phosphorylated oligo duplex  (New England Biolabs)

Journal: iScience

Article Title: A calcium-sensing receptor dileucine motif directs internalization to spatially distinct endosomal signaling pathways

doi: 10.1016/j.isci.2025.112651

Figure Lengend Snippet: CaSR traffics to Rab4 pathways and recycles by Rab4 and Rab11 (A and B) BRET between Venus-Rab4 and CaSR-Nluc-WT or CaSR-Nluc-AA ( N = 7), or CaSR-Nluc-WT in HEK-AP2σ-WT or HEK-AP2σ-R15H cells ( N = 9), with (B) AUC. (C) BRET between Rab4-Venus and CaSR-Nluc-WT, with vehicle (DMSO) or Dyngo-4a. N = 5. (D) SIM of SNAP-CaSR-647 and Venus-Rab4. Scale, 5 μm. Arrows show colocalization. N = 23 (0 mM), N = 22 (3 mM) from N = 4 biological replicates. (E) SIM images of cells exposed to FLAG antibody and either non-permeabilized (total) or exposed to 0 or 3 mM Ca 2+ e for 30 min or 30 min with 3-h recovery, before acid strip to remove cell surface FLAG. Internalization occurs in cells stimulated with 3 mM Ca 2+ e conditions other than plates incubated at 4°C to block endocytosis. Cells transfected with Rab4 siRNA and Rab11-DN have reduced receptor recycling (seen as FLAG expression at cell surfaces) than cells transfected with scrambled siRNA or Rab11-WT. Scale, 5 μm. (F) Non-permeabilized cells incubated with FLAG antibody and exposed to 0 or 3 mM Ca 2+ e for 30 min or 30 min with 3-h recovery to monitor recycling. Comparisons to 3 mM (30 min) in black, to 3 mM (30 min with recovery) in red, and to 0 mM (30 min with recovery) in blue. N = 5. (G) Antibody feeding in cells transfected with scrambled siRNA, Rab4 siRNA, or Rab11a-dominant negative (DN). Comparisons to 0 min (blue). N = 5. (H) Antibody feeding assays in which permeabilized cells were incubated with FLAG antibody and exposed to 0 or 3 mM Ca 2+ e for 30 min or 30 min with 3-h recovery. Statistics show comparisons to 3 mM (30 min) in black, to 3 mM (30 min with recovery) in red, and to 0 mM (30 min with recovery) in blue. N = 5. (I) Antibody feeding assays in cells transfected with scrambled siRNA, Rab4 siRNA, or Rab11a-DN and permeabilized to monitor total CaSR. Statistical analyses show comparisons to 0 min in blue and between 0 and 3 mM in each condition in black. N = 4. Statistical analyses were performed using one-way ANOVA with Tukey’s multiple-comparisons test for (B), unpaired t test in (C), and one-way ANOVA with Sidak’s multiple-comparisons test in (F)–(I). ∗∗∗∗ p < 0.0001, ∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05. Data shows mean + SEM in A–C,and F–I.

Article Snippet: Rab4 (RAB4A) Human siRNA Oligo Duplex (Locus ID 5867) , Origene , Catalog. no. SR303946.

Techniques: Stripping Membranes, Incubation, Blocking Assay, Transfection, Expressing, Dominant Negative Mutation

Journal: iScience

Article Title: A calcium-sensing receptor dileucine motif directs internalization to spatially distinct endosomal signaling pathways

doi: 10.1016/j.isci.2025.112651

Figure Lengend Snippet: CaSR signaling from Rab4- and Rab9-positive endosomes contributes to sustained signaling (A) BRET between Venus-Rab4 and Nluc-mGq in HEK-CaSR. Calcium ( N = 9), spermine ( N = 6). (B) Ca 2+ -induced BRET with vehicle (DMSO) or Dyngo-4a. N = 8. (C and D) BRET between Venus-Rab4 and CaSR-Nluc-WT or CaSR-Nluc-AA or (D) CaSR-Nluc-WT in HEK-AP2σ-WT or HEK-AP2σ-R15H cells. N = 7. (E) AUC of ligand-induced BRET between Nluc-GRK3 and Venus-Gβγ in HEK-CaSR overexpressing Gα11, with scrambled or Rab4 siRNA. N = 8. (F) AUC of NanoBiT IP 3 responses with scrambled or Rab4 siRNA. N = 6. (G) Western blot of p -ERK1/2 in HEK-CaSR with scrambled or Rab4 siRNA following a 5-min pulse of 3 mM Ca 2+ then 0.1 mM Ca 2+ . Quantification of densitometry from five western blots is shown below. (H) BRET between Venus-Rab9 and Nluc-mGq in HEK-CaSR. Calcium ( N = 9), spermine ( N = 6). (I) BRET with vehicle (DMSO) or Dyngo-4a. N = 8. (J and K) Ca 2+ -induced BRET in (J) cells transfected with CaSR-Nluc-WT or CaSR-Nluc-AA ( N = 5) or in (K) HEK-AP2σ-WT or HEK-AP2σ-R15H cells ( N = 10). (L) BRET between Nluc-GRK3 and Venus-Gβγ in HEK-CaSR overexpressing Gα11, with scrambled or Rab9 siRNA. N = 8. (M) NanoBiT IP 3 responses with scrambled or Rab9 siRNA. N = 6. (N) p -ERK1/2 responses with scrambled or Rab4 siRNA and exposed to a 5-min pulse of 3 mM Ca 2+ e . Quantification of densitometry from four western blots is shown below. Data shows mean ± SEM. ∗∗∗∗ p < 0.0001, ∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05. Black asterisks show vehicle vs. agonist. Statistical analyses were performed using one-way ANOVA with Sidak’s multiple comparisons test for (A), (B), (C), (D), (G), (H), (I), (J), (K), and (N), and two-way ANOVA with Sidak’s test for (E), (F), (L), and (M).

Article Snippet: Rab4 (RAB4A) Human siRNA Oligo Duplex (Locus ID 5867) , Origene , Catalog. no. SR303946.

Techniques: Western Blot, Transfection

Journal: iScience

Article Title: A calcium-sensing receptor dileucine motif directs internalization to spatially distinct endosomal signaling pathways

doi: 10.1016/j.isci.2025.112651

Figure Lengend Snippet: Cinacalcet increases CaSR signaling in FHH3-associated AP2σ-R15H mutant cells by enhancing sustained signaling (A) BRET between Venus-Rab4 and CaSR-Nluc with vehicle or cinacalcet. (B and C) Dose response of BRET responses with increasing cinacalcet concentrations with (C) pEC50 values. N = 4. (D and E) BRET between Venus-Rab4 and CaSR-Nluc with vehicle or NPS-2143, with (E) dose response. N = 6. (F and G) BRET between Venus-Rab4 and Nluc-mGq with increasing cinacalcet concentrations, with (G) pEC50. N = 4. (H) BRET between Nluc-mGq and Venus-Rab4 with NPS-2143. N = 12. (I–K) BRET between Nluc-mGq and Venus-Rab9 with cinacalcet, with (J) increasing cinacalcet concentrations, and (K) pEC50. N = 5. (L and M) BRET between Nluc-mGq and Venus-Rab9 in cells with increasing concentrations of cinacalcet, with (M) pEC50. N = 5. (N) BRET between Nluc-CaSR and Venus-Rab9 with NPS-2143. N = 12. (O) BRET between Nluc-mGq and Venus-Rab9 with NPS-2143. N = 11. (P–R) BRET between Nluc-mGq and (P) Rab5 ( N = 6), (Q) Rab4 ( N = 4), (R) Rab9 ( N = 5), in AP2σ-WT or AP2σ-R15H cells expressing CaSR with vehicle or cinacalcet. (S and T) Cinacalcet effect on responses in cells expressing a siRNA-resistant AP2σ-R15H plasmid combined with AP2σ-siRNA to knockdown endogenous protein or an AP2σ-R15H plasmid. N = 5. Statistics show: WT vs. AP2σ-R15H (red), AP2σ-R15H with vehicle or cinacalcet (blue) in (P)–(R). Statistical analyses were performed using two-way ANOVA with Dunnett’s test in (E), and Sidak’s test in (H,) (N), (O), (P), (Q), (R), (S), and (T), and one-way ANOVA with Dunnett’s test in (C), (G), (K), and (M). ∗∗∗∗ p < 0.0001, ∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05. (A), (D), and (I) show examples of BRET data. Data shows mean ± SEM.

Article Snippet: Rab4 (RAB4A) Human siRNA Oligo Duplex (Locus ID 5867) , Origene , Catalog. no. SR303946.

Techniques: Mutagenesis, Expressing, Plasmid Preparation, Knockdown

Journal: Aging (Albany NY)

Article Title: Decreased mitochondrial NAD+ in WRN deficient cells links to dysfunctional proliferation

doi: 10.18632/aging.206236

Figure Lengend Snippet: WRN regulates main NAD + synthetic proteins and shares many transcription targets with NMNAT1-3. ( A ) Representative western blots of RIPA extracts from HEK293 parental cells with either 30 nM Scramble siRNA (Scr) or 30 nM WRN siRNA ( WRN -KD) followed by 24 h 1 mM NR treatment . ( B – F ) Quantification of western blots from 4–6 biological repeats. Statistical analysis was performed in GraphPad Prism with either One-way ANOVA with Šidák multiple comparison’s test or Student’s t -test. ( C ) NMNAT1 was significantly decreased in WRN -KD cells compared to Scr (Veh) and Scr (NR) (One-way ANOVA, Tukey’s multiple comparisons test, p -value = 0.0108, student’s t -test p -value = 0.0163), ( D ) NAMPT was significantly increased in WRN -KD (Veh) cells compared to Scr (Veh) (One-way ANOVA, Tukey’s multiple comparisons test, p -value = 0.0492, Student’s t -test, p -value = 0.0010) and ( E ) NADSYN1 was upregulated in WRN -KD (Veh) compared to Scr (Veh) (Student’s t -test, p -value = 0.0442). ( F – I ) Venn diagram showing the shared transcription targets of WRN and NMNAT1-3 based on the ENCODE transcription database.

Article Snippet: To induce knockdown of WRN , cells were treated with 30 nM WRN siRNA oligo duplex for 6 h (#SR322215, OriGene) in OPTI-MEM media, followed by addition of normal growth media.

Techniques: Western Blot

Journal: Aging (Albany NY)

Article Title: Decreased mitochondrial NAD+ in WRN deficient cells links to dysfunctional proliferation

doi: 10.18632/aging.206236

Figure Lengend Snippet: Reduced mitochondrial NAD + in WRN depleted cells. ( A , B ) WRN -KD in the HEK293-mitoPARP reporter cell line led to decreased mitochondrial NAD + compared to Scr as measured by PAR signal relative to β-Actin. Cells were grown in the presence of the PARP inhibitor 3-AB (1 mM) and collected at the designated timepoints after removal of 3-AB from the media (0, 3, 6 h). ( A ) Quantification of 6 biological repeats. Scramble (Scr) is paired with WRN -KD from the same biological repeat with a black line in the graph. Statistical significance was found between 0 h (right after the release of 3-AB treatment) compared to 3 h and 6 h (Two-way ANOVA, Tukey’s multiple comparisons test. Scr (Veh): 0 h to 3 h and 6 h p -values < 0.0001. WRN -KD (Veh): 0 h to 3 h p -value = 0.001; 0 h to 6 h p -value < 0.0001). Further increased PARylation signal, resembling increased mitochondrial NAD + levels, was observed in Scr between 3 h and 6 h after 3-AB release (Two-way ANOVA, Tukey’s multiple comparisons, p -value = 0.0030), however no change was observed in WRN -KD cells between 3 h and 6 h, suggesting less available mitochondrial NAD + (Two-way ANOVA, Tukey’s multiple comparisons, p -value = 0.8310). Moreover, there was a significant reduced level of PAR in WRN -KD cells compared to Scr at 6 h after 3-AB release (Two-ANOVA, Tukey’s multiple comparisons, p -value = 0.0321). ( B ) Representative western blot of PAR and β-Actin signal. ( C , D ) WRN -KD leads to decreased colony formation in HEK293 cells (Two-way ANOVA, Tukey’s multiple comparisons, p -value = 0.012), which was not rescued by overexpression of the human mitochondrial NAD + transporter SLC25A51 or 1 mM NR treatment for 24 h. Overexpression of SLC25A51 in HEK293 (HEK293-SLC25A51) did on the other hand significantly increase colony formation in Scr cells (Two-way ANOVA, Tukey’s multiple comparisons, p -value = 0.0049). Colony formation assay was performed for Scr or WRN -KD (30 nM siRNA) in three different HEK293 cell lines: Parental HEK293, HEK293-mitoPARP and HEK293-SLC25A51. The HEK293-mitoPARP cells were exposed to 6 h 3-AB release before seeding out for colonies in medium containing 1 mM 3-AB for 11 days. ( C ) Representative images of stained colonies. ( D ) quantification of 3 biological replicates with 3 technical repeats. ( E ) Confirmation of WRN -KD was shown to be 90–100% in all biological replicates. Western blotting of GFP-PARP1 and FLAG confirmed the specific expression of the mitoPARP and SLC25A51-constructs, respectively. The dotted line indicates that the representative blots are from two different blots with their respective loading controls. Statistics were performed with GraphPad Prism using Two-way ANOVA with Tukey’s multiple comparison.

Article Snippet: To induce knockdown of WRN , cells were treated with 30 nM WRN siRNA oligo duplex for 6 h (#SR322215, OriGene) in OPTI-MEM media, followed by addition of normal growth media.

Techniques: Western Blot, Over Expression, Colony Assay, Staining, Expressing, Construct, Comparison

Journal: The EMBO Journal

Article Title: SDCBP/Syntenin-1 stabilizes BACH1 by disassembling the SCF FBXO22 –BACH1 complex in triple-negative breast cancer

doi: 10.1038/s44318-025-00440-1

Figure Lengend Snippet: ( A ) TCGA data analysis showing the correlation between SDCBP mRNA and BACH1 mRNA expression in GSE142102 ( n = 226) dataset of TNBC patients (Pearson correlation coefficient r = 0.3245, P < 0.0001). ( B ) TCGA data analysis showing the correlation between SDCBP mRNA and BACH1 mRNA expression in GSE103091 ( n = 238) dataset of TNBC patients (Pearson correlation coefficient r = 0.2120, P < 0.001). ( C ) Western blot showing SDCBP, BACH1, and HO-1 protein expression in MDA-MB-231, MDA-MB-468, Hs578T, MCF-7, and T47D cells. ( D ) The expression levels of SDCBP and BACH1 protein in Fig. EV1C were quantified using densitometry and normalized to the housekeeping protein α-tubulin ( n = 3). ( E ) Real-time qPCR showing SDCBP and BACH1 mRNA expression in MDA-MB-231, MDA-MB-468, Hs578T, MCF-7, and T47D cells ( n = 3). Quantitative data were normalized to β-actin expression. ( F ) Western blot showing SDCBP and HO-1 protein expression in MDA-MB-231 cells transfected with scramble or BACH1 siRNA. ( G ) Left, western blot showing the protein expression of SDCBP in the scramble and in several SDCBP-KO MDA-MB-231 subclones generated using CRISPR-Cas9 system; Right, real-time qPCR showing the SDCBP mRNA expression in scramble and in SDCBP-KO MDA-MB-231 subclones ( n = 3). ( H ) Real-time qPCR showing the mRNA expression of BACH1 in MDA-MB-231 cells, in scramble and in SDCBP-KO MDA-MB-231 subclone#2 and subclone#12 ( n = 3). ( I ) Immunofluorescence staining was used to visualize SDCBP (green color) and BACH1 (red color) in scramble and in SDCBP-KO MDA-MB-231 cells. DAPI (blue color) was used to stain the nucleus ( n = 3); Representative confocal immunofluorescence images are shown. Scale bar = 20 µm. ( J ) Western blot showing BACH1 and HO-1 protein expression in 4T1 cells infected with scramble or adenoviral SDCBP shRNA. ( K ) Real-time qPCR showing the mRNA expression of BACH1-regulated antioxidant genes ( HMOX1, NQO1 , and GLCL ) in 4T1 cells transfected with scramble or SDCBP siRNA ( n = 3); mRNA expression of KEAP1 was the negative control. Data are expressed as the mean ± SEM and analyzed using one-way ANOVA ( D , E , G , H ) or two-way ANOVA ( K ). P values less than 0.05 were considered statistically significant. All experiments were repeated at least three times unless otherwise indicated.

Article Snippet: Briefly, cells were initially transfected with SDCBP siRNA oligo duplex (CAT#SR321723, OriGene, USA) and then resuspended at a density of 2 × 10 4 cells/well into XFe24V7 PS cell culture microplates.

Techniques: Expressing, Western Blot, Transfection, Generated, CRISPR, Immunofluorescence, Staining, Infection, shRNA, Negative Control

Journal: The EMBO Journal

Article Title: SDCBP/Syntenin-1 stabilizes BACH1 by disassembling the SCF FBXO22 –BACH1 complex in triple-negative breast cancer

doi: 10.1038/s44318-025-00440-1

Figure Lengend Snippet: ( A ) Immunohistochemistry staining against the SDCBP and BACH1 protein in human TNBC-derived tissue microarray sections ( n = 78). Representative images showing the co-expression of SDCBP and BACH1 in the same section. Normal breast cancer tissues were considered as the negative control. Scale bar = 20 µm. ( B ) Pearson correlation coefficient (r = 0.5772, P < 0.0001) between SDCBP and BACH1 expression in ( A ). ( C ) Western blot showing BACH1 and HO-1 protein expression in Hs578T cells transfected with control vector or Myc-SDCBP-expressing vector. ( D ) Real-time qPCR showing BACH1 mRNA expression in Hs578T cells transfected with a control vector or a Myc-SDCBP-expressing vector ( n = 3). ( E ) Real-time qPCR showing the mRNA expression of BACH1-regulated antioxidant genes ( HMOX1 and NQO1 ) and BACH1-regulated metastatic genes ( HK2, MMP1 , and CXCR4 ) in Hs578T cells transfected with control vector or Myc-SDCBP-expressing vector ( n = 3). ( F ) Western blot showing BACH1 protein expression in MDA-MB-231 infected with lentiviral scramble or SDCBP shRNA. ( G ) Real-time qPCR showing BACH1 mRNA expression in MDA-MB-231 infected with lentiviral scramble or SDCBP shRNA ( n = 3). ( H ) Left, Representative images of immunofluorescence staining to visualize SDCBP ( green color ) and BACH1 ( red color ) expression in MDA-MB-231 cells transfected with a scramble siRNA or SDCBP siRNA. DAPI ( blue color) was used to stain the nucleus ( n = 3); Scale bar = 50 µm. Right, fluorescence levels of SDCBP and BACH1 were quantified based on their spectral densities. ( I ) Real-time qPCR showing the mRNA expression of BACH1-regulated antioxidant genes ( HMOX1 and NQO1 ) and BACH1-regulated metastatic genes ( HK2, MMP1, MMP13 , and CXCR4 ) in MDA-MB-231 cells transfected with scramble siRNA or SDCBP siRNA ( n = 3). ( J ) Western blot showing SDCBP and BACH1 protein expression in scramble control and two SDCBP-KO MDA-MB-231 clones (KO#2, KO#12) generated using CRISPR-Cas9 system ( n = 3). ( K ) Real-time qPCR showing the mRNA expression of BACH1-regulated metastatic genes ( HK2 , MMP1 , CXCR4, GAPDH , and VEGF ) in scramble control and SDCBP-KO MDA-MB-231 cells. ( L ) The reconstitution of SDCBP recovers BACH1 protein expression in SDCBP-KO MDA-MB-231 cells. Western blot showing BACH1 protein expression in scramble control and SDCBP-KO MDA-MB-231 cells transfected with control vector or Myc-SDCBP-expressing vector. The arrows indicate the endogenous (Endo) and exogenous (Exo) SDCBP. Data are expressed as the mean ± SEM and analyzed using two-tailed Student’s t test with Welch’s correction ( D , E , G , I ), two-way ANOVA ( H ), or one-way ANOVA ( K ). P values less than 0.05 were considered statistically significant. All experiments were repeated at least three times unless otherwise indicated. .

Article Snippet: Briefly, cells were initially transfected with SDCBP siRNA oligo duplex (CAT#SR321723, OriGene, USA) and then resuspended at a density of 2 × 10 4 cells/well into XFe24V7 PS cell culture microplates.

Techniques: Immunohistochemistry, Staining, Derivative Assay, Microarray, Expressing, Negative Control, Western Blot, Transfection, Control, Plasmid Preparation, Infection, shRNA, Immunofluorescence, Fluorescence, Clone Assay, Generated, CRISPR, Two Tailed Test

Journal: The EMBO Journal

Article Title: SDCBP/Syntenin-1 stabilizes BACH1 by disassembling the SCF FBXO22 –BACH1 complex in triple-negative breast cancer

doi: 10.1038/s44318-025-00440-1

Figure Lengend Snippet: ( A ) Western blot showing BACH1 protein expression in Hs578T cells transfected with Myc-SDCBP-expressing vector with or without BACH1 siRNA. ( B ) Colony formation of Hs578T cells transfected with Myc-SDCBP-expressing vector with or without BACH1 siRNA ( n = 3); See also Fig. . Migration of Hs578T cells transfected with Myc-SDCBP-expressing vector with or without BACH1 siRNA ( n = 3); See also Fig. . ( D ) Western blot showing SDCBP and Flag-BACH1 protein expression in MDA-MB-231 cells transfected with SDCBP siRNA with or without a Flag-BACH1-expressing vector. Cell proliferation of MDA-MB-231 cells transfected with SDCBP siRNA with or without Flag-BACH1-expressing vector ( n = 3). ( F ) Colony formation of MDA-MB-231 cells transfected with SDCBP siRNA with or without Flag-BACH1-expressing vector ( n = 3); See also Fig. . ( G ) Wound closure of scratched MDA-MB-231 cells transfected with SDCBP siRNA with or without a Flag-BACH1-expressing vector ( n = 3); See also Fig. . ( H ) Schematic of various SDCBP mutant constructs generated using the Myc-SDCBP plasmid. ( I ) Left, western blot showing BACH1 protein expression in Hs578T cells transfected with the indicted SDCBP constructs. Right, quantification of BACH1 levels using densitometry ( n = 3). ( J ) Top, schematic of the PDZ1 construct. Bottom, western blot showing BACH1 protein expression in Hs578T cells transfected with Myc-SDCBP or Myc-SDCBP_PDZ1 plasmid. ( K ) Migration of Hs578T cells transfected with Myc-SDCBP, Myc-SDCBP_Δ4, or Myc-SDCBP_PDZ1 plasmid ( n = 3); See also Fig. . ( L ) Colony formation of Hs578T cells transfected with Myc-SDCBP, Myc-SDCBP_Δ4, or Myc-SDCBP_PDZ1 plasmid ( n = 3); See also Fig. . ( M ) Tumor volumes from athymic BALB/c nude mice 6 weeks after mammary fat-pad injection of the scramble control or SDCBP-KO MDA-MB-231 cells (1 × 10 5 cells/mouse; n = 5 or 7 mice/group). ( N ) Tumor weights in Fig. 2M ( n = 7 mice/group); See also Fig. . ( O ) Tumor volumes from athymic BALB/c nude mice 25 days after mammary fat-pad injection of the scramble control, SDCBP-KO MDA-MB-231 cells, and SDCBP-KO MDA-MB-231 cells stably transfected with Flag-SDCBP (1 × 10 5 cells/mouse; n = 5 mice/group); See also Fig. – , . Data are expressed as the mean ± SEM and analyzed using one-way ANOVA ( B , , I , K , L , N ), two-tailed Student’s t test ( , F , O ), or two-way ANOVA ( G , M ). P values less than 0.05 were considered statistically significant. All experiments were repeated at least three times unless otherwise indicated. .

Article Snippet: Briefly, cells were initially transfected with SDCBP siRNA oligo duplex (CAT#SR321723, OriGene, USA) and then resuspended at a density of 2 × 10 4 cells/well into XFe24V7 PS cell culture microplates.

Techniques: Western Blot, Expressing, Transfection, Plasmid Preparation, Migration, Mutagenesis, Construct, Generated, Injection, Control, Stable Transfection, Two Tailed Test

Journal: The EMBO Journal

Article Title: SDCBP/Syntenin-1 stabilizes BACH1 by disassembling the SCF FBXO22 –BACH1 complex in triple-negative breast cancer

doi: 10.1038/s44318-025-00440-1

Figure Lengend Snippet: ( A ) Western blot showing the expression of SDCBP and Flag-SDCBP in Fig. . ( B ) Tumor weights in Fig. ( n = 5 mice/group). ( C ) Tumor volume in Fig. ( n = 5 mice/group). ( D ) Representative images of immunohistochemistry staining against SDCBP, BACH1, and Ki67 protein for xenografted tumors isolated from athymic BALB/c nude mice 6 weeks after mammary fat-pad injection of the scramble control or SDCBP-KO MDA-MB-231 cells (1 × 10 5 cells/mouse; n = 7 mice/group). Representative images of IHC staining are shown. Scale bar = 200 µm (upper) and 50 µm (lower), respectively. ( E ) Representative images of immunohistochemistry staining against SDCBP, BACH1, and Ki67 protein for xenografted tumors isolated from athymic BALB/c nude mice 25 days after mammary fat-pad injection of the scramble control, SDCBP-KO MDA-MB-231 cells, or SDCBP-KO MDA-MB-231 cells stably transfected with Flag-SDCBP (1 × 10 5 cells/mouse; n = 5 mice/group). Scale bar = 200 µm (upper) and 50 µm (lower), respectively. ( F ) TCGA data analysis showing association between SDCBP mRNA expression and overall survival ( n = 392) and lymph node status ( n = 98) of TNBC patients. ( G ) TCGA data analysis showing association between BACH1 mRNA expression and overall survival ( n = 2032) and lymph node status ( n = 98) of TNBC patients. Data are expressed as the mean ± SEM and analyzed using two-way ANOVA ( B ) or two-tailed Student’s t test ( C ). P values less than 0.05 were considered statistically significant. All experiments were repeated at least three times unless otherwise indicated.

Article Snippet: Briefly, cells were initially transfected with SDCBP siRNA oligo duplex (CAT#SR321723, OriGene, USA) and then resuspended at a density of 2 × 10 4 cells/well into XFe24V7 PS cell culture microplates.

Techniques: Western Blot, Expressing, Immunohistochemistry, Staining, Isolation, Injection, Control, Stable Transfection, Transfection, Two Tailed Test

Journal: The EMBO Journal

Article Title: SDCBP/Syntenin-1 stabilizes BACH1 by disassembling the SCF FBXO22 –BACH1 complex in triple-negative breast cancer

doi: 10.1038/s44318-025-00440-1

Figure Lengend Snippet: ( A ) Representative images of colony formation in Fig. . ( B ) Representative images of the migrated cells in Fig. . Scale bar = 200 µm. ( C ) Representative images of colony formation in Fig. . ( D ) Representative images of wounding migration in Fig. . Scale bar = 200 µm. ( E ) Cell proliferation of MDA-MB-231 cells transfected with scramble or BACH1 siRNA. Cell proliferation was estimated by an automatic cell counter at the indicated time points ( n = 3). ( F ) Colony formation of MDA-MB-231 cells transfected with scramble or BACH1 siRNA. The clonogenic ability was assessed and quantified based on the absorbance at 600 nm and normalized to the control ( n = 3). ( G ) Migration of MDA-MB-231 cells transfected with scramble or BACH1 siRNA. The number of migrated cells were counted and expressed as percentages ( n = 3). ( H ) Representative images of the migrated cells in Fig. . Scale bar = 500 µm. ( I ) Representative images of colony formation in Fig. . Scale bar = 1000 µm. Data are expressed as the mean ± SEM and analyzed using two-tailed Student’s t test with Welch’s correction ( E – G ). P values less than 0.05 were considered statistically significant. All experiments were repeated at least three times unless otherwise indicated.

Article Snippet: Briefly, cells were initially transfected with SDCBP siRNA oligo duplex (CAT#SR321723, OriGene, USA) and then resuspended at a density of 2 × 10 4 cells/well into XFe24V7 PS cell culture microplates.

Techniques: Migration, Transfection, Control, Two Tailed Test

Journal: The EMBO Journal

Article Title: SDCBP/Syntenin-1 stabilizes BACH1 by disassembling the SCF FBXO22 –BACH1 complex in triple-negative breast cancer

doi: 10.1038/s44318-025-00440-1

Figure Lengend Snippet: ( A ) Western blot showing BACH1 protein expression in MDA-MB-231 cells transfected with SDCBP siRNA with or without MG132 proteasome inhibitor. ( B ) Left, western blot showing BACH1 protein expression in Hs578T cells transfected with control vector or Myc-SDCBP-expressing vector in the presence of CHX protein synthesis inhibitor at various time points. Right, quantification of BACH1 protein levels using densitometry ( n = 3). ( C ) Left, western blot showing BACH1 protein expression in MDA-MB-231 cells transfected with scramble or FBXO22 siRNA in the presence of CHX protein synthesis inhibitor at various time points. Right, quantification of BACH1 protein levels using densitometry ( n = 3). ( D ) Western blot showing BACH1, FBXO22, and SDCBP protein expression in MDA-MB-231 cells transfected with scramble or FBXO22 siRNA. ( E ) Western blot showing BACH1 protein expression in MDA-MB-231 cells transfected with HA-FBXO22-expressiong vector with or without Myc-SDCBP-expressing vector. ( F ) In vitro ubiquitylation assay of the recombinant human BACH1 protein mediated by the FBXO22 complex. Active recombinant human UbcH5a protein was used as the E2 ubiquitin-conjugating enzyme for FBXO22 complex-mediated BACH1 degradative polyubiquitylation. ( G ) In vivo ubiquitylation assay showing the decrease in the K48-linked polyubiquitylation of BACH1 by SDCBP overexpression in HEK293 cells transfected with the indicated plasmids. ( H ) In vivo ubiquitylation assay showing the increase in the K48-linked polyubiquitylation of BACH1 by SDCBP KD in MDA-MB-231 cells transfected with the indicated plasmids. ( I ) In vitro ubiquitylation assay showing the inhibitory effect of SDCBP on the polyubiquitylation of BACH1 mediated by the FBXO22 complex. Active recombinant human protein UbcH5a and immunocomplex FBXO22 were added as described above. Recombinant human BACH1 and recombinant human SDCBP proteins were added at ratios of 1:1 (+) and 1:2 (++). Data are expressed as the mean ± SEM and analyzed using two-tailed Student’s t test with Welch’s correction ( B , C ). P values less than 0.05 were considered statistically significant. All experiments were repeated at least three times unless otherwise indicated. .

Article Snippet: Briefly, cells were initially transfected with SDCBP siRNA oligo duplex (CAT#SR321723, OriGene, USA) and then resuspended at a density of 2 × 10 4 cells/well into XFe24V7 PS cell culture microplates.

Techniques: Western Blot, Expressing, Transfection, Control, Plasmid Preparation, In Vitro, Ubiquitin Assay, Recombinant, Ubiquitin Proteomics, In Vivo, Over Expression, Two Tailed Test

Journal: The EMBO Journal

Article Title: SDCBP/Syntenin-1 stabilizes BACH1 by disassembling the SCF FBXO22 –BACH1 complex in triple-negative breast cancer

doi: 10.1038/s44318-025-00440-1

Figure Lengend Snippet: ( A ) Left, Western blot showing BACH1 protein expression in MDA-MB-231 cells transfected with control vector or Myc-SDCBP-expressing vector in the presence of CHX protein synthesis inhibitor at various time points. Right, quantification of BACH1 protein levels using densitometry ( n = 3). ( B ) Free heme level in Hs578T cells transfected with control vector or Myc-SDCBP-expressing vector ( n = 3). ( C ) Free heme level in scramble and in SDCBP - KO MDA-MB-231 cells ( n = 3). ( D ) Western blot showing BACH1 protein expression in MDA-MB-231 cells transfected with control vector or NRF2 (encoded by NFE2L2 )-expressing vector. HO-1 protein expression was considered as the positive control. ( E ) Western blot showing BACH1 protein expression in MDA-MB-231 cells transfected with a control or a HO-1 (encoded by HMOX1 )-expressing vector. ( F ) Western blot showing BACH1 protein expression in Hs578T cells transfected with scramble or HO-1 siRNA. ( G ) Western blot showing BACH1 protein expression in MDA-MB-231 cells transfected with the HO-1 or the catalytic inactive HO-1 mutant (H25A) plasmid. ( H ) Western blot showing BACH1 and SDCBP protein expression in MDA-MB-231 cells transfected with scramble or HOIL1 siRNA. ( I ) Western blot showing endogenous FBXO22 protein expression in several breast cancer cells. ( J ) Immunoprecipitation showing the interaction of BACH1 with FBXO22 in HEK293 cells transfected with the indicated plasmids. An arrow indicates the specific signal for HA-FBXO22. ns: none specific. ( K ) In vivo ubiquitylation assay showing the increase in the polyubiquitylation of BACH1 by FBXO22 overexpression in HEK293 cells transfected with the indicated plasmids. ( L ) In vivo ubiquitylation assay showing the increase in the polyubiquitylation of BACH1 by FBXO22 overexpression in MDA-MB-231 cells transfected with the indicated plasmids. ( M ) In vivo ubiquitylation assay showing the decrease in FBXO22-mediated polyubiquitylation of BACH1 by SDCBP overexpression in HEK293 cells transfected with the indicated plasmids. Data are expressed as the mean ± SEM and analyzed using two-tailed Student’s t test with Welch’s correction ( A – C ). All experiments were repeated at least three times unless otherwise indicated. P values less than 0.05 were considered statistically significant.

Article Snippet: Briefly, cells were initially transfected with SDCBP siRNA oligo duplex (CAT#SR321723, OriGene, USA) and then resuspended at a density of 2 × 10 4 cells/well into XFe24V7 PS cell culture microplates.

Techniques: Western Blot, Expressing, Transfection, Control, Plasmid Preparation, Positive Control, Mutagenesis, Immunoprecipitation, In Vivo, Ubiquitin Assay, Over Expression, Two Tailed Test

Journal: The EMBO Journal

Article Title: SDCBP/Syntenin-1 stabilizes BACH1 by disassembling the SCF FBXO22 –BACH1 complex in triple-negative breast cancer

doi: 10.1038/s44318-025-00440-1

Figure Lengend Snippet: ( A ) Immunoprecipitation showing the interaction of FBXO22 with SDCBP in HEK293 cells transfected with the indicated plasmids. An arrow indicates the specific signal for HA-FBXO22. ( B ) Immunoprecipitation showing the interaction of SDCBP with FBXO22 in HEK293 cells transfected with the indicated plasmids. An arrow indicates the specific signal for HA-FBXO22. ( C ) Co-immunoprecipitation showing the interaction of FBXO22 with BACH1 in HEK293 cells with or without SDCBP after the indicated transfections. ( D ) Schematic of experimental design to investigate the assembly of SCF FBXO22 –BACH1 complex via His Pull-down assay and endogenous IP assay in Fig. D– . ( E ) His-pulldown assay showing the interaction of FBXO22 with SKP1 in HEK293 cells with control vector or Myc-SDCBP-expressing vector after the indicated transfections. See also Appendix Fig. S . ( F ) Western blot showing BACH1, PTEN, and PD-L1 protein expression in scramble and in SDCBP-KO MDA-MB-231 cells. Western blot showing BACH1, PTEN, and PD-L1 protein expression in A549 cells transfected with scramble or SDCBP siRNA. ( H ) Western blot showing BACH1 and PD-L1 protein expression in NCI-H1299 cells transfected with scramble or SDCBP siRNA. ( I ) Western blot showing BACH1, PTEN, and PD-L1 protein expression in Hs578T cells transfected with control vector or Myc-SDCBP-expressing vector. ( J ) In vivo ubiquitylation assay showing the inhibitory effect of SDCBP on SCF FBXO22 -mediated K48-linked polyubiquitylation of BACH1 in HEK293 cells transfected with the indicated plasmids.

Article Snippet: Briefly, cells were initially transfected with SDCBP siRNA oligo duplex (CAT#SR321723, OriGene, USA) and then resuspended at a density of 2 × 10 4 cells/well into XFe24V7 PS cell culture microplates.

Techniques: Immunoprecipitation, Transfection, Pull Down Assay, Control, Plasmid Preparation, Expressing, Western Blot, In Vivo, Ubiquitin Assay

Journal: The EMBO Journal

Article Title: SDCBP/Syntenin-1 stabilizes BACH1 by disassembling the SCF FBXO22 –BACH1 complex in triple-negative breast cancer

doi: 10.1038/s44318-025-00440-1

Figure Lengend Snippet: ( A ) Crosslink immunoprecipitation showing an interaction of FBXO22 with SDCBP in Hs578T cells transfected with Myc-SDCBP-expressing vector or Myc-SDCBP_Δ4-expressing vector. Schematic showing the FBXO22 mutant constructs generated using the HA-FBXO22 plasmid. ( C ) Immunoprecipitation showing SDCBP interactions with FBXO22 and its mutant constructs in HEK293 cells transfected with the indicated plasmids. ns indicates non-specific bands. See also Fig. , . ( D ) His-Pulldown assay showing the effect of SDCBP on SKP1-CUL1-FBXO22 complex formation after the indicated transfections in HEK293 cells. See also Appendix Fig. S . ( E ) His-Pulldown assay showing the effect of SDCBP KO on the SCF FBXO22 –BACH1 complex formation in the scramble control and SDCBP-KO MDA-MB-231 cells transfected with control vector or His-SKP1-expressing vector. See also Appendix Fig. S . ( F ) Immunoprecipitation showing the effect of SDCBP KD on the SCF FBXO22 –BACH1 complex formation in MDA-MB-231 cells transfected with scramble or SDCBP siRNA. ( G ) Immunoprecipitation showing the effect of SDCBP overexpression on the SCF FBXO22 –BACH1 complex formation in Hs578T cells transfected with control vector or Myc-SDCBP-overexpressing vector. ( H ) Immunoprecipitation showing the effect of SDCBP PDZ1 domain on the SCF FBXO22 –BACH1 complex formation in Hs578T cells transfected with a control vector or a Myc-SDCBP-PDZ1-overexpressing vector. .

Article Snippet: Briefly, cells were initially transfected with SDCBP siRNA oligo duplex (CAT#SR321723, OriGene, USA) and then resuspended at a density of 2 × 10 4 cells/well into XFe24V7 PS cell culture microplates.

Techniques: Immunoprecipitation, Transfection, Expressing, Plasmid Preparation, Mutagenesis, Construct, Generated, Control, Over Expression

Journal: The EMBO Journal

Article Title: SDCBP/Syntenin-1 stabilizes BACH1 by disassembling the SCF FBXO22 –BACH1 complex in triple-negative breast cancer

doi: 10.1038/s44318-025-00440-1

Figure Lengend Snippet: ( A ) Real-time qPCR showing the mRNA expression of BACH1-regulated ETC genes ( NDUFA4 , NDUFA4L2 , NDUFC2 , and COX6B2 ) in scramble and SDCBP-KO MDA-MB-231 cells ( n = 3); See also Appendix Fig. S , . Real-time qPCR showing the mRNA expression of NDUFA4 and COX6B2 in scramble, SDCBP-KO MDA-MB-231 cells, and SDCBP-KO MDA-MB-231 cells transfected with SDCBP ( n = 3). ( C ) Western blots showing the expression of mitochondrial proteins NDUFA4 and COX6B2 in MDA-MB-231 cells transfected with SDCBP siRNA in the presence or absence of FLAG-BACH1-expressing vector. ( D ) ChIP-qPCR showing BACH1 enrichments in the promoter regions of NDUFA4 and COX6B2 in the scramble and SDCBP-KO MDA-MB-231 cells. Quantitative data were normalized to IgG binding expression ( n = 3); See also Appendix Fig. S . ( E ) Left, flow cytometry histogram showing the mitochondrial membrane potentials using TMRE (tetramethylrhodamine ethyl ester) staining in MDA-MB-231 cells transfected with a scramble or SDCBP siRNA after the indicated treatments. FCCP (trifluoromethoxy carbonylcyanide phenylhydrazone). Right, quantification of TMRE fluorescence intensity ( n = 3). ( F ) Left, immunofluorescence staining and confocal imaging of the fluorescent signals for TMRE (orange-red color) in the scramble control and SDCBP-KO MDA-MB-231 cells after incubation with TMRE. DAPI (blue color) was used to stain the nucleus ( n = 7); Representative confocal images are shown; scale bars = 20 µm and 5 µm. Right, fluorescent levels of the TMRE were quantified based on their spectral densities. ( G ) Representative images of immunohistochemistry staining against the NDUFA4, BACH1, and SDCBP proteins showing a negative correlation between the expression of NDUFA4 and SDCBP in the same sections of TNBC tumor tissues. Scale bar = 20 µm. ( H ) Pearson correlation coefficients between SDCBP and NDUFA4 protein expression ( n = 64), and between BACH1 and NDUFA4 protein expression ( n = 60) in Fig. 5G. Data are expressed as the mean ± SEM and analyzed using two-way ANOVA ( A , D , F ), two-tailed Student’s t test , or one-way ANOVA ( E ). P values less than 0.05 were considered statistically significant. All experiments were repeated at least three times unless otherwise indicated. .

Article Snippet: Briefly, cells were initially transfected with SDCBP siRNA oligo duplex (CAT#SR321723, OriGene, USA) and then resuspended at a density of 2 × 10 4 cells/well into XFe24V7 PS cell culture microplates.

Techniques: Expressing, Transfection, Western Blot, Plasmid Preparation, ChIP-qPCR, Binding Assay, Flow Cytometry, Membrane, Staining, Fluorescence, Immunofluorescence, Imaging, Control, Incubation, Immunohistochemistry, Two Tailed Test

Journal: The EMBO Journal

Article Title: SDCBP/Syntenin-1 stabilizes BACH1 by disassembling the SCF FBXO22 –BACH1 complex in triple-negative breast cancer

doi: 10.1038/s44318-025-00440-1

Figure Lengend Snippet: ( A ) Cell proliferation of MDA-MB-231 cells transfected with a scramble or SDCBP siRNA after treatment with metformin for the indicated periods of time ( n = 3). ( B ) Cell viability of MDA-MB-231 cells transfected with a scramble or SDCBP siRNA after treatment with the indicated concentrations of metformin for 96 h ( n = 5). ( C ) Colony formation for MDA-MB-231 cells transfected with a scramble or SDCBP siRNA after treatment with the indicated concentrations of metformin. Colony numbers were counted and converted to percentages by normalizing with the control groups ( n = 3). ( D – F ) Effect of the indicated treatment on 4T1 tumor growth. Tumor growth was monitored in BALB/c mice bearing 4T1 cells after mammary fat-pad injections. When the average tumor volumes reached 100 mm 3 , the mice ( n = 7 mice/group) were administered with 100 mg/kg metformin (once a day) and/or adenoviral SDCBP shRNA (1 × 10 9 PFU/mice). Black arrows indicate the day of adenoviral SDCBP shRNA injection. Final tumor volume ( E ) and weight ( F ) are shown. ( G ) Immunohistochemistry staining against SDCBP, BACH1, Ki67, NDUFA4, and COX6B2 protein in 4T1 tumors from BALB/c mice in Fig. 6A. Representative images of the IHC staining are shown. Scale bar = 50 µm for low (left) and high (right) magnification. Data are expressed as the mean ± SEM and analyzed using two-way ANOVA ( A – C ), one-way ANOVA ( E ), or two-tailed Student’s t test ( F ). P values less than 0.05 were considered statistically significant. All experiments were repeated at least three times unless otherwise indicated. .

Article Snippet: Briefly, cells were initially transfected with SDCBP siRNA oligo duplex (CAT#SR321723, OriGene, USA) and then resuspended at a density of 2 × 10 4 cells/well into XFe24V7 PS cell culture microplates.

Techniques: Transfection, Control, shRNA, Injection, Immunohistochemistry, Staining, Two Tailed Test

Journal: The EMBO Journal

Article Title: SDCBP/Syntenin-1 stabilizes BACH1 by disassembling the SCF FBXO22 –BACH1 complex in triple-negative breast cancer

doi: 10.1038/s44318-025-00440-1

Figure Lengend Snippet: Schematic showing the novel oncogenic roles of SDCBP in promoting aggressiveness and mitochondrial inhibitor resistance in TNBCs. An abundance of SDCBP stabilizes the BACH1 protein by blocking E3 ubiquitin ligase SCF FBXO22 complex-targeted BACH1 for degradative ubiquitination. Mechanistically, SDCBP binds to different members of the SCF FBXO22 complex, including SKP1 and FBXO22, via its PDZ1 domain and induces SCF FBXO22 complex disassociation, suggesting that SDCBP is a key adapter regulating the activity of the E3 ubiquitin ligase SCF FBXO22 complex in the proteasomal pathway. SDCBP-induced BACH1 accumulation upregulates several pro-metastatic genes and downregulates numerous mitochondrial ETC genes, resulting in tumor progression and high resistance to metformin treatment in TNBCs. Targeting SDCBP switches the SCF FBXO22 complex to degrade BACH1 protein via the proteasome, reducing tumor aggressiveness and boosting the anti-tumor effect of metformin administration in TNBCs. .

Article Snippet: Briefly, cells were initially transfected with SDCBP siRNA oligo duplex (CAT#SR321723, OriGene, USA) and then resuspended at a density of 2 × 10 4 cells/well into XFe24V7 PS cell culture microplates.

Techniques: Blocking Assay, Ubiquitin Proteomics, Activity Assay

Journal: The EMBO Journal

Article Title: SDCBP/Syntenin-1 stabilizes BACH1 by disassembling the SCF FBXO22 –BACH1 complex in triple-negative breast cancer

doi: 10.1038/s44318-025-00440-1

Figure Lengend Snippet: ( A ) TCGA data analysis showing the correlation between SDCBP mRNA and BACH1 mRNA expression in GSE142102 ( n = 226) dataset of TNBC patients (Pearson correlation coefficient r = 0.3245, P < 0.0001). ( B ) TCGA data analysis showing the correlation between SDCBP mRNA and BACH1 mRNA expression in GSE103091 ( n = 238) dataset of TNBC patients (Pearson correlation coefficient r = 0.2120, P < 0.001). ( C ) Western blot showing SDCBP, BACH1, and HO-1 protein expression in MDA-MB-231, MDA-MB-468, Hs578T, MCF-7, and T47D cells. ( D ) The expression levels of SDCBP and BACH1 protein in Fig. EV1C were quantified using densitometry and normalized to the housekeeping protein α-tubulin ( n = 3). ( E ) Real-time qPCR showing SDCBP and BACH1 mRNA expression in MDA-MB-231, MDA-MB-468, Hs578T, MCF-7, and T47D cells ( n = 3). Quantitative data were normalized to β-actin expression. ( F ) Western blot showing SDCBP and HO-1 protein expression in MDA-MB-231 cells transfected with scramble or BACH1 siRNA. ( G ) Left, western blot showing the protein expression of SDCBP in the scramble and in several SDCBP-KO MDA-MB-231 subclones generated using CRISPR-Cas9 system; Right, real-time qPCR showing the SDCBP mRNA expression in scramble and in SDCBP-KO MDA-MB-231 subclones ( n = 3). ( H ) Real-time qPCR showing the mRNA expression of BACH1 in MDA-MB-231 cells, in scramble and in SDCBP-KO MDA-MB-231 subclone#2 and subclone#12 ( n = 3). ( I ) Immunofluorescence staining was used to visualize SDCBP (green color) and BACH1 (red color) in scramble and in SDCBP-KO MDA-MB-231 cells. DAPI (blue color) was used to stain the nucleus ( n = 3); Representative confocal immunofluorescence images are shown. Scale bar = 20 µm. ( J ) Western blot showing BACH1 and HO-1 protein expression in 4T1 cells infected with scramble or adenoviral SDCBP shRNA. ( K ) Real-time qPCR showing the mRNA expression of BACH1-regulated antioxidant genes ( HMOX1, NQO1 , and GLCL ) in 4T1 cells transfected with scramble or SDCBP siRNA ( n = 3); mRNA expression of KEAP1 was the negative control. Data are expressed as the mean ± SEM and analyzed using one-way ANOVA ( D , E , G , H ) or two-way ANOVA ( K ). P values less than 0.05 were considered statistically significant. All experiments were repeated at least three times unless otherwise indicated.

Article Snippet: Mouse SDCBP siRNA Oligo Duplex (Locus ID53378) , Origene , Cat#SR426294.

Techniques: Expressing, Western Blot, Transfection, Generated, CRISPR, Immunofluorescence, Staining, Infection, shRNA, Negative Control

Journal: The EMBO Journal

Article Title: SDCBP/Syntenin-1 stabilizes BACH1 by disassembling the SCF FBXO22 –BACH1 complex in triple-negative breast cancer

doi: 10.1038/s44318-025-00440-1

Figure Lengend Snippet: ( A ) Immunohistochemistry staining against the SDCBP and BACH1 protein in human TNBC-derived tissue microarray sections ( n = 78). Representative images showing the co-expression of SDCBP and BACH1 in the same section. Normal breast cancer tissues were considered as the negative control. Scale bar = 20 µm. ( B ) Pearson correlation coefficient (r = 0.5772, P < 0.0001) between SDCBP and BACH1 expression in ( A ). ( C ) Western blot showing BACH1 and HO-1 protein expression in Hs578T cells transfected with control vector or Myc-SDCBP-expressing vector. ( D ) Real-time qPCR showing BACH1 mRNA expression in Hs578T cells transfected with a control vector or a Myc-SDCBP-expressing vector ( n = 3). ( E ) Real-time qPCR showing the mRNA expression of BACH1-regulated antioxidant genes ( HMOX1 and NQO1 ) and BACH1-regulated metastatic genes ( HK2, MMP1 , and CXCR4 ) in Hs578T cells transfected with control vector or Myc-SDCBP-expressing vector ( n = 3). ( F ) Western blot showing BACH1 protein expression in MDA-MB-231 infected with lentiviral scramble or SDCBP shRNA. ( G ) Real-time qPCR showing BACH1 mRNA expression in MDA-MB-231 infected with lentiviral scramble or SDCBP shRNA ( n = 3). ( H ) Left, Representative images of immunofluorescence staining to visualize SDCBP ( green color ) and BACH1 ( red color ) expression in MDA-MB-231 cells transfected with a scramble siRNA or SDCBP siRNA. DAPI ( blue color) was used to stain the nucleus ( n = 3); Scale bar = 50 µm. Right, fluorescence levels of SDCBP and BACH1 were quantified based on their spectral densities. ( I ) Real-time qPCR showing the mRNA expression of BACH1-regulated antioxidant genes ( HMOX1 and NQO1 ) and BACH1-regulated metastatic genes ( HK2, MMP1, MMP13 , and CXCR4 ) in MDA-MB-231 cells transfected with scramble siRNA or SDCBP siRNA ( n = 3). ( J ) Western blot showing SDCBP and BACH1 protein expression in scramble control and two SDCBP-KO MDA-MB-231 clones (KO#2, KO#12) generated using CRISPR-Cas9 system ( n = 3). ( K ) Real-time qPCR showing the mRNA expression of BACH1-regulated metastatic genes ( HK2 , MMP1 , CXCR4, GAPDH , and VEGF ) in scramble control and SDCBP-KO MDA-MB-231 cells. ( L ) The reconstitution of SDCBP recovers BACH1 protein expression in SDCBP-KO MDA-MB-231 cells. Western blot showing BACH1 protein expression in scramble control and SDCBP-KO MDA-MB-231 cells transfected with control vector or Myc-SDCBP-expressing vector. The arrows indicate the endogenous (Endo) and exogenous (Exo) SDCBP. Data are expressed as the mean ± SEM and analyzed using two-tailed Student’s t test with Welch’s correction ( D , E , G , I ), two-way ANOVA ( H ), or one-way ANOVA ( K ). P values less than 0.05 were considered statistically significant. All experiments were repeated at least three times unless otherwise indicated. .

Article Snippet: Mouse SDCBP siRNA Oligo Duplex (Locus ID53378) , Origene , Cat#SR426294.

Techniques: Immunohistochemistry, Staining, Derivative Assay, Microarray, Expressing, Negative Control, Western Blot, Transfection, Control, Plasmid Preparation, Infection, shRNA, Immunofluorescence, Fluorescence, Clone Assay, Generated, CRISPR, Two Tailed Test

Journal: The EMBO Journal

Article Title: SDCBP/Syntenin-1 stabilizes BACH1 by disassembling the SCF FBXO22 –BACH1 complex in triple-negative breast cancer

doi: 10.1038/s44318-025-00440-1

Figure Lengend Snippet: ( A ) Western blot showing BACH1 protein expression in Hs578T cells transfected with Myc-SDCBP-expressing vector with or without BACH1 siRNA. ( B ) Colony formation of Hs578T cells transfected with Myc-SDCBP-expressing vector with or without BACH1 siRNA ( n = 3); See also Fig. . Migration of Hs578T cells transfected with Myc-SDCBP-expressing vector with or without BACH1 siRNA ( n = 3); See also Fig. . ( D ) Western blot showing SDCBP and Flag-BACH1 protein expression in MDA-MB-231 cells transfected with SDCBP siRNA with or without a Flag-BACH1-expressing vector. Cell proliferation of MDA-MB-231 cells transfected with SDCBP siRNA with or without Flag-BACH1-expressing vector ( n = 3). ( F ) Colony formation of MDA-MB-231 cells transfected with SDCBP siRNA with or without Flag-BACH1-expressing vector ( n = 3); See also Fig. . ( G ) Wound closure of scratched MDA-MB-231 cells transfected with SDCBP siRNA with or without a Flag-BACH1-expressing vector ( n = 3); See also Fig. . ( H ) Schematic of various SDCBP mutant constructs generated using the Myc-SDCBP plasmid. ( I ) Left, western blot showing BACH1 protein expression in Hs578T cells transfected with the indicted SDCBP constructs. Right, quantification of BACH1 levels using densitometry ( n = 3). ( J ) Top, schematic of the PDZ1 construct. Bottom, western blot showing BACH1 protein expression in Hs578T cells transfected with Myc-SDCBP or Myc-SDCBP_PDZ1 plasmid. ( K ) Migration of Hs578T cells transfected with Myc-SDCBP, Myc-SDCBP_Δ4, or Myc-SDCBP_PDZ1 plasmid ( n = 3); See also Fig. . ( L ) Colony formation of Hs578T cells transfected with Myc-SDCBP, Myc-SDCBP_Δ4, or Myc-SDCBP_PDZ1 plasmid ( n = 3); See also Fig. . ( M ) Tumor volumes from athymic BALB/c nude mice 6 weeks after mammary fat-pad injection of the scramble control or SDCBP-KO MDA-MB-231 cells (1 × 10 5 cells/mouse; n = 5 or 7 mice/group). ( N ) Tumor weights in Fig. 2M ( n = 7 mice/group); See also Fig. . ( O ) Tumor volumes from athymic BALB/c nude mice 25 days after mammary fat-pad injection of the scramble control, SDCBP-KO MDA-MB-231 cells, and SDCBP-KO MDA-MB-231 cells stably transfected with Flag-SDCBP (1 × 10 5 cells/mouse; n = 5 mice/group); See also Fig. – , . Data are expressed as the mean ± SEM and analyzed using one-way ANOVA ( B , , I , K , L , N ), two-tailed Student’s t test ( , F , O ), or two-way ANOVA ( G , M ). P values less than 0.05 were considered statistically significant. All experiments were repeated at least three times unless otherwise indicated. .

Article Snippet: Mouse SDCBP siRNA Oligo Duplex (Locus ID53378) , Origene , Cat#SR426294.

Techniques: Western Blot, Expressing, Transfection, Plasmid Preparation, Migration, Mutagenesis, Construct, Generated, Injection, Control, Stable Transfection, Two Tailed Test

Journal: The EMBO Journal

Article Title: SDCBP/Syntenin-1 stabilizes BACH1 by disassembling the SCF FBXO22 –BACH1 complex in triple-negative breast cancer

doi: 10.1038/s44318-025-00440-1

Figure Lengend Snippet: ( A ) Western blot showing the expression of SDCBP and Flag-SDCBP in Fig. . ( B ) Tumor weights in Fig. ( n = 5 mice/group). ( C ) Tumor volume in Fig. ( n = 5 mice/group). ( D ) Representative images of immunohistochemistry staining against SDCBP, BACH1, and Ki67 protein for xenografted tumors isolated from athymic BALB/c nude mice 6 weeks after mammary fat-pad injection of the scramble control or SDCBP-KO MDA-MB-231 cells (1 × 10 5 cells/mouse; n = 7 mice/group). Representative images of IHC staining are shown. Scale bar = 200 µm (upper) and 50 µm (lower), respectively. ( E ) Representative images of immunohistochemistry staining against SDCBP, BACH1, and Ki67 protein for xenografted tumors isolated from athymic BALB/c nude mice 25 days after mammary fat-pad injection of the scramble control, SDCBP-KO MDA-MB-231 cells, or SDCBP-KO MDA-MB-231 cells stably transfected with Flag-SDCBP (1 × 10 5 cells/mouse; n = 5 mice/group). Scale bar = 200 µm (upper) and 50 µm (lower), respectively. ( F ) TCGA data analysis showing association between SDCBP mRNA expression and overall survival ( n = 392) and lymph node status ( n = 98) of TNBC patients. ( G ) TCGA data analysis showing association between BACH1 mRNA expression and overall survival ( n = 2032) and lymph node status ( n = 98) of TNBC patients. Data are expressed as the mean ± SEM and analyzed using two-way ANOVA ( B ) or two-tailed Student’s t test ( C ). P values less than 0.05 were considered statistically significant. All experiments were repeated at least three times unless otherwise indicated.

Article Snippet: Mouse SDCBP siRNA Oligo Duplex (Locus ID53378) , Origene , Cat#SR426294.

Techniques: Western Blot, Expressing, Immunohistochemistry, Staining, Isolation, Injection, Control, Stable Transfection, Transfection, Two Tailed Test

Journal: The EMBO Journal

Article Title: SDCBP/Syntenin-1 stabilizes BACH1 by disassembling the SCF FBXO22 –BACH1 complex in triple-negative breast cancer

doi: 10.1038/s44318-025-00440-1

Figure Lengend Snippet: ( A ) Representative images of colony formation in Fig. . ( B ) Representative images of the migrated cells in Fig. . Scale bar = 200 µm. ( C ) Representative images of colony formation in Fig. . ( D ) Representative images of wounding migration in Fig. . Scale bar = 200 µm. ( E ) Cell proliferation of MDA-MB-231 cells transfected with scramble or BACH1 siRNA. Cell proliferation was estimated by an automatic cell counter at the indicated time points ( n = 3). ( F ) Colony formation of MDA-MB-231 cells transfected with scramble or BACH1 siRNA. The clonogenic ability was assessed and quantified based on the absorbance at 600 nm and normalized to the control ( n = 3). ( G ) Migration of MDA-MB-231 cells transfected with scramble or BACH1 siRNA. The number of migrated cells were counted and expressed as percentages ( n = 3). ( H ) Representative images of the migrated cells in Fig. . Scale bar = 500 µm. ( I ) Representative images of colony formation in Fig. . Scale bar = 1000 µm. Data are expressed as the mean ± SEM and analyzed using two-tailed Student’s t test with Welch’s correction ( E – G ). P values less than 0.05 were considered statistically significant. All experiments were repeated at least three times unless otherwise indicated.

Article Snippet: Mouse SDCBP siRNA Oligo Duplex (Locus ID53378) , Origene , Cat#SR426294.

Techniques: Migration, Transfection, Control, Two Tailed Test

Journal: The EMBO Journal

Article Title: SDCBP/Syntenin-1 stabilizes BACH1 by disassembling the SCF FBXO22 –BACH1 complex in triple-negative breast cancer

doi: 10.1038/s44318-025-00440-1

Figure Lengend Snippet: ( A ) Western blot showing BACH1 protein expression in MDA-MB-231 cells transfected with SDCBP siRNA with or without MG132 proteasome inhibitor. ( B ) Left, western blot showing BACH1 protein expression in Hs578T cells transfected with control vector or Myc-SDCBP-expressing vector in the presence of CHX protein synthesis inhibitor at various time points. Right, quantification of BACH1 protein levels using densitometry ( n = 3). ( C ) Left, western blot showing BACH1 protein expression in MDA-MB-231 cells transfected with scramble or FBXO22 siRNA in the presence of CHX protein synthesis inhibitor at various time points. Right, quantification of BACH1 protein levels using densitometry ( n = 3). ( D ) Western blot showing BACH1, FBXO22, and SDCBP protein expression in MDA-MB-231 cells transfected with scramble or FBXO22 siRNA. ( E ) Western blot showing BACH1 protein expression in MDA-MB-231 cells transfected with HA-FBXO22-expressiong vector with or without Myc-SDCBP-expressing vector. ( F ) In vitro ubiquitylation assay of the recombinant human BACH1 protein mediated by the FBXO22 complex. Active recombinant human UbcH5a protein was used as the E2 ubiquitin-conjugating enzyme for FBXO22 complex-mediated BACH1 degradative polyubiquitylation. ( G ) In vivo ubiquitylation assay showing the decrease in the K48-linked polyubiquitylation of BACH1 by SDCBP overexpression in HEK293 cells transfected with the indicated plasmids. ( H ) In vivo ubiquitylation assay showing the increase in the K48-linked polyubiquitylation of BACH1 by SDCBP KD in MDA-MB-231 cells transfected with the indicated plasmids. ( I ) In vitro ubiquitylation assay showing the inhibitory effect of SDCBP on the polyubiquitylation of BACH1 mediated by the FBXO22 complex. Active recombinant human protein UbcH5a and immunocomplex FBXO22 were added as described above. Recombinant human BACH1 and recombinant human SDCBP proteins were added at ratios of 1:1 (+) and 1:2 (++). Data are expressed as the mean ± SEM and analyzed using two-tailed Student’s t test with Welch’s correction ( B , C ). P values less than 0.05 were considered statistically significant. All experiments were repeated at least three times unless otherwise indicated. .

Article Snippet: Mouse SDCBP siRNA Oligo Duplex (Locus ID53378) , Origene , Cat#SR426294.

Techniques: Western Blot, Expressing, Transfection, Control, Plasmid Preparation, In Vitro, Ubiquitin Assay, Recombinant, Ubiquitin Proteomics, In Vivo, Over Expression, Two Tailed Test

Journal: The EMBO Journal

Article Title: SDCBP/Syntenin-1 stabilizes BACH1 by disassembling the SCF FBXO22 –BACH1 complex in triple-negative breast cancer

doi: 10.1038/s44318-025-00440-1

Figure Lengend Snippet: ( A ) Left, Western blot showing BACH1 protein expression in MDA-MB-231 cells transfected with control vector or Myc-SDCBP-expressing vector in the presence of CHX protein synthesis inhibitor at various time points. Right, quantification of BACH1 protein levels using densitometry ( n = 3). ( B ) Free heme level in Hs578T cells transfected with control vector or Myc-SDCBP-expressing vector ( n = 3). ( C ) Free heme level in scramble and in SDCBP - KO MDA-MB-231 cells ( n = 3). ( D ) Western blot showing BACH1 protein expression in MDA-MB-231 cells transfected with control vector or NRF2 (encoded by NFE2L2 )-expressing vector. HO-1 protein expression was considered as the positive control. ( E ) Western blot showing BACH1 protein expression in MDA-MB-231 cells transfected with a control or a HO-1 (encoded by HMOX1 )-expressing vector. ( F ) Western blot showing BACH1 protein expression in Hs578T cells transfected with scramble or HO-1 siRNA. ( G ) Western blot showing BACH1 protein expression in MDA-MB-231 cells transfected with the HO-1 or the catalytic inactive HO-1 mutant (H25A) plasmid. ( H ) Western blot showing BACH1 and SDCBP protein expression in MDA-MB-231 cells transfected with scramble or HOIL1 siRNA. ( I ) Western blot showing endogenous FBXO22 protein expression in several breast cancer cells. ( J ) Immunoprecipitation showing the interaction of BACH1 with FBXO22 in HEK293 cells transfected with the indicated plasmids. An arrow indicates the specific signal for HA-FBXO22. ns: none specific. ( K ) In vivo ubiquitylation assay showing the increase in the polyubiquitylation of BACH1 by FBXO22 overexpression in HEK293 cells transfected with the indicated plasmids. ( L ) In vivo ubiquitylation assay showing the increase in the polyubiquitylation of BACH1 by FBXO22 overexpression in MDA-MB-231 cells transfected with the indicated plasmids. ( M ) In vivo ubiquitylation assay showing the decrease in FBXO22-mediated polyubiquitylation of BACH1 by SDCBP overexpression in HEK293 cells transfected with the indicated plasmids. Data are expressed as the mean ± SEM and analyzed using two-tailed Student’s t test with Welch’s correction ( A – C ). All experiments were repeated at least three times unless otherwise indicated. P values less than 0.05 were considered statistically significant.

Article Snippet: Mouse SDCBP siRNA Oligo Duplex (Locus ID53378) , Origene , Cat#SR426294.

Techniques: Western Blot, Expressing, Transfection, Control, Plasmid Preparation, Positive Control, Mutagenesis, Immunoprecipitation, In Vivo, Ubiquitin Assay, Over Expression, Two Tailed Test

Journal: The EMBO Journal

Article Title: SDCBP/Syntenin-1 stabilizes BACH1 by disassembling the SCF FBXO22 –BACH1 complex in triple-negative breast cancer

doi: 10.1038/s44318-025-00440-1

Figure Lengend Snippet: ( A ) Immunoprecipitation showing the interaction of FBXO22 with SDCBP in HEK293 cells transfected with the indicated plasmids. An arrow indicates the specific signal for HA-FBXO22. ( B ) Immunoprecipitation showing the interaction of SDCBP with FBXO22 in HEK293 cells transfected with the indicated plasmids. An arrow indicates the specific signal for HA-FBXO22. ( C ) Co-immunoprecipitation showing the interaction of FBXO22 with BACH1 in HEK293 cells with or without SDCBP after the indicated transfections. ( D ) Schematic of experimental design to investigate the assembly of SCF FBXO22 –BACH1 complex via His Pull-down assay and endogenous IP assay in Fig. D– . ( E ) His-pulldown assay showing the interaction of FBXO22 with SKP1 in HEK293 cells with control vector or Myc-SDCBP-expressing vector after the indicated transfections. See also Appendix Fig. S . ( F ) Western blot showing BACH1, PTEN, and PD-L1 protein expression in scramble and in SDCBP-KO MDA-MB-231 cells. Western blot showing BACH1, PTEN, and PD-L1 protein expression in A549 cells transfected with scramble or SDCBP siRNA. ( H ) Western blot showing BACH1 and PD-L1 protein expression in NCI-H1299 cells transfected with scramble or SDCBP siRNA. ( I ) Western blot showing BACH1, PTEN, and PD-L1 protein expression in Hs578T cells transfected with control vector or Myc-SDCBP-expressing vector. ( J ) In vivo ubiquitylation assay showing the inhibitory effect of SDCBP on SCF FBXO22 -mediated K48-linked polyubiquitylation of BACH1 in HEK293 cells transfected with the indicated plasmids.

Article Snippet: Mouse SDCBP siRNA Oligo Duplex (Locus ID53378) , Origene , Cat#SR426294.

Techniques: Immunoprecipitation, Transfection, Pull Down Assay, Control, Plasmid Preparation, Expressing, Western Blot, In Vivo, Ubiquitin Assay

Journal: The EMBO Journal

Article Title: SDCBP/Syntenin-1 stabilizes BACH1 by disassembling the SCF FBXO22 –BACH1 complex in triple-negative breast cancer

doi: 10.1038/s44318-025-00440-1

Figure Lengend Snippet: ( A ) Crosslink immunoprecipitation showing an interaction of FBXO22 with SDCBP in Hs578T cells transfected with Myc-SDCBP-expressing vector or Myc-SDCBP_Δ4-expressing vector. Schematic showing the FBXO22 mutant constructs generated using the HA-FBXO22 plasmid. ( C ) Immunoprecipitation showing SDCBP interactions with FBXO22 and its mutant constructs in HEK293 cells transfected with the indicated plasmids. ns indicates non-specific bands. See also Fig. , . ( D ) His-Pulldown assay showing the effect of SDCBP on SKP1-CUL1-FBXO22 complex formation after the indicated transfections in HEK293 cells. See also Appendix Fig. S . ( E ) His-Pulldown assay showing the effect of SDCBP KO on the SCF FBXO22 –BACH1 complex formation in the scramble control and SDCBP-KO MDA-MB-231 cells transfected with control vector or His-SKP1-expressing vector. See also Appendix Fig. S . ( F ) Immunoprecipitation showing the effect of SDCBP KD on the SCF FBXO22 –BACH1 complex formation in MDA-MB-231 cells transfected with scramble or SDCBP siRNA. ( G ) Immunoprecipitation showing the effect of SDCBP overexpression on the SCF FBXO22 –BACH1 complex formation in Hs578T cells transfected with control vector or Myc-SDCBP-overexpressing vector. ( H ) Immunoprecipitation showing the effect of SDCBP PDZ1 domain on the SCF FBXO22 –BACH1 complex formation in Hs578T cells transfected with a control vector or a Myc-SDCBP-PDZ1-overexpressing vector. .

Article Snippet: Mouse SDCBP siRNA Oligo Duplex (Locus ID53378) , Origene , Cat#SR426294.

Techniques: Immunoprecipitation, Transfection, Expressing, Plasmid Preparation, Mutagenesis, Construct, Generated, Control, Over Expression

Journal: The EMBO Journal

Article Title: SDCBP/Syntenin-1 stabilizes BACH1 by disassembling the SCF FBXO22 –BACH1 complex in triple-negative breast cancer

doi: 10.1038/s44318-025-00440-1

Figure Lengend Snippet: ( A ) Real-time qPCR showing the mRNA expression of BACH1-regulated ETC genes ( NDUFA4 , NDUFA4L2 , NDUFC2 , and COX6B2 ) in scramble and SDCBP-KO MDA-MB-231 cells ( n = 3); See also Appendix Fig. S , . Real-time qPCR showing the mRNA expression of NDUFA4 and COX6B2 in scramble, SDCBP-KO MDA-MB-231 cells, and SDCBP-KO MDA-MB-231 cells transfected with SDCBP ( n = 3). ( C ) Western blots showing the expression of mitochondrial proteins NDUFA4 and COX6B2 in MDA-MB-231 cells transfected with SDCBP siRNA in the presence or absence of FLAG-BACH1-expressing vector. ( D ) ChIP-qPCR showing BACH1 enrichments in the promoter regions of NDUFA4 and COX6B2 in the scramble and SDCBP-KO MDA-MB-231 cells. Quantitative data were normalized to IgG binding expression ( n = 3); See also Appendix Fig. S . ( E ) Left, flow cytometry histogram showing the mitochondrial membrane potentials using TMRE (tetramethylrhodamine ethyl ester) staining in MDA-MB-231 cells transfected with a scramble or SDCBP siRNA after the indicated treatments. FCCP (trifluoromethoxy carbonylcyanide phenylhydrazone). Right, quantification of TMRE fluorescence intensity ( n = 3). ( F ) Left, immunofluorescence staining and confocal imaging of the fluorescent signals for TMRE (orange-red color) in the scramble control and SDCBP-KO MDA-MB-231 cells after incubation with TMRE. DAPI (blue color) was used to stain the nucleus ( n = 7); Representative confocal images are shown; scale bars = 20 µm and 5 µm. Right, fluorescent levels of the TMRE were quantified based on their spectral densities. ( G ) Representative images of immunohistochemistry staining against the NDUFA4, BACH1, and SDCBP proteins showing a negative correlation between the expression of NDUFA4 and SDCBP in the same sections of TNBC tumor tissues. Scale bar = 20 µm. ( H ) Pearson correlation coefficients between SDCBP and NDUFA4 protein expression ( n = 64), and between BACH1 and NDUFA4 protein expression ( n = 60) in Fig. 5G. Data are expressed as the mean ± SEM and analyzed using two-way ANOVA ( A , D , F ), two-tailed Student’s t test , or one-way ANOVA ( E ). P values less than 0.05 were considered statistically significant. All experiments were repeated at least three times unless otherwise indicated. .

Article Snippet: Mouse SDCBP siRNA Oligo Duplex (Locus ID53378) , Origene , Cat#SR426294.

Techniques: Expressing, Transfection, Western Blot, Plasmid Preparation, ChIP-qPCR, Binding Assay, Flow Cytometry, Membrane, Staining, Fluorescence, Immunofluorescence, Imaging, Control, Incubation, Immunohistochemistry, Two Tailed Test

Journal: The EMBO Journal

Article Title: SDCBP/Syntenin-1 stabilizes BACH1 by disassembling the SCF FBXO22 –BACH1 complex in triple-negative breast cancer

doi: 10.1038/s44318-025-00440-1

Figure Lengend Snippet: ( A ) Cell proliferation of MDA-MB-231 cells transfected with a scramble or SDCBP siRNA after treatment with metformin for the indicated periods of time ( n = 3). ( B ) Cell viability of MDA-MB-231 cells transfected with a scramble or SDCBP siRNA after treatment with the indicated concentrations of metformin for 96 h ( n = 5). ( C ) Colony formation for MDA-MB-231 cells transfected with a scramble or SDCBP siRNA after treatment with the indicated concentrations of metformin. Colony numbers were counted and converted to percentages by normalizing with the control groups ( n = 3). ( D – F ) Effect of the indicated treatment on 4T1 tumor growth. Tumor growth was monitored in BALB/c mice bearing 4T1 cells after mammary fat-pad injections. When the average tumor volumes reached 100 mm 3 , the mice ( n = 7 mice/group) were administered with 100 mg/kg metformin (once a day) and/or adenoviral SDCBP shRNA (1 × 10 9 PFU/mice). Black arrows indicate the day of adenoviral SDCBP shRNA injection. Final tumor volume ( E ) and weight ( F ) are shown. ( G ) Immunohistochemistry staining against SDCBP, BACH1, Ki67, NDUFA4, and COX6B2 protein in 4T1 tumors from BALB/c mice in Fig. 6A. Representative images of the IHC staining are shown. Scale bar = 50 µm for low (left) and high (right) magnification. Data are expressed as the mean ± SEM and analyzed using two-way ANOVA ( A – C ), one-way ANOVA ( E ), or two-tailed Student’s t test ( F ). P values less than 0.05 were considered statistically significant. All experiments were repeated at least three times unless otherwise indicated. .

Article Snippet: Mouse SDCBP siRNA Oligo Duplex (Locus ID53378) , Origene , Cat#SR426294.

Techniques: Transfection, Control, shRNA, Injection, Immunohistochemistry, Staining, Two Tailed Test

Journal: The EMBO Journal

Article Title: SDCBP/Syntenin-1 stabilizes BACH1 by disassembling the SCF FBXO22 –BACH1 complex in triple-negative breast cancer

doi: 10.1038/s44318-025-00440-1

Figure Lengend Snippet: Schematic showing the novel oncogenic roles of SDCBP in promoting aggressiveness and mitochondrial inhibitor resistance in TNBCs. An abundance of SDCBP stabilizes the BACH1 protein by blocking E3 ubiquitin ligase SCF FBXO22 complex-targeted BACH1 for degradative ubiquitination. Mechanistically, SDCBP binds to different members of the SCF FBXO22 complex, including SKP1 and FBXO22, via its PDZ1 domain and induces SCF FBXO22 complex disassociation, suggesting that SDCBP is a key adapter regulating the activity of the E3 ubiquitin ligase SCF FBXO22 complex in the proteasomal pathway. SDCBP-induced BACH1 accumulation upregulates several pro-metastatic genes and downregulates numerous mitochondrial ETC genes, resulting in tumor progression and high resistance to metformin treatment in TNBCs. Targeting SDCBP switches the SCF FBXO22 complex to degrade BACH1 protein via the proteasome, reducing tumor aggressiveness and boosting the anti-tumor effect of metformin administration in TNBCs. .

Article Snippet: Mouse SDCBP siRNA Oligo Duplex (Locus ID53378) , Origene , Cat#SR426294.

Techniques: Blocking Assay, Ubiquitin Proteomics, Activity Assay