Journal: Neurotoxicity Research

Article Title: Perinatal Asphyxia Leads to PARP-1 Overactivity, p65 Translocation, IL-1β and TNF-α Overexpression, and Apoptotic-Like Cell Death in Mesencephalon of Neonatal Rats: Prevention by Systemic Neonatal Nicotinamide Administration

doi: 10.1007/s12640-015-9517-0

Figure Lengend Snippet: Effect of perinatal asphyxia on IκBα, nuclear translocation of p65, IL-1β and TNF-α mRNA levels in mesencephalon of rat neonates, 1–24 h after birth. Caesarean-delivered control (CS) and asphyxia-exposed (AS) rats were euthanized 1–24 h after delivery, and brain tissue sampled and treated for Western blots or RT-qPCR. a Representative immunoblots for IκBα (35–41 kDa) and β-actin (42 kDa) levels. b IκBα levels (normalized to β-actin; A.U. arbitrary units) (CS open columns , AS grey columns ). c Representative immunoblots for p65 (65 kDa), α-tubulin (45 kDa) and histone H4 (15–11 kDa) levels, measured in cytoplasmic ( c ) and nuclear ( n ) protein extracts. d Nuclear p65 levels normalized to total p65. e , f IL-1β (E) and TNF-α f mRNA levels measured by RT-qPCR with specific primers; analysed in triplicates with MxPro software and normalized to GAPDH mRNA levels. Pair-wise comparisons analysed with Student t test (* p < 0.05, *** p < 0.0005; n = 5–7, for each condition and experiment)

Article Snippet: Membranes were blocked with Tris-buffered saline (TBS) containing 0.1 % Tween-20 and 5 % (w/v) non-fat dry milk at room temperature for 1 h, and incubated with (i) mouse anti-pADPr (170–70 kDa) (3H2844) (sc-71848, Santa Cruz Biotechnology); (ii) goat anti-PARP-1 (113 kDa) (sc-F0908, Santa Cruz Biotechnology); (iii) Rabbit anti-p65 (65 kDa) (sc-372, Santa Cruz Biotechnology); (iv) Mouse anti-iKB-α (35–41 kDa) (sc-1643, Santa Cruz Biotechnology); (v) Mouse anti-β-actin (42 kDa) (Sigma A5316); (vi) Rabbit anti-histone H4 (13–15 kDa) (Abcam 1261) and (vii) mouse anti-α-tubulin (45 kDa) (Sigma T8203).

Techniques: Translocation Assay, Western Blot, Quantitative RT-PCR, Software

Journal: Neurotoxicity Research

Article Title: Perinatal Asphyxia Leads to PARP-1 Overactivity, p65 Translocation, IL-1β and TNF-α Overexpression, and Apoptotic-Like Cell Death in Mesencephalon of Neonatal Rats: Prevention by Systemic Neonatal Nicotinamide Administration

doi: 10.1007/s12640-015-9517-0

Figure Lengend Snippet: Effect of nicotinamide or saline on p65 translocation; on p65, IL-1β and TNF-α mRNA levels, and on ELISA TNF-α protein levels in mesencephalon of asphyxia-exposed and control rats. Caesarean-delivered control (CS) and asphyxia-exposed (AS) rats were treated with a single dose of nicotinamide (0.8 mmol/kg, i.p.) (CNam, ANam) or saline (0.1 ml, i.p.) (CSal, ASal), 1 h after delivery, euthanized at 8 or 24 h after birth. Selected brain tissue was treated for Western blots, RT-qPCR or ELISA. a Representative immunoblots for p65, α-tubulin and histone H4 levels, measured in cytoplasmic ( c ) and nuclear ( n ) protein extracts. b Nuclear p65 levels normalized to total p65. c De novo synthesis of p65, d IL-1β and e TNF-α, measured by RT-qPCR 8 h and/or 24 h after delivery (data analysed in triplicates with MxPro software, normalized to GAPDH mRNA levels). f TNF-α protein levels measured in total protein extracts with Quantikine ® ELISA Rat (normalized by mg of protein for each sample, in duplicate) (CSal open columns , CNam dashed columns , ASal grey columns , ANam doubled dashed columns ). Pair-wise comparisons analysed with Student t test (* p < 0.05, ** p < 0.005)

Article Snippet: Membranes were blocked with Tris-buffered saline (TBS) containing 0.1 % Tween-20 and 5 % (w/v) non-fat dry milk at room temperature for 1 h, and incubated with (i) mouse anti-pADPr (170–70 kDa) (3H2844) (sc-71848, Santa Cruz Biotechnology); (ii) goat anti-PARP-1 (113 kDa) (sc-F0908, Santa Cruz Biotechnology); (iii) Rabbit anti-p65 (65 kDa) (sc-372, Santa Cruz Biotechnology); (iv) Mouse anti-iKB-α (35–41 kDa) (sc-1643, Santa Cruz Biotechnology); (v) Mouse anti-β-actin (42 kDa) (Sigma A5316); (vi) Rabbit anti-histone H4 (13–15 kDa) (Abcam 1261) and (vii) mouse anti-α-tubulin (45 kDa) (Sigma T8203).

Techniques: Translocation Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Quantitative RT-PCR, Software

Journal: International journal of biological sciences

Article Title: Prolonged DEHP exposure enhances the stemness and metastatic potential of TNBC cells in an MSI2-dependent manner.

doi: 10.7150/ijbs.101598

Figure Lengend Snippet: Figure 4. Prolonged DEHP exposure induces EMT and stemness in MDA-MB-231 cells in an MSI2-dependent manner. (A) Changes in the expression of the EMT markers α-SMA, β-catenin, SNAI1, and vimentin, as evaluated by western blotting. (B) Evaluation of the effect of MSI2 knockdown on the expression of EMT markers by western blotting. (C) Anchorage-independent growth/spheroid formation as evaluated by the soft agar colony formation assay. Scale bar = 50 µm. (D-E) Quantitative analysis of the number and size of colonies/spheroids originating from untreated and DEHP-exposed MDA-MB-231 cells. (F) Assessment of anchorage-independent growth/spheroid formation by soft agar colony formation assays in Scr-treated and MSI2-depleted cells. Scale bar = 50 µm. (G-H) Quantitative analysis of colony number and colony size in the soft agar colony formation assay (mean ± SD). (I) Changes in the expression of the stemness-related markers CD133, cMyc, and SOX-2 in untreated and DEHP-exposed clones evaluated by western blotting. (J) Evaluation of changes in the expression of stemness-related markers in Scr-treated and MSI2-depleted MDA-MB-231 cells and in DEHP-exposed clones; **P < 0.001.

Article Snippet: Primary antibodies targeting the following proteins were used: MSI2 (37 kDa, Proteintech, 10770-1-AP), β-catenin (92 kDa, Proteintech, 51067-2-AP), vimentin (54 kDa, Gentex, GTX110619), α-SMA (46 kDa, Abcam, ab32575), SNAI 1 (28 kDa, CusAb, CSB-PA021867LA01HU), CD133 (115 kDa, Proteintech, 18470-1-AP), c-Myc (69 kDa, Abcam, ab32072), SOX2 (35 kDa, CusAb, PA16539AORb), HLA class I ABC (41 kDa, Proteintech, 66013-1-Ig), β-actin (45 kDa, Santa Cruz, SC-47778), phospho-PI3K p85 (Tyr458) (85 kDa, Cell Int.

Techniques: Expressing, Western Blot, Knockdown, Soft Agar Assay, Clone Assay

Journal: Neurotoxicity Research

Article Title: Perinatal Asphyxia Leads to PARP-1 Overactivity, p65 Translocation, IL-1β and TNF-α Overexpression, and Apoptotic-Like Cell Death in Mesencephalon of Neonatal Rats: Prevention by Systemic Neonatal Nicotinamide Administration

doi: 10.1007/s12640-015-9517-0

Figure Lengend Snippet: Effect of perinatal asphyxia on pADPr, PARP-1 levels and PARP-1 activity in mesencephalon of rat neonates, 0–24 h after delivery. Caesarean-delivered control (CS) and asphyxia-exposed (AS) rats were euthanized immediately after delivery (0 h), or following re-oxygenation (1–24 h), brain tissue sampled and treated for Western blots. a Representative immunoblots for pADPr (170–70 kDa), PARP-1 (113 kDa) and β-actin (42 kDa) levels following hypoxia and/or re-oxygenation. b pADPr levels (expressed as arbitrary units, A.U.) (CS open columns , AS grey columns ). c PARP-1 levels, normalized to β-actin, A.U.). d PARP-1 activity (pADPr/PARP-1 ratio) (CS open circles , AS filled triangles ). Pair-wise comparisons were analysed with Student t test (* p < 0.05, ** p < 0.005; n = 5–7, for each condition and experiment)

Article Snippet: Membranes were blocked with Tris-buffered saline (TBS) containing 0.1 % Tween-20 and 5 % (w/v) non-fat dry milk at room temperature for 1 h, and incubated with (i) mouse anti-pADPr (170–70 kDa) (3H2844) (sc-71848, Santa Cruz Biotechnology); (ii) goat anti-PARP-1 (113 kDa) (sc-F0908, Santa Cruz Biotechnology); (iii) Rabbit anti-p65 (65 kDa) (sc-372, Santa Cruz Biotechnology); (iv) Mouse anti-iKB-α (35–41 kDa) (sc-1643, Santa Cruz Biotechnology); (v) Mouse anti-β-actin (42 kDa) (Sigma A5316); (vi) Rabbit anti-histone H4 (13–15 kDa) (Abcam 1261) and (vii) mouse anti-α-tubulin (45 kDa) (Sigma T8203).

Techniques: Activity Assay, Western Blot

Journal: Neurotoxicity Research

Article Title: Perinatal Asphyxia Leads to PARP-1 Overactivity, p65 Translocation, IL-1β and TNF-α Overexpression, and Apoptotic-Like Cell Death in Mesencephalon of Neonatal Rats: Prevention by Systemic Neonatal Nicotinamide Administration

doi: 10.1007/s12640-015-9517-0

Figure Lengend Snippet: Effect of perinatal asphyxia on IκBα, nuclear translocation of p65, IL-1β and TNF-α mRNA levels in mesencephalon of rat neonates, 1–24 h after birth. Caesarean-delivered control (CS) and asphyxia-exposed (AS) rats were euthanized 1–24 h after delivery, and brain tissue sampled and treated for Western blots or RT-qPCR. a Representative immunoblots for IκBα (35–41 kDa) and β-actin (42 kDa) levels. b IκBα levels (normalized to β-actin; A.U. arbitrary units) (CS open columns , AS grey columns ). c Representative immunoblots for p65 (65 kDa), α-tubulin (45 kDa) and histone H4 (15–11 kDa) levels, measured in cytoplasmic ( c ) and nuclear ( n ) protein extracts. d Nuclear p65 levels normalized to total p65. e , f IL-1β (E) and TNF-α f mRNA levels measured by RT-qPCR with specific primers; analysed in triplicates with MxPro software and normalized to GAPDH mRNA levels. Pair-wise comparisons analysed with Student t test (* p < 0.05, *** p < 0.0005; n = 5–7, for each condition and experiment)

Article Snippet: Membranes were blocked with Tris-buffered saline (TBS) containing 0.1 % Tween-20 and 5 % (w/v) non-fat dry milk at room temperature for 1 h, and incubated with (i) mouse anti-pADPr (170–70 kDa) (3H2844) (sc-71848, Santa Cruz Biotechnology); (ii) goat anti-PARP-1 (113 kDa) (sc-F0908, Santa Cruz Biotechnology); (iii) Rabbit anti-p65 (65 kDa) (sc-372, Santa Cruz Biotechnology); (iv) Mouse anti-iKB-α (35–41 kDa) (sc-1643, Santa Cruz Biotechnology); (v) Mouse anti-β-actin (42 kDa) (Sigma A5316); (vi) Rabbit anti-histone H4 (13–15 kDa) (Abcam 1261) and (vii) mouse anti-α-tubulin (45 kDa) (Sigma T8203).

Techniques: Translocation Assay, Western Blot, Quantitative RT-PCR, Software

Journal: Neurotoxicity Research

Article Title: Perinatal Asphyxia Leads to PARP-1 Overactivity, p65 Translocation, IL-1β and TNF-α Overexpression, and Apoptotic-Like Cell Death in Mesencephalon of Neonatal Rats: Prevention by Systemic Neonatal Nicotinamide Administration

doi: 10.1007/s12640-015-9517-0

Figure Lengend Snippet: Effect of nicotinamide or saline on PARP-1 activity in mesencephalon of asphyxia-exposed and control rats. Caesarean-delivered control (CS) and asphyxia-exposed (AS) rats were treated 1 h after birth with a single dose of Nam (0.8 mmol/kg, i.p.) (CNam, ANam) or saline (0.1 ml, i.p.) (CSal, ASal), and euthanized 2 h after delivery. Brain tissue sampled and treated for Western blots. pADPr and PARP-1 protein levels were measured in total protein extracts. PARP-1 activity was estimated as the pADPr/PARP-1 ratio. a Representative immunoblots for pADPr, PARP-1 and β-actin levels; b pADPr levels (expressed as arbitrary units, A.U.); c PARP-1 levels (normalized to β-actin, A.U.); d PARP-1 activity (pADPr/PARP-1 ratio) (CSal open columns , CNam dashed columns , ASal grey columns , ANam double dashed columns ). Pair-wise comparisons analysed with Student t test (* p < 0.05, ** p < 0.005, *** p < 0.0005; n = 4–5, for each condition and experiment)

Article Snippet: Membranes were blocked with Tris-buffered saline (TBS) containing 0.1 % Tween-20 and 5 % (w/v) non-fat dry milk at room temperature for 1 h, and incubated with (i) mouse anti-pADPr (170–70 kDa) (3H2844) (sc-71848, Santa Cruz Biotechnology); (ii) goat anti-PARP-1 (113 kDa) (sc-F0908, Santa Cruz Biotechnology); (iii) Rabbit anti-p65 (65 kDa) (sc-372, Santa Cruz Biotechnology); (iv) Mouse anti-iKB-α (35–41 kDa) (sc-1643, Santa Cruz Biotechnology); (v) Mouse anti-β-actin (42 kDa) (Sigma A5316); (vi) Rabbit anti-histone H4 (13–15 kDa) (Abcam 1261) and (vii) mouse anti-α-tubulin (45 kDa) (Sigma T8203).

Techniques: Activity Assay, Western Blot

Journal: International journal of biological sciences

Article Title: Prolonged DEHP exposure enhances the stemness and metastatic potential of TNBC cells in an MSI2-dependent manner.

doi: 10.7150/ijbs.101598

Figure Lengend Snippet: Figure 2. Involvement of MSI2 in DEHP-induced migration and invasion as predicted by NGS analysis. (A) Heatmap of DEGs in control and DEHP-exposed cells. (B) Venn diagram of total DEGs overlapping among different samples; volcano plot of the DEGs; pie chart of common differentially expressed GO terms in control and DEHP-exposed clones #1 and #2. (C) Bar chart of significantly enriched GO terms and the number of DEGs enriched in biological processes (green) and cellular components (orange); (* significant enrichment). (D) IPA-derived heatmap analysis of cellular movement under various conditions and the functions of the DEGs involved. (E) Downstream analysis of genes involved in the migration and invasion of tumor cell lines, highlighting increased MSI2 expression.

Article Snippet: Primary antibodies targeting the following proteins were used: MSI2 (37 kDa, Proteintech, 10770-1-AP), β-catenin (92 kDa, Proteintech, 51067-2-AP), vimentin (54 kDa, Gentex, GTX110619), α-SMA (46 kDa, Abcam, ab32575), SNAI 1 (28 kDa, CusAb, CSB-PA021867LA01HU), CD133 (115 kDa, Proteintech, 18470-1-AP), c-Myc (69 kDa, Abcam, ab32072), SOX2 (35 kDa, CusAb, PA16539AORb), HLA class I ABC (41 kDa, Proteintech, 66013-1-Ig), β-actin (45 kDa, Santa Cruz, SC-47778), phospho-PI3K p85 (Tyr458) (85 kDa, Cell Int.

Techniques: Migration, Control, Clone Assay, Derivative Assay, Expressing

Journal: International journal of biological sciences

Article Title: Prolonged DEHP exposure enhances the stemness and metastatic potential of TNBC cells in an MSI2-dependent manner.

doi: 10.7150/ijbs.101598

Figure Lengend Snippet: Figure 3. MSI2 knockdown reversed DEHP-induced migration and invasion in vitro and in vivo. (A) Cell morphology of Scr-treated and MSI2-silenced MDA-MB-231 and DEHP-exposed clones as observed by light microscopy. Scale bar = 150 µm. (B) Quantitative analysis of cell length (means ±SDs). (C) A wound healing assay was performed to validate the role of MSI2 in DEHP-induced migration in Scr- and MSI2-silenced MDA-MB-231 cells and in DEHP-exposed clones. (D) Quantitative analysis of cell migration at 20 h after wound formation (mean ± SD). (E-F) Matrigel-coated Transwell assay to evaluate the effect of MSI2 knockdown on cell invasion and quantitative analysis of cell invasion for (E) 12h and (F) 24h. (G) Zebrafish xenograft assay to evaluate the cell migration of Scr-treated and MSI2-silenced MDA-MB-231- and DEHP-exposed clones in 48 hpf Tg (fli1:EGFP) zebrafish embryos (fluorescence image captured at 24 hpi). (H-I) Quantification of embryos showing metastasis to SIV (mean ±SD, n=50) and fluorescence intensity analysis of cells that migrated to SIV in zebrafish embryos (fluorescence intensity reflects the cell number, fold change vs. control); ** P < 0.001.

Article Snippet: Primary antibodies targeting the following proteins were used: MSI2 (37 kDa, Proteintech, 10770-1-AP), β-catenin (92 kDa, Proteintech, 51067-2-AP), vimentin (54 kDa, Gentex, GTX110619), α-SMA (46 kDa, Abcam, ab32575), SNAI 1 (28 kDa, CusAb, CSB-PA021867LA01HU), CD133 (115 kDa, Proteintech, 18470-1-AP), c-Myc (69 kDa, Abcam, ab32072), SOX2 (35 kDa, CusAb, PA16539AORb), HLA class I ABC (41 kDa, Proteintech, 66013-1-Ig), β-actin (45 kDa, Santa Cruz, SC-47778), phospho-PI3K p85 (Tyr458) (85 kDa, Cell Int.

Techniques: Knockdown, Migration, In Vitro, In Vivo, Clone Assay, Light Microscopy, Wound Healing Assay, Transwell Assay, Xenograft Assay, Fluorescence, Control

Journal: International journal of biological sciences

Article Title: Prolonged DEHP exposure enhances the stemness and metastatic potential of TNBC cells in an MSI2-dependent manner.

doi: 10.7150/ijbs.101598

Figure Lengend Snippet: Figure 4. Prolonged DEHP exposure induces EMT and stemness in MDA-MB-231 cells in an MSI2-dependent manner. (A) Changes in the expression of the EMT markers α-SMA, β-catenin, SNAI1, and vimentin, as evaluated by western blotting. (B) Evaluation of the effect of MSI2 knockdown on the expression of EMT markers by western blotting. (C) Anchorage-independent growth/spheroid formation as evaluated by the soft agar colony formation assay. Scale bar = 50 µm. (D-E) Quantitative analysis of the number and size of colonies/spheroids originating from untreated and DEHP-exposed MDA-MB-231 cells. (F) Assessment of anchorage-independent growth/spheroid formation by soft agar colony formation assays in Scr-treated and MSI2-depleted cells. Scale bar = 50 µm. (G-H) Quantitative analysis of colony number and colony size in the soft agar colony formation assay (mean ± SD). (I) Changes in the expression of the stemness-related markers CD133, cMyc, and SOX-2 in untreated and DEHP-exposed clones evaluated by western blotting. (J) Evaluation of changes in the expression of stemness-related markers in Scr-treated and MSI2-depleted MDA-MB-231 cells and in DEHP-exposed clones; **P < 0.001.

Article Snippet: Primary antibodies targeting the following proteins were used: MSI2 (37 kDa, Proteintech, 10770-1-AP), β-catenin (92 kDa, Proteintech, 51067-2-AP), vimentin (54 kDa, Gentex, GTX110619), α-SMA (46 kDa, Abcam, ab32575), SNAI 1 (28 kDa, CusAb, CSB-PA021867LA01HU), CD133 (115 kDa, Proteintech, 18470-1-AP), c-Myc (69 kDa, Abcam, ab32072), SOX2 (35 kDa, CusAb, PA16539AORb), HLA class I ABC (41 kDa, Proteintech, 66013-1-Ig), β-actin (45 kDa, Santa Cruz, SC-47778), phospho-PI3K p85 (Tyr458) (85 kDa, Cell Int.

Techniques: Expressing, Western Blot, Knockdown, Soft Agar Assay, Clone Assay

Journal: International journal of biological sciences

Article Title: Prolonged DEHP exposure enhances the stemness and metastatic potential of TNBC cells in an MSI2-dependent manner.

doi: 10.7150/ijbs.101598

Figure Lengend Snippet: Figure 5. MSI2 regulates the PI3K/Akt/NFκB signaling axis. (A) Heatmap of DEGs from the co-IP protein complexes in control and DEHP-exposed cells. (B) Volcano plot of total DEGs in control and DEHP-exposed cells (red: upregulated; green: downregulated). (C) KEGG pathway analysis identified the 5 most significantly enriched pathways with DEG annotations, p values, and q values. (D-E) GSEA of TNFα and Akt signaling revealed a positive correlation in untreated MDA-MB-231 cells and DEHP-exposed clone #1 cells (enrichment score). (F) Evaluation of the expression of the PI3K/Akt/NFκB signaling markers PI3K p85, p-PI3K, Akt, p-Akt, Ikkα, Ikkβ, Ikkε, p-Ikkα/β, and NFκB in untreated MDA-MB-231 cells and DEHP-exposed clones.

Article Snippet: Primary antibodies targeting the following proteins were used: MSI2 (37 kDa, Proteintech, 10770-1-AP), β-catenin (92 kDa, Proteintech, 51067-2-AP), vimentin (54 kDa, Gentex, GTX110619), α-SMA (46 kDa, Abcam, ab32575), SNAI 1 (28 kDa, CusAb, CSB-PA021867LA01HU), CD133 (115 kDa, Proteintech, 18470-1-AP), c-Myc (69 kDa, Abcam, ab32072), SOX2 (35 kDa, CusAb, PA16539AORb), HLA class I ABC (41 kDa, Proteintech, 66013-1-Ig), β-actin (45 kDa, Santa Cruz, SC-47778), phospho-PI3K p85 (Tyr458) (85 kDa, Cell Int.

Techniques: Co-Immunoprecipitation Assay, Control, Expressing, Clone Assay

Journal: International journal of biological sciences

Article Title: Prolonged DEHP exposure enhances the stemness and metastatic potential of TNBC cells in an MSI2-dependent manner.

doi: 10.7150/ijbs.101598

Figure Lengend Snippet: Figure 6. NFκB controls DEHP-induced cell migration via MMP-9 regulation. (A) Total and nuclear NFκB p65 expression levels in Scr-treated and MSI2-depleted MDA-MB-231 and DEHP-exposed clones were evaluated by western blotting. (B) Gelatin zymography analysis of MMP-9 expression in Scr-treated and MSI2-depleted MDA-MB-231 cells and DEHP-exposed clones. (C) IF analysis of intracellular NFκB p65 (red) localization and expression. Nuclear staining (blue). Scale bar = 100 µm. (D-E) Evaluation of total and nuclear NFκB p65 expression levels following BAY 11--7082 treatment (10 µM, 24 h) by western blotting in MDA-MB-231 and DEHP-exposed clones. (F) MMP-9 expression/activity analysis by gelatin zymography in BAY 11-7082 (10 µM, 24 h)-treated MDA-MB-231 cells and DEHP-exposed clones. (G) IF analysis of intracellular NFκB p65 (red) localization and expression following BAY 11--7082 (10 µM, 24 h) treatment. Nuclear staining (blue). Scale bar = 100 µm. (H) Effect of BAY 11-7082 (10 µM, 24 h) treatment on cell migration as evaluated by a wound healing assay for 24 h. (I) Quantitative analysis of cell migration at 24 h after wound formation (mean ± SD); **P < 0.001.

Article Snippet: Primary antibodies targeting the following proteins were used: MSI2 (37 kDa, Proteintech, 10770-1-AP), β-catenin (92 kDa, Proteintech, 51067-2-AP), vimentin (54 kDa, Gentex, GTX110619), α-SMA (46 kDa, Abcam, ab32575), SNAI 1 (28 kDa, CusAb, CSB-PA021867LA01HU), CD133 (115 kDa, Proteintech, 18470-1-AP), c-Myc (69 kDa, Abcam, ab32072), SOX2 (35 kDa, CusAb, PA16539AORb), HLA class I ABC (41 kDa, Proteintech, 66013-1-Ig), β-actin (45 kDa, Santa Cruz, SC-47778), phospho-PI3K p85 (Tyr458) (85 kDa, Cell Int.

Techniques: Migration, Expressing, Clone Assay, Western Blot, Zymography, Staining, Activity Assay, Wound Healing Assay

Journal: International journal of biological sciences

Article Title: Prolonged DEHP exposure enhances the stemness and metastatic potential of TNBC cells in an MSI2-dependent manner.

doi: 10.7150/ijbs.101598

Figure Lengend Snippet: Figure 7. MSI2 interacts with vimentin and regulates its expression and subcellular distribution. (A) SDS–PAGE analysis of total cell lysates, Flag-MSI2 co-IP eluates, and antibodies (IgG). (B) LC/MS/MS analysis and protein identification evaluation (unique and shared proteins) of Flag-MSI2 co-IP protein complexes in untreated MDA-MB-231 and DEHP-exposed clone #1 cells performed by Proteome Discoverer software. (C) Unique peptide identification of LC/MS/MS vimentin (FANYIDK) and (D) MSI2 (IFVGGLSANTVVEDVKQYFEQFGK). x-axis: mass/charge ratio (m/z), y-axis: intensity of peak [count]. (E-F) Evaluation of MSI2 and vimentin coexpression in cell lysates and co-IP products by western blotting. (G) IF analysis of the intracellular localization and expression of MSI2 (red) and vimentin (green). Nuclear staining (blue). Scale bar = 50 µm. (H-I) Quantitative analysis of MSI2 (H) and vimentin (I) expression in Scr-treated and MSI2-depleted MDA-MB-231 cells and DEHP-exposed clone #1 cells (means ± SDs); **P < 0.001.

Article Snippet: Primary antibodies targeting the following proteins were used: MSI2 (37 kDa, Proteintech, 10770-1-AP), β-catenin (92 kDa, Proteintech, 51067-2-AP), vimentin (54 kDa, Gentex, GTX110619), α-SMA (46 kDa, Abcam, ab32575), SNAI 1 (28 kDa, CusAb, CSB-PA021867LA01HU), CD133 (115 kDa, Proteintech, 18470-1-AP), c-Myc (69 kDa, Abcam, ab32072), SOX2 (35 kDa, CusAb, PA16539AORb), HLA class I ABC (41 kDa, Proteintech, 66013-1-Ig), β-actin (45 kDa, Santa Cruz, SC-47778), phospho-PI3K p85 (Tyr458) (85 kDa, Cell Int.

Techniques: Expressing, SDS Page, Co-Immunoprecipitation Assay, Liquid Chromatography with Mass Spectroscopy, Software, Western Blot, Staining

Journal: International journal of biological sciences

Article Title: Prolonged DEHP exposure enhances the stemness and metastatic potential of TNBC cells in an MSI2-dependent manner.

doi: 10.7150/ijbs.101598

Figure Lengend Snippet: Figure 8. miR-155-5p negatively regulates MSI2 expression and MSI2-induced migration. (A) Evaluation of prediction-based MSI2-targeting miRNAs via the TargetScanHuman (Release 7.2) and miRBD miRNA target prediction databases. Conserved sites, site length and predicted position of the miR-155-5p binding site in the MSI2 3′UTR. (B) Evaluation of miR-155-5p levels by qPCR in untreated MDA-MB-231 cells and DEHP-exposed clones. (C) Effects of miR-155-5p mimic treatment (10, 25, or 50 nM) on cell migration, as evaluated by a wound healing assay 20 h after scratching. (D) Quantitative analysis of cell migration at 20 h after wound formation (means ± SDsSDs). (E) Evaluation of changes in the expression of MSI2 and vimentin in miR-155-5p mimic-treated (10, 25, 50 nMol) DEHP-exposed clones. (F-G) Evaluation of the effect of the miR-155-5p mimic on MSI2 (F) and vimentin (G) mRNA levels in DEHP-exposed clone #1 cells (means ± SDsSD). (H) Evaluation of miR-155-5p specificity with respect to the MSI2 3′UTR via a luciferase reporter assay via MSI2 3′UTR luciferase expression clone transfection in 293T cells (mean ±SD); **P < 0.001.

Article Snippet: Primary antibodies targeting the following proteins were used: MSI2 (37 kDa, Proteintech, 10770-1-AP), β-catenin (92 kDa, Proteintech, 51067-2-AP), vimentin (54 kDa, Gentex, GTX110619), α-SMA (46 kDa, Abcam, ab32575), SNAI 1 (28 kDa, CusAb, CSB-PA021867LA01HU), CD133 (115 kDa, Proteintech, 18470-1-AP), c-Myc (69 kDa, Abcam, ab32072), SOX2 (35 kDa, CusAb, PA16539AORb), HLA class I ABC (41 kDa, Proteintech, 66013-1-Ig), β-actin (45 kDa, Santa Cruz, SC-47778), phospho-PI3K p85 (Tyr458) (85 kDa, Cell Int.

Techniques: Expressing, Migration, Binding Assay, Clone Assay, Wound Healing Assay, Luciferase, Reporter Assay, Transfection

Journal: International journal of biological sciences

Article Title: Prolonged DEHP exposure enhances the stemness and metastatic potential of TNBC cells in an MSI2-dependent manner.

doi: 10.7150/ijbs.101598

Figure Lengend Snippet: Figure 9. MSI2 knockdown reduces DEHP-induced breast cancer metastasis in vivo. (A) The effect of prolonged DEHP treatment on TNBC cell metastasis was evaluated in a mouse metastasis model. Untreated and DEHP-exposed MDA-MB-231 cells (Scr and MSI2 knockdown) were implanted into the mammary fat pads of 8-week-old female BALB/c nude mice. The average tumor size was recorded. The mice were sacrificed and processed for evaluation of metastasis. (B) Evaluation of average tumor sizes in different groups (mean ±SD). (C-D) Evaluation of tumor growth and size measurements of excised tumors (means ± SDs). (E) Evaluation of lung metastasis by morphological changes and changes in the size of the excised lungs. (F) Lung metastasis evaluation via IHC analysis of the metastasis-associated markers vimentin, N-cadherin, E-cadherin, and HLA-ABC. Scale bar = 30 µm. *P < 0.05, **P < 0.001.

Article Snippet: Primary antibodies targeting the following proteins were used: MSI2 (37 kDa, Proteintech, 10770-1-AP), β-catenin (92 kDa, Proteintech, 51067-2-AP), vimentin (54 kDa, Gentex, GTX110619), α-SMA (46 kDa, Abcam, ab32575), SNAI 1 (28 kDa, CusAb, CSB-PA021867LA01HU), CD133 (115 kDa, Proteintech, 18470-1-AP), c-Myc (69 kDa, Abcam, ab32072), SOX2 (35 kDa, CusAb, PA16539AORb), HLA class I ABC (41 kDa, Proteintech, 66013-1-Ig), β-actin (45 kDa, Santa Cruz, SC-47778), phospho-PI3K p85 (Tyr458) (85 kDa, Cell Int.

Techniques: Knockdown, In Vivo

Journal: International journal of biological sciences

Article Title: Prolonged DEHP exposure enhances the stemness and metastatic potential of TNBC cells in an MSI2-dependent manner.

doi: 10.7150/ijbs.101598

Figure Lengend Snippet: Figure 10. Schematic representation of DEHP-induced TNBC progression. Low-dose and prolonged DEHP exposure results in MSI2 overexpression in MDA-MB-231 cells. Increased MSI2 expression initiates the PI3K/Akt/NF-κB/MMP-9 axis and regulates vimentin to enhance EMT, which facilitates migration, invasion, and metastasis. In addition, MSI2 promotes TNBC stemness. Interestingly, miR-155-5p negatively regulates the expression of MSI2, while miRNA-155-5p is downregulated under DEHP treatment.

Article Snippet: Primary antibodies targeting the following proteins were used: MSI2 (37 kDa, Proteintech, 10770-1-AP), β-catenin (92 kDa, Proteintech, 51067-2-AP), vimentin (54 kDa, Gentex, GTX110619), α-SMA (46 kDa, Abcam, ab32575), SNAI 1 (28 kDa, CusAb, CSB-PA021867LA01HU), CD133 (115 kDa, Proteintech, 18470-1-AP), c-Myc (69 kDa, Abcam, ab32072), SOX2 (35 kDa, CusAb, PA16539AORb), HLA class I ABC (41 kDa, Proteintech, 66013-1-Ig), β-actin (45 kDa, Santa Cruz, SC-47778), phospho-PI3K p85 (Tyr458) (85 kDa, Cell Int.

Techniques: Over Expression, Expressing, Migration

Journal: International journal of biological sciences

Article Title: Prolonged DEHP exposure enhances the stemness and metastatic potential of TNBC cells in an MSI2-dependent manner.

doi: 10.7150/ijbs.101598

Figure Lengend Snippet: Figure 4. Prolonged DEHP exposure induces EMT and stemness in MDA-MB-231 cells in an MSI2-dependent manner. (A) Changes in the expression of the EMT markers α-SMA, β-catenin, SNAI1, and vimentin, as evaluated by western blotting. (B) Evaluation of the effect of MSI2 knockdown on the expression of EMT markers by western blotting. (C) Anchorage-independent growth/spheroid formation as evaluated by the soft agar colony formation assay. Scale bar = 50 µm. (D-E) Quantitative analysis of the number and size of colonies/spheroids originating from untreated and DEHP-exposed MDA-MB-231 cells. (F) Assessment of anchorage-independent growth/spheroid formation by soft agar colony formation assays in Scr-treated and MSI2-depleted cells. Scale bar = 50 µm. (G-H) Quantitative analysis of colony number and colony size in the soft agar colony formation assay (mean ± SD). (I) Changes in the expression of the stemness-related markers CD133, cMyc, and SOX-2 in untreated and DEHP-exposed clones evaluated by western blotting. (J) Evaluation of changes in the expression of stemness-related markers in Scr-treated and MSI2-depleted MDA-MB-231 cells and in DEHP-exposed clones; **P < 0.001.

Article Snippet: Primary antibodies targeting the following proteins were used: MSI2 (37 kDa, Proteintech, 10770-1-AP), β-catenin (92 kDa, Proteintech, 51067-2-AP), vimentin (54 kDa, Gentex, GTX110619), α-SMA (46 kDa, Abcam, ab32575), SNAI 1 (28 kDa, CusAb, CSB-PA021867LA01HU), CD133 (115 kDa, Proteintech, 18470-1-AP), c-Myc (69 kDa, Abcam, ab32072), SOX2 (35 kDa, CusAb, PA16539AORb), HLA class I ABC (41 kDa, Proteintech, 66013-1-Ig), β-actin (45 kDa, Santa Cruz, SC-47778), phospho-PI3K p85 (Tyr458) (85 kDa, Cell Int.

Techniques: Expressing, Western Blot, Knockdown, Soft Agar Assay, Clone Assay