anti-oct-3/4 sc-5279  (Santa Cruz Biotechnology)

Journal: Molecular Therapy Oncology

Article Title: hsa-miR-5688 inhibits FOXC1-OCT4/SOX2 feedforward loop that drives chemoresistance in breast cancer stem cells

doi: 10.1016/j.omton.2025.200982

Figure Lengend Snippet: Inherently chemo-resistant CSCs acquire more resistance during chemotherapy (A) Representative flow-cytometric plots depicting the gating strategy for CD44 + /CD24 − CSC (P2) and CD44 + /CD24 + , CD44 - /CD24 - , CD44 − CD24 + NSCC (P3) populations in MCF-7 cell (left panel). Bar diagrams depicting percentage of annexin-V-FITC + apoptotic cells in MCF-7/MDA-MB-231/MDA-MB-468 cell-derived gated NSCC and CSC populations in the presence of 2.5-μM Dox after 24 h of treatment (right panel). (B) Bar diagrams illustrating relative mean fluorescence intensity (MFI) of drug-resistance markers ABCG2 (left panel), MDR1 (middle panel), and MRP1 (right panel) in cells vs. respective 2 0 spheres of MCF-7, MDA-MB-231, and MDA-MB-468, as analyzed by flow cytometry. (C) Bar diagrams showing relative MFI of stemness factors OCT4 (left panel), SOX2 (middle left panel), NANOG (middle right panel), and ABCG2 (right panel) as evaluated using flow cytometry in MCF-7, MDA-MB-231, and MDA-MB-468 CSCs in presence or absence of Dox, after 24 h of treatment. Data are mean ± SE or representative of three independent experiments unless otherwise noted. p = ns (non-significant), ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001 by unpaired Student’s t test.

Article Snippet: The antibodies used are FOXC1 (Novus Biologicals, Thermo Fisher Scientific), SOX2 and OCT4 (Cell Signaling Technology).

Techniques: Derivative Assay, Fluorescence, Flow Cytometry

Journal: Molecular Therapy Oncology

Article Title: hsa-miR-5688 inhibits FOXC1-OCT4/SOX2 feedforward loop that drives chemoresistance in breast cancer stem cells

doi: 10.1016/j.omton.2025.200982

Figure Lengend Snippet: Identification of OCT4 as a positive regulator of FOXC1 in CSCs (A) Pearson correlation plots showing correlation between FOXC1 vs. OCT4 , FOXC1 vs. SOX2 , FOXC1 vs. NANOG , FOXC1 vs. ABCG2 , FOXC1 vs. MRP1 , and FOXC1 vs. MDR1 from GSE25066 breast cancer patient dataset. (B) In-silico prediction of putative binding sites of OCT4 on FOXC1 promoter and (C) SOX2 on FOXC1 promoter, from JASPAR database. (D) Bar diagrams depicting fold change of OCT4 mRNA expression in control vs. OCT4-shRNA-treated (left panel), SOX2 mRNA expression in control vs. SOX2-shRNA-treated (middle panel), and FOXC1 mRNA expression in control vs. OCT4-shRNA vs. SOX2-shRNA-treated (right panel) MDA-MB-468 CSCs. (E) Western blot images showing protein expression of OCT4, SOX2, and FOXC1 upon transient knock-down of OCT4 and SOX2 by treatment with their respective shRNA in MDA-MB-468 CSCs. βACTIN was used as internal loading control. (F) In-silico docking interaction of OCT4 on FOXC1 promoter (left panel) and SOX2 on FOXC1 promoter (right panel). (G) Representative semi-quantitative PCR data showing occupancy of OCT4 on FOXC1 promoter in MDA-MB-468 CSCs using ChIP assay. Data in (D) is mean ± SE or representative of three independent experiments unless otherwise noted. p = ns, ∗ p < 0.05, ∗∗∗ p < 0.01, and ∗∗∗∗ p < 0.0001 by unpaired Student’s t test (D).

Article Snippet: The antibodies used are FOXC1 (Novus Biologicals, Thermo Fisher Scientific), SOX2 and OCT4 (Cell Signaling Technology).

Techniques: In Silico, Binding Assay, Expressing, Control, shRNA, Western Blot, Knockdown, Real-time Polymerase Chain Reaction

Journal: Molecular Therapy Oncology

Article Title: hsa-miR-5688 inhibits FOXC1-OCT4/SOX2 feedforward loop that drives chemoresistance in breast cancer stem cells

doi: 10.1016/j.omton.2025.200982

Figure Lengend Snippet: FOXC1 transactivates OCT4 and other stemness and drug-resistance factors in CSCs (A) Bar diagrams depicting fold change in mRNA expression levels of FOXC1 , OCT4 , and SOX2 in control vs. FOXC1-shRNA-treated MDA-MB-468 CSCs. (B) Western blot images showing protein expression of FOXC1 (left panel), OCT4 (middle panel), and SOX2 (right panel) upon transient knockdown of FOXC1 by shRNA in MDA-MB-468 CSCs. βACTIN was used as internal loading control. (C) In-silico prediction of putative binding sites of FOXC1 on OCT4 and SOX2 promoters from JASPAR database. (D) In-silico docking interaction of FOXC1 on OCT4 and SOX2 promoters. (E) Representative data showing occupancy of FOXC1 on the promoters of OCT4 and SOX2 in MDA-MB-468 CSCs using ChIP assay followed by semi-quantitative PCR. (F) Bar diagrams portraying fold changes in the mRNA expression levels of NANOG , and ABCG2 in control vs. FOXC1-shRNA-treated MDA-MB-468 CSCs. (G) In-silico prediction of putative binding sites of FOXC1 on NANOG and ABCG2 promoters from JASPAR database. (H) In-silico docking interactions of FOXC1 on NANOG and FOXC1 on ABCG2 promoter sequences. (I) Representative data of FOXC1 occupancy on the promoters of NANOG , and ABCG2 in MDA-MB-468 CSCs using ChIP assay followed by semi-quantitative PCR. Data in (A) and (F) are mean ± SE or representative of three independent experiments unless otherwise noted. ∗∗∗ p < 0.001 and ∗∗∗∗ p < 0.0001 by unpaired Student’s t test (A) and (F).

Article Snippet: The antibodies used are FOXC1 (Novus Biologicals, Thermo Fisher Scientific), SOX2 and OCT4 (Cell Signaling Technology).

Techniques: Expressing, Control, shRNA, Western Blot, Knockdown, In Silico, Binding Assay, Real-time Polymerase Chain Reaction

Journal: Molecular Therapy Oncology

Article Title: hsa-miR-5688 inhibits FOXC1-OCT4/SOX2 feedforward loop that drives chemoresistance in breast cancer stem cells

doi: 10.1016/j.omton.2025.200982

Figure Lengend Snippet: ChIP assay determines FOXC1-OCT4 reciprocal feedforward transactivation loop in CSCs (A) Bar diagrams illustrating fold change in binding of OCT4 on FOXC1 promoter (left panel) in control vs. OCT4-shRNA-treated and FOXC1 on OCT4 promoter (right panel) in control vs. FOXC1-shRNA-treated MDA-MB-468 CSCs. (B) Bar diagrams portraying fold change in binding of: FOXC1 on SOX2 (left panel), NANOG (middle panel), and FOXC1 on ABCG2 (right panel) promoters in control vs. FOXC1-shRNA-treated MDA-MB-468 CSCs. (C) Bar diagrams depicting fold change of FOXC1-binding on OCT4 promoter (left panel), FOXC1-binding on SOX2 promoter (middle panel), and OCT4-binding on FOXC1 promoter in control vs. Dox-treated MDA-MB-468 CSCs. (D) Bar diagrams showing difference in fold change of FOXC1-binding on NANOG (left panel) and ABCG2 (right panel) promoters in control vs. Dox-treated MDA-MB-468 CSCs. Data are mean ± SE or representative of three independent experiments unless otherwise noted. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001 by unpaired Student’s t test.

Article Snippet: The antibodies used are FOXC1 (Novus Biologicals, Thermo Fisher Scientific), SOX2 and OCT4 (Cell Signaling Technology).

Techniques: Binding Assay, Control, shRNA

Journal: Molecular Therapy Oncology

Article Title: hsa-miR-5688 inhibits FOXC1-OCT4/SOX2 feedforward loop that drives chemoresistance in breast cancer stem cells

doi: 10.1016/j.omton.2025.200982

Figure Lengend Snippet: Downregulation of FOXC1 sensitizes CSCs toward chemotherapeutic drug doxorubicin (A) Representative flow cytometry plots of annexin-V-FITC vs. propidium iodide (left panel), and bar diagram depicting percentage cell apoptosis (right panel) in MDA-MB-468 CSCs under the conditions: control, Dox-treated, FOXC1-shRNA - transfected, and FOXC1-shRNA-transfected + Dox-treated. (B) Bar diagrams depicting annexin-V-FITC + apoptotic cells following three cycles of Dox therapy in control vs. 3 cycles of Dox-treated NSCC (left panel) and CSC (right panel) subpopulations of MDA-MB-468 1 0 spheres. (C) Bar diagrams showing percentage of annexin-V-FITC + apoptotic cells in MDA-MB-468 CSCs under the conditions of control vs. Dox vs. FOXC1-shRNA vs. FOXC1-shRNA + Dox during cycles 1–3 (left panel). Line plot portraying percentage of annexin-V-FITC + apoptotic cells for 1–3 cycles of Dox in control and FOXC1-shRNA-transfected MDA-MB-468 CSCs (right panel). The NSCC and CSC populations were selected after differential gating using FACS. (D) Representative image showing volumes of tumor generated in BALB/c mice (left panel), and bar diagrams depicting difference in their tumor volumes (right panel) in control vs. Dox vs. Foxc1-shRNA vs. Foxc1-shRNA+Dox-treated mice. (E) Bar diagram demonstrating fold change of Foxc1 (left panel), Oct4 (middle panel), and Sox2 (right panel) mRNA expression in the tumor tissues of control vs. Dox vs. Foxc1-shRNA vs. Foxc1-shRNA+Dox-treated BALB/c mice. (F) Bar diagrams signifying fold change in mRNA expression levels of Nanog and Abcg2 in tumors derived from control vs. Dox vs. Foxc1-shRNA vs. Foxc1-shRNA+Dox-treated BALB/c mice. Data are mean ± SE or representative of three independent experiments unless otherwise noted. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001 by unpaired Student’s t test.

Article Snippet: The antibodies used are FOXC1 (Novus Biologicals, Thermo Fisher Scientific), SOX2 and OCT4 (Cell Signaling Technology).

Techniques: Flow Cytometry, Control, shRNA, Transfection, Generated, Expressing, Derivative Assay

Journal: Molecular Therapy Oncology

Article Title: hsa-miR-5688 inhibits FOXC1-OCT4/SOX2 feedforward loop that drives chemoresistance in breast cancer stem cells

doi: 10.1016/j.omton.2025.200982

Figure Lengend Snippet: Downregulation of FOXC1 inhibits recurrence after chemotherapy (A) Bar diagrams showing relative MFI of FOXC1 (left panel), OCT4 (middle panel), and SOX2 (right panel) in control vs. 3rd cycle of Dox vs. FOXC1-shRNA vs. FOXC1-shRNA + 3rd cycle of Dox-treated MDA-MB-468 CSCs, as determined by flow cytometry. (B) Bar diagrams showing relative MFI of NANOG (left panel) and ABCG2 (right panel) in control vs. 3rd cycle of Dox-treated vs. FOXC1-shRNA vs. FOXC1-shRNA + 3rd cycle of Dox-treated MDA-MB-468 CSCs, as evaluated by flow cytometry. (C) Phase contrast images of sphere formation assay of control vs. Dox vs. FOXC1-shRNA vs. FOXC1-shRNA + Dox-treated MDA-MB-468 CSCs after 3rd cycle of consecutive Dox treatment. (D) Phase contrast images showing recurrent colony formation of control vs. Dox vs. FOXC1-shRNA vs. FOXC1-shRNA+Dox-treated MDA-MB-468 CSCs after cycle 3 of Dox treatment subjected to serum-containing differentiation media (left panel). Bar diagram depicting area of recurrent colonies formed in μm 2 (right panel). Data in (A), (B), and (D) are mean ± SE or representative of three independent experiments unless otherwise noted. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001 by unpaired Student’s t test.

Article Snippet: The antibodies used are FOXC1 (Novus Biologicals, Thermo Fisher Scientific), SOX2 and OCT4 (Cell Signaling Technology).

Techniques: Control, shRNA, Flow Cytometry, Tube Formation Assay

Journal: Molecular Therapy Oncology

Article Title: hsa-miR-5688 inhibits FOXC1-OCT4/SOX2 feedforward loop that drives chemoresistance in breast cancer stem cells

doi: 10.1016/j.omton.2025.200982

Figure Lengend Snippet: Identification of novel miRNA targeting FOXC1 expression for sensitizing chemo-resistant CSCs (A) KM plot of triple negative breast cancer patients correlating higher probability of OS with higher expression of hsa-miR-5688. (B) Bar graphs illustrating the difference in expression levels of hsa-miR-5688 between MACS-sorted MDA-MB-468 NSCCs and CSCs and (C) breast cancer patient-derived NSCCs vs. CSCs ( n = 5). (D) Bar diagrams portraying fold change in expression levels of hsa-miR-5688, FOXC1 , OCT4 , and SOX2 in control vs. mimic-treated vs. decoy-treated MDA-MB-468 CSCs. (E) Bar diagrams depicting difference in fold change of FOXC1-binding on OCT4 and SOX2 promoters in control vs. mimic-treated vs. decoy-treated MDA-MB-468 CSCs. (F) Bar diagrams showing percentage of annexin-V-FITC + apoptotic cells in control vs. 1–3 cycles of Dox-treated vs . mimic vs. mimic + 1–3 cycles of Dox-treated MDA-MB-468 CSCs (left panel). Line plot indicating the same in 1–3 cycles of Dox-treated vs . mimic vs. mimic + 1–3 cycles of Dox-treated MDA-MB-468 CSCs (right panel). (G) Bar diagrams showing relative MFI of FOXC1 (left panel), OCT4 (middle panel) and SOX2 (right panel) in control vs. 3rd cycle Dox-treated vs. mimic vs. mimic + 3rd cycle Dox-treated MDA-MB-468 CSCs, as validated using flow cytometry. (H) Phase contrast images of sphere formation assay of control vs. 3rd cycle Dox-treated vs. mimic vs. mimic + 3rd cycle Dox-treated MDA-MB-468 CSCs. (I) Phase contrast images of recurrent colony formation assay in control vs. 3rd cycle Dox-treated vs. mimic vs. mimic + 3rd cycle Dox-treated MDA-MB-468 CSCs when exposed to serum-containing differentiation media (left panel). Bar diagram depicting area of recurrent colonies formed per μm 2 (right panel). Data in (B) to (G), and (I) are mean ± SE or representative of three independent experiments unless otherwise noted. p = ns, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001 by unpaired Student’s t test (B) to (G), and (I).

Article Snippet: The antibodies used are FOXC1 (Novus Biologicals, Thermo Fisher Scientific), SOX2 and OCT4 (Cell Signaling Technology).

Techniques: Expressing, Derivative Assay, Control, Binding Assay, Flow Cytometry, Tube Formation Assay, Colony Assay

Journal: bioRxiv

Article Title: Pasta, an age-shift transcriptomic clock, maps the chemical and genetic determinants of aging and rejuvenation

doi: 10.1101/2025.06.04.657785

Figure Lengend Snippet: ( a ) Representative images from Senescence Associated Beta Galactosidase assay performed on A-375 cells exposed to Pralatrexate (Prltx). Doxorubicin (Doxo) was used as positive control for senescence induction. Scale Bar: 10 μm. ( b ) qPCR analysis of CDKN1A and IL6 mRNA levels in A-375 cells in response to Prtlx and Doxo.; N=3 biological replicates; *** p< 0.001 (one-way ANOVA). ( c ) Cell proliferation assay by Edu incorporation performed in A-375 cells exposed to Prltx and Doxo (one representative experiment with n=3 replicates/condition). *** p< 0.001 (one-way ANOVA, calculated on Edu + cells). ( d ) Immunoblotting showing LMNB1, CDKN1A and GAPDH protein levels in A-375 cells exposed to Prltx and Doxo (N=3 biological replicates). ( e ) Representative images from Senescence Associated Beta Galactosidase assay performed on MDA-231 cells exposed to Pralatrexate (Prltx). Doxorubicin (Doxo) was used as positive control for senescence induction. Scale Bar: 10 μm. ( f ) qPCR analysis of CDKN1A and IL6 mRNA levels in MDA-231 cells in response to Prltx and Doxo.; N=3 biological replicates; *p< 0.05 (one-way ANOVA). ( g ) Cell proliferation assay by Edu incorporation performed in MDA-231 cells exposed to Prltx and Doxo (one representative experiment with n=3 replicates/condition). ** p< 0.01; *** p< 0.001 (one-way ANOVA, calculated on Edu + cells). ( h ) Immunoblotting showing LMNB1, CDKN1A and GAPDH protein levels in A-375 cells exposed to Prltx and Doxo (N=3 biological replicates). (i) qPCR analysis of Oct4, Sox2 and Nanog levels in PC3 cells exposed to Piperlongumine (Piplng). * p< 0.05 Student’s t test. (j) Immunoblotting showing OCT3/4 and alpha-tubulin protein levels in PC3 cells incubated with Piplng..

Article Snippet: Non-specific binding sites were saturated by incubating membranes for 1□ h in 0.05% Tween 20 (#P9416, Sigma Aldrich) v-v in Tris-buffered saline (TBS) supplemented with 5% BSA (Sigma-Aldrich, #10735078001, w:v in TBS), followed by an overnight incubation with primary antibodies specific for Waf1/Cip1/CDKN1A p21 (Santa Cruz Biotechnology, #sc-6246), LaminB1 (12987-1-AP, Proteintech) and Oct3/4 (sc-5279, Santa Cruz Biotechnology).

Techniques: β-Gal Assay, Positive Control, Proliferation Assay, Western Blot, Incubation