GGAGUAGGGAGCAAACCUAUAGGAA-3′, R 5′-UUCCUAUAGGUUUGCUCCCUACUCC-3′; pcDNA3.1, F 5′-CTAGAGAACCCACTGCTTAC-3′, R 5′-TAGAAGGCACAGTCGAGG-3′) using Lipofectamine ® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C for 24 h. All siRNAs and pcDNAs were purchased from Guangzhou RiboBio Co., Ltd. After 24 h post-transfection, cells were treated with 10 ng/ml IL-1β for 24 h at 37°C.

    Techniques: Transfection, Expressing, Western Blot, MTT Assay, EdU Assay, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Activity Assay, Negative Control

    Effects of the SP1/ADAMTS5 axis on the activity of the Wnt/β-catenin pathway. (A) western blotting was used to measure the protein expression of β-catenin and Wnt3a in IL-1β-induced CHON-001 cells transfected with si-NC/si-ADAMTS5. (B) Protein expression of β-catenin and Wnt3a was detected by western blotting in IL-1β-induced CHON-001 cells transfected with si-NC/si-SP1/pcDNA/ADAMTS5. **P<0.01, ***P<0.001 and ****P<0.0001. NC, negative control; SP1, specific protein 1.

    Journal: Molecular Medicine Reports

    Article Title: SP1‑mediated ADAMTS5 transcription promotes IL‑1β‑induced chondrocyte injury via Wnt/β‑catenin pathway in osteoarthritis

    doi: 10.3892/mmr.2024.13273

    Figure Lengend Snippet: Effects of the SP1/ADAMTS5 axis on the activity of the Wnt/β-catenin pathway. (A) western blotting was used to measure the protein expression of β-catenin and Wnt3a in IL-1β-induced CHON-001 cells transfected with si-NC/si-ADAMTS5. (B) Protein expression of β-catenin and Wnt3a was detected by western blotting in IL-1β-induced CHON-001 cells transfected with si-NC/si-SP1/pcDNA/ADAMTS5. **P<0.01, ***P<0.001 and ****P<0.0001. NC, negative control; SP1, specific protein 1.

    Article Snippet: CHON-001 cells were stimulated with different concentrations (0, 5, 10 and 15 ng/ml) of IL-1β at 37°C for 24 h to establish the OA cell model. For transfection, cells were transfected with 50 nM ADAMTS5 siRNA [si-ADAMTS5, forward (F) 5′-AAAAUGUUUGGAUUCGUGCUC-3′; reverse (R) 5′-GCACGAAUCCAAACAUUUUCC-3′], 50 nM SP1 siRNA (si-SP1, F 5′-ACUUGAUACUGAAUAUUAGGC-3′; R 5′-CUAAUAUUCAGUAUCAAGUAA-3′), 4.0 µg pcDNA3.1 ADAMTS5 overexpression vector (F 5′-AAAGGGGAGAATCTGCCTGC-3′; R 5′-CCAAGATCCCCAGTTGCCAT-3′), 4.0 µg pcDNA3.1 SP1 overexpression vector (F 5′-GTCCGCCCTCTGACCAAG-3′; R 5′-AAGGCACCACCACCATTACC-3′) or negative controls (si-NC, F 5′-GGAGUAGGGAGCAAACCUAUAGGAA-3′, R 5′-UUCCUAUAGGUUUGCUCCCUACUCC-3′; pcDNA3.1, F 5′-CTAGAGAACCCACTGCTTAC-3′, R 5′-TAGAAGGCACAGTCGAGG-3′) using Lipofectamine ® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C for 24 h. All siRNAs and pcDNAs were purchased from Guangzhou RiboBio Co., Ltd. After 24 h post-transfection, cells were treated with 10 ng/ml IL-1β for 24 h at 37°C.

    Techniques: Activity Assay, Western Blot, Expressing, Transfection, Negative Control

    Summary diagram of the present study. In IL-1β-induced chondrocytes, the SP1/ADAMTS5/Wnt/β-catenin pathway suppressed proliferation and accelerated apoptosis, ECM degradation, inflammation and oxidative stress to promote OA progression. OA, osteoarthritis; ECM, extracellular matrix; SP1, specific protein 1; MDA, malondialdehyde; SOD, superoxide dismutase.

    Journal: Molecular Medicine Reports

    Article Title: SP1‑mediated ADAMTS5 transcription promotes IL‑1β‑induced chondrocyte injury via Wnt/β‑catenin pathway in osteoarthritis

    doi: 10.3892/mmr.2024.13273

    Figure Lengend Snippet: Summary diagram of the present study. In IL-1β-induced chondrocytes, the SP1/ADAMTS5/Wnt/β-catenin pathway suppressed proliferation and accelerated apoptosis, ECM degradation, inflammation and oxidative stress to promote OA progression. OA, osteoarthritis; ECM, extracellular matrix; SP1, specific protein 1; MDA, malondialdehyde; SOD, superoxide dismutase.

    Article Snippet: CHON-001 cells were stimulated with different concentrations (0, 5, 10 and 15 ng/ml) of IL-1β at 37°C for 24 h to establish the OA cell model. For transfection, cells were transfected with 50 nM ADAMTS5 siRNA [si-ADAMTS5, forward (F) 5′-AAAAUGUUUGGAUUCGUGCUC-3′; reverse (R) 5′-GCACGAAUCCAAACAUUUUCC-3′], 50 nM SP1 siRNA (si-SP1, F 5′-ACUUGAUACUGAAUAUUAGGC-3′; R 5′-CUAAUAUUCAGUAUCAAGUAA-3′), 4.0 µg pcDNA3.1 ADAMTS5 overexpression vector (F 5′-AAAGGGGAGAATCTGCCTGC-3′; R 5′-CCAAGATCCCCAGTTGCCAT-3′), 4.0 µg pcDNA3.1 SP1 overexpression vector (F 5′-GTCCGCCCTCTGACCAAG-3′; R 5′-AAGGCACCACCACCATTACC-3′) or negative controls (si-NC, F 5′-GGAGUAGGGAGCAAACCUAUAGGAA-3′, R 5′-UUCCUAUAGGUUUGCUCCCUACUCC-3′; pcDNA3.1, F 5′-CTAGAGAACCCACTGCTTAC-3′, R 5′-TAGAAGGCACAGTCGAGG-3′) using Lipofectamine ® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C for 24 h. All siRNAs and pcDNAs were purchased from Guangzhou RiboBio Co., Ltd. After 24 h post-transfection, cells were treated with 10 ng/ml IL-1β for 24 h at 37°C.

    Techniques:

    Primer sequences used for RT-qPCR.

    Journal: Molecular Medicine Reports

    Article Title: SP1‑mediated ADAMTS5 transcription promotes IL‑1β‑induced chondrocyte injury via Wnt/β‑catenin pathway in osteoarthritis

    doi: 10.3892/mmr.2024.13273

    Figure Lengend Snippet: Primer sequences used for RT-qPCR.

    Article Snippet: The 293T cells (American Type Culture Collection) were co-transfected with the pcDNA3.1 SP1 overexpression vector and the aforementioned vectors (WT, MUT1, MUT3 and MUT1+3) using Lipofectamine ® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), and the relative luciferase activity (Firefly luciferase/Renilla luciferase) was detected by Dual-Lucy Detection Assay Kit (Beyotime Institute of Biotechnology) after 48 h.

    Techniques: Sequencing

    SP1 activated the transcription of ADAMTS5. (A) Jaspar software identified the binding sites of SP1 on the promoter region of ADAMTS5. (B) ChIP was used to confirm the binding ability of SP1 with the ADAMTS5 promoter. (C) SP1 protein expression was detected by western blotting in 293T cells transfected with pcDNA/SP1. (D) Dual-luciferase reporter assay was used to verify the binding of SP1 with the WT of the ADAMTS5 promoter. (E) Transfection efficiency of si-SP1 was confirmed by western blotting. (F) ADAMTS5 protein expression was examined by western blotting in CHON-001 cells transfected with si-NC/si-SP1. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. SP1, specific protein 1; WT, wild-type; MUT, mutant.

    Journal: Molecular Medicine Reports

    Article Title: SP1‑mediated ADAMTS5 transcription promotes IL‑1β‑induced chondrocyte injury via Wnt/β‑catenin pathway in osteoarthritis

    doi: 10.3892/mmr.2024.13273

    Figure Lengend Snippet: SP1 activated the transcription of ADAMTS5. (A) Jaspar software identified the binding sites of SP1 on the promoter region of ADAMTS5. (B) ChIP was used to confirm the binding ability of SP1 with the ADAMTS5 promoter. (C) SP1 protein expression was detected by western blotting in 293T cells transfected with pcDNA/SP1. (D) Dual-luciferase reporter assay was used to verify the binding of SP1 with the WT of the ADAMTS5 promoter. (E) Transfection efficiency of si-SP1 was confirmed by western blotting. (F) ADAMTS5 protein expression was examined by western blotting in CHON-001 cells transfected with si-NC/si-SP1. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. SP1, specific protein 1; WT, wild-type; MUT, mutant.

    Article Snippet: The 293T cells (American Type Culture Collection) were co-transfected with the pcDNA3.1 SP1 overexpression vector and the aforementioned vectors (WT, MUT1, MUT3 and MUT1+3) using Lipofectamine ® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), and the relative luciferase activity (Firefly luciferase/Renilla luciferase) was detected by Dual-Lucy Detection Assay Kit (Beyotime Institute of Biotechnology) after 48 h.

    Techniques: Software, Binding Assay, Expressing, Western Blot, Transfection, Luciferase, Reporter Assay, Mutagenesis

    Effects of si-SP1 and ADAMTS5 on IL-1β-induced chondrocyte injury. CHON-001 cells were transfected with si-NC/si-SP1/pcDNA/ADAMTS5 followed by treatment with IL-1β. (A) ADAMTS5 protein expression was assessed using western blotting. Cell proliferation and apoptosis were determined by (B) MTT assay, (C) EdU assay and (D) flow cytometry. (E) Protein expression of aggrecan and collagen II was evaluated using western blotting. (F) Concentrations of IL-6 and TNF-α were assessed by ELISA. Corresponding kits were used to test (G) SOD activity and (H) MDA levels. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. MDA, malondialdehyde; SOD, superoxide dismutase; NC, negative control; SP1, specific protein 1.

    Journal: Molecular Medicine Reports

    Article Title: SP1‑mediated ADAMTS5 transcription promotes IL‑1β‑induced chondrocyte injury via Wnt/β‑catenin pathway in osteoarthritis

    doi: 10.3892/mmr.2024.13273

    Figure Lengend Snippet: Effects of si-SP1 and ADAMTS5 on IL-1β-induced chondrocyte injury. CHON-001 cells were transfected with si-NC/si-SP1/pcDNA/ADAMTS5 followed by treatment with IL-1β. (A) ADAMTS5 protein expression was assessed using western blotting. Cell proliferation and apoptosis were determined by (B) MTT assay, (C) EdU assay and (D) flow cytometry. (E) Protein expression of aggrecan and collagen II was evaluated using western blotting. (F) Concentrations of IL-6 and TNF-α were assessed by ELISA. Corresponding kits were used to test (G) SOD activity and (H) MDA levels. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. MDA, malondialdehyde; SOD, superoxide dismutase; NC, negative control; SP1, specific protein 1.

    Article Snippet: The 293T cells (American Type Culture Collection) were co-transfected with the pcDNA3.1 SP1 overexpression vector and the aforementioned vectors (WT, MUT1, MUT3 and MUT1+3) using Lipofectamine ® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), and the relative luciferase activity (Firefly luciferase/Renilla luciferase) was detected by Dual-Lucy Detection Assay Kit (Beyotime Institute of Biotechnology) after 48 h.

    Techniques: Transfection, Expressing, Western Blot, MTT Assay, EdU Assay, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Activity Assay, Negative Control

    Effects of the SP1/ADAMTS5 axis on the activity of the Wnt/β-catenin pathway. (A) western blotting was used to measure the protein expression of β-catenin and Wnt3a in IL-1β-induced CHON-001 cells transfected with si-NC/si-ADAMTS5. (B) Protein expression of β-catenin and Wnt3a was detected by western blotting in IL-1β-induced CHON-001 cells transfected with si-NC/si-SP1/pcDNA/ADAMTS5. **P<0.01, ***P<0.001 and ****P<0.0001. NC, negative control; SP1, specific protein 1.

    Journal: Molecular Medicine Reports

    Article Title: SP1‑mediated ADAMTS5 transcription promotes IL‑1β‑induced chondrocyte injury via Wnt/β‑catenin pathway in osteoarthritis

    doi: 10.3892/mmr.2024.13273

    Figure Lengend Snippet: Effects of the SP1/ADAMTS5 axis on the activity of the Wnt/β-catenin pathway. (A) western blotting was used to measure the protein expression of β-catenin and Wnt3a in IL-1β-induced CHON-001 cells transfected with si-NC/si-ADAMTS5. (B) Protein expression of β-catenin and Wnt3a was detected by western blotting in IL-1β-induced CHON-001 cells transfected with si-NC/si-SP1/pcDNA/ADAMTS5. **P<0.01, ***P<0.001 and ****P<0.0001. NC, negative control; SP1, specific protein 1.

    Article Snippet: The 293T cells (American Type Culture Collection) were co-transfected with the pcDNA3.1 SP1 overexpression vector and the aforementioned vectors (WT, MUT1, MUT3 and MUT1+3) using Lipofectamine ® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), and the relative luciferase activity (Firefly luciferase/Renilla luciferase) was detected by Dual-Lucy Detection Assay Kit (Beyotime Institute of Biotechnology) after 48 h.

    Techniques: Activity Assay, Western Blot, Expressing, Transfection, Negative Control

    Summary diagram of the present study. In IL-1β-induced chondrocytes, the SP1/ADAMTS5/Wnt/β-catenin pathway suppressed proliferation and accelerated apoptosis, ECM degradation, inflammation and oxidative stress to promote OA progression. OA, osteoarthritis; ECM, extracellular matrix; SP1, specific protein 1; MDA, malondialdehyde; SOD, superoxide dismutase.

    Journal: Molecular Medicine Reports

    Article Title: SP1‑mediated ADAMTS5 transcription promotes IL‑1β‑induced chondrocyte injury via Wnt/β‑catenin pathway in osteoarthritis

    doi: 10.3892/mmr.2024.13273

    Figure Lengend Snippet: Summary diagram of the present study. In IL-1β-induced chondrocytes, the SP1/ADAMTS5/Wnt/β-catenin pathway suppressed proliferation and accelerated apoptosis, ECM degradation, inflammation and oxidative stress to promote OA progression. OA, osteoarthritis; ECM, extracellular matrix; SP1, specific protein 1; MDA, malondialdehyde; SOD, superoxide dismutase.

    Article Snippet: The 293T cells (American Type Culture Collection) were co-transfected with the pcDNA3.1 SP1 overexpression vector and the aforementioned vectors (WT, MUT1, MUT3 and MUT1+3) using Lipofectamine ® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), and the relative luciferase activity (Firefly luciferase/Renilla luciferase) was detected by Dual-Lucy Detection Assay Kit (Beyotime Institute of Biotechnology) after 48 h.

    Techniques:

    Primer sequences used for RT-qPCR.

    Journal: Molecular Medicine Reports

    Article Title: SP1‑mediated ADAMTS5 transcription promotes IL‑1β‑induced chondrocyte injury via Wnt/β‑catenin pathway in osteoarthritis

    doi: 10.3892/mmr.2024.13273

    Figure Lengend Snippet: Primer sequences used for RT-qPCR.

    Article Snippet: The membrane was blocked with 5% skimmed milk at 4°C for 2 h and incubated with the following primary antibodies (all Abcam): Anti-Aggrecan (1:1,000; cat. no. ab36861), anti-Collagen II (1:1,000; cat. no. ab34712), anti-ADAMTS5 (1:250; cat. no. ab41037), anti-SP1 (1:10,000; cat. no. ab227383), anti-Wnt3a (1:1,000; cat. no. ab28472), anti-β-catenin (1:4,000; cat. no. ab16051) and anti-GAPDH (1:2,500; cat. no. ab9485) at 4°C overnight.

    Techniques: Sequencing

    SP1 activated the transcription of ADAMTS5. (A) Jaspar software identified the binding sites of SP1 on the promoter region of ADAMTS5. (B) ChIP was used to confirm the binding ability of SP1 with the ADAMTS5 promoter. (C) SP1 protein expression was detected by western blotting in 293T cells transfected with pcDNA/SP1. (D) Dual-luciferase reporter assay was used to verify the binding of SP1 with the WT of the ADAMTS5 promoter. (E) Transfection efficiency of si-SP1 was confirmed by western blotting. (F) ADAMTS5 protein expression was examined by western blotting in CHON-001 cells transfected with si-NC/si-SP1. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. SP1, specific protein 1; WT, wild-type; MUT, mutant.

    Journal: Molecular Medicine Reports

    Article Title: SP1‑mediated ADAMTS5 transcription promotes IL‑1β‑induced chondrocyte injury via Wnt/β‑catenin pathway in osteoarthritis

    doi: 10.3892/mmr.2024.13273

    Figure Lengend Snippet: SP1 activated the transcription of ADAMTS5. (A) Jaspar software identified the binding sites of SP1 on the promoter region of ADAMTS5. (B) ChIP was used to confirm the binding ability of SP1 with the ADAMTS5 promoter. (C) SP1 protein expression was detected by western blotting in 293T cells transfected with pcDNA/SP1. (D) Dual-luciferase reporter assay was used to verify the binding of SP1 with the WT of the ADAMTS5 promoter. (E) Transfection efficiency of si-SP1 was confirmed by western blotting. (F) ADAMTS5 protein expression was examined by western blotting in CHON-001 cells transfected with si-NC/si-SP1. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. SP1, specific protein 1; WT, wild-type; MUT, mutant.

    Article Snippet: The membrane was blocked with 5% skimmed milk at 4°C for 2 h and incubated with the following primary antibodies (all Abcam): Anti-Aggrecan (1:1,000; cat. no. ab36861), anti-Collagen II (1:1,000; cat. no. ab34712), anti-ADAMTS5 (1:250; cat. no. ab41037), anti-SP1 (1:10,000; cat. no. ab227383), anti-Wnt3a (1:1,000; cat. no. ab28472), anti-β-catenin (1:4,000; cat. no. ab16051) and anti-GAPDH (1:2,500; cat. no. ab9485) at 4°C overnight.

    Techniques: Software, Binding Assay, Expressing, Western Blot, Transfection, Luciferase, Reporter Assay, Mutagenesis

    Effects of si-SP1 and ADAMTS5 on IL-1β-induced chondrocyte injury. CHON-001 cells were transfected with si-NC/si-SP1/pcDNA/ADAMTS5 followed by treatment with IL-1β. (A) ADAMTS5 protein expression was assessed using western blotting. Cell proliferation and apoptosis were determined by (B) MTT assay, (C) EdU assay and (D) flow cytometry. (E) Protein expression of aggrecan and collagen II was evaluated using western blotting. (F) Concentrations of IL-6 and TNF-α were assessed by ELISA. Corresponding kits were used to test (G) SOD activity and (H) MDA levels. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. MDA, malondialdehyde; SOD, superoxide dismutase; NC, negative control; SP1, specific protein 1.

    Journal: Molecular Medicine Reports

    Article Title: SP1‑mediated ADAMTS5 transcription promotes IL‑1β‑induced chondrocyte injury via Wnt/β‑catenin pathway in osteoarthritis

    doi: 10.3892/mmr.2024.13273

    Figure Lengend Snippet: Effects of si-SP1 and ADAMTS5 on IL-1β-induced chondrocyte injury. CHON-001 cells were transfected with si-NC/si-SP1/pcDNA/ADAMTS5 followed by treatment with IL-1β. (A) ADAMTS5 protein expression was assessed using western blotting. Cell proliferation and apoptosis were determined by (B) MTT assay, (C) EdU assay and (D) flow cytometry. (E) Protein expression of aggrecan and collagen II was evaluated using western blotting. (F) Concentrations of IL-6 and TNF-α were assessed by ELISA. Corresponding kits were used to test (G) SOD activity and (H) MDA levels. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. MDA, malondialdehyde; SOD, superoxide dismutase; NC, negative control; SP1, specific protein 1.

    Article Snippet: The membrane was blocked with 5% skimmed milk at 4°C for 2 h and incubated with the following primary antibodies (all Abcam): Anti-Aggrecan (1:1,000; cat. no. ab36861), anti-Collagen II (1:1,000; cat. no. ab34712), anti-ADAMTS5 (1:250; cat. no. ab41037), anti-SP1 (1:10,000; cat. no. ab227383), anti-Wnt3a (1:1,000; cat. no. ab28472), anti-β-catenin (1:4,000; cat. no. ab16051) and anti-GAPDH (1:2,500; cat. no. ab9485) at 4°C overnight.

    Techniques: Transfection, Expressing, Western Blot, MTT Assay, EdU Assay, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Activity Assay, Negative Control

    Effects of the SP1/ADAMTS5 axis on the activity of the Wnt/β-catenin pathway. (A) western blotting was used to measure the protein expression of β-catenin and Wnt3a in IL-1β-induced CHON-001 cells transfected with si-NC/si-ADAMTS5. (B) Protein expression of β-catenin and Wnt3a was detected by western blotting in IL-1β-induced CHON-001 cells transfected with si-NC/si-SP1/pcDNA/ADAMTS5. **P<0.01, ***P<0.001 and ****P<0.0001. NC, negative control; SP1, specific protein 1.

    Journal: Molecular Medicine Reports

    Article Title: SP1‑mediated ADAMTS5 transcription promotes IL‑1β‑induced chondrocyte injury via Wnt/β‑catenin pathway in osteoarthritis

    doi: 10.3892/mmr.2024.13273

    Figure Lengend Snippet: Effects of the SP1/ADAMTS5 axis on the activity of the Wnt/β-catenin pathway. (A) western blotting was used to measure the protein expression of β-catenin and Wnt3a in IL-1β-induced CHON-001 cells transfected with si-NC/si-ADAMTS5. (B) Protein expression of β-catenin and Wnt3a was detected by western blotting in IL-1β-induced CHON-001 cells transfected with si-NC/si-SP1/pcDNA/ADAMTS5. **P<0.01, ***P<0.001 and ****P<0.0001. NC, negative control; SP1, specific protein 1.

    Article Snippet: The membrane was blocked with 5% skimmed milk at 4°C for 2 h and incubated with the following primary antibodies (all Abcam): Anti-Aggrecan (1:1,000; cat. no. ab36861), anti-Collagen II (1:1,000; cat. no. ab34712), anti-ADAMTS5 (1:250; cat. no. ab41037), anti-SP1 (1:10,000; cat. no. ab227383), anti-Wnt3a (1:1,000; cat. no. ab28472), anti-β-catenin (1:4,000; cat. no. ab16051) and anti-GAPDH (1:2,500; cat. no. ab9485) at 4°C overnight.

    Techniques: Activity Assay, Western Blot, Expressing, Transfection, Negative Control

    Summary diagram of the present study. In IL-1β-induced chondrocytes, the SP1/ADAMTS5/Wnt/β-catenin pathway suppressed proliferation and accelerated apoptosis, ECM degradation, inflammation and oxidative stress to promote OA progression. OA, osteoarthritis; ECM, extracellular matrix; SP1, specific protein 1; MDA, malondialdehyde; SOD, superoxide dismutase.

    Journal: Molecular Medicine Reports

    Article Title: SP1‑mediated ADAMTS5 transcription promotes IL‑1β‑induced chondrocyte injury via Wnt/β‑catenin pathway in osteoarthritis

    doi: 10.3892/mmr.2024.13273

    Figure Lengend Snippet: Summary diagram of the present study. In IL-1β-induced chondrocytes, the SP1/ADAMTS5/Wnt/β-catenin pathway suppressed proliferation and accelerated apoptosis, ECM degradation, inflammation and oxidative stress to promote OA progression. OA, osteoarthritis; ECM, extracellular matrix; SP1, specific protein 1; MDA, malondialdehyde; SOD, superoxide dismutase.

    Article Snippet: The membrane was blocked with 5% skimmed milk at 4°C for 2 h and incubated with the following primary antibodies (all Abcam): Anti-Aggrecan (1:1,000; cat. no. ab36861), anti-Collagen II (1:1,000; cat. no. ab34712), anti-ADAMTS5 (1:250; cat. no. ab41037), anti-SP1 (1:10,000; cat. no. ab227383), anti-Wnt3a (1:1,000; cat. no. ab28472), anti-β-catenin (1:4,000; cat. no. ab16051) and anti-GAPDH (1:2,500; cat. no. ab9485) at 4°C overnight.

    Techniques: