Journal: Cell Stem Cell

Article Title: Androgen Signaling Regulates SARS-CoV-2 Receptor Levels and Is Associated with Severe COVID-19 Symptoms in Men

doi: 10.1016/j.stem.2020.11.009

Figure Lengend Snippet:

Article Snippet: Human embryonic stem cells, H9 , WiCell , WA09.

Techniques: Recombinant, Purification, SYBR Green Assay, Staining, Gene Knockout, Transfection, RNA Sequencing Assay, Plasmid Preparation, Expressing, Software, Generated

Journal: bioRxiv

Article Title: Lysine63-linked ubiquitin chains earmark GPCRs for BBSome-mediated removal from cilia

doi: 10.1101/2020.03.04.977090

Figure Lengend Snippet: A. SSTR3 fused to the fluorescent protein mNeonGreen (NG) at its intracellular C-terminus and a biotinylation Acceptor Peptide (AP) tag at its extracellular N-terminus was expressed under the control of an attenuated EF1α promoter (pEF1α Δ ) in wild type (WT) and Arl6 -/- IMCD3 cells. Cells were treated with or without somatostatin-14 (sst) for 2 h, then fixed and stained for acetylated tubulin (acTub, magenta) and ubiquitin (Ub, yellow). AP SSTR3 NG (cyan) was imaged through the intrinsic fluorescence of NG. Channels are shifted in the insets to facilitate visualization of overlapping ciliary signals. Scale bar: 5μm (main panel), 2μm (inset). In WT cells, the ciliary SSTR3 signal decreases over the experimental time course. In Arl6 -/- cells, SSTR3 fails to exit cilia and an increase in the ciliary Ub level is detected. As a control, Arl6 -/- cells that did not express SSTR3-NG were tested; no increase in ciliary Ub levels was observed upon addition of sst. B. The fluorescence intensity of the Ub channel in the cilium was measured in each condition and the data are represented as violin plots. The thick bar indicates the median and the dotted lines the first and third quartiles. An 11-fold increase in ciliary Ub signal is observed upon addition of sst to SSTR3-expressing cells. Asterisks indicate ANOVA significance value. ****, p= <0.0001. C. WT or Arl6 -/- IMCD3 cells stably expressing AP SSTR3 NG and the biotin ligase BirA targeted to the ER lumen (BirA-ER) were transiently transfected with HA-tagged ubiquitin (HA-Ub). 10 µM biotin was added to cells 24 h post transfection for maximal biotinylation of AP SSTR3 NG and, after another 18 h, cells were treated with sst (10μM) for indicated times. Cells were lysed under denaturing conditions and biotinylated SSTR3 was captured on streptavidin resin. Eluates were probed for HA via immunoblotting and for biotin via streptavidin-HRP. Two major biotinylated proteins endogenous to cells are marked by asterisks. Whole cell lysates were probed for Arl6 and, as a loading control, actin. A non-specific band cross-reacting with the anti-Arl6 antibody is marked with a dot. D. Quantitation of SSTR3 ubiquitination. The signals of HA-Ub conjugated to SSTR3 in the streptavidin eluates were measured. The experiment shown in C was repeated three times and for each experiment, Ub-SSTR3 signals were normalized to the value in Arl6 -/- cells at t = 0 of sst stimulation and plotted as grey circles. The horizontal blue lines represent mean values. E. IMCD3 cells of the indicated genotypes were treated with the Smoothened agonist SAG or the vehicle DMSO for 2h. Cells were then fixed and stained for acTub and Ub. Channels are shifted in the insets to facilitate visualization of overlapping ciliary signals. Scale bar 5μm (main panel), 2μm (inset). Activation of Hh signaling promotes a detectable increase in ciliary Ub levels only in Arl6 -/- cells. F. Violin plots of the fluorescence intensity of the Ub channel in the cilium in each condition are shown. Asterisks indicate ANOVA significance value. ****, p= <0.0001.

Article Snippet: The following monoclonal antibodies were used for immunoblotting: anti-ubiquitin (mouse; clone P4D1; 3936; Cell Signaling; 1:1000), anti-α tubulin (mouse; clone DM1A; MS-581-P1ABX; Thermo Scientific;1:1000), anti-HA (mouse; clone 16B12; 901501; Biolegend;1:500), streptavidin-HRP (Pierce-21140;Thermo Scientific; 1:10000).

Techniques: Control, Staining, Ubiquitin Proteomics, Fluorescence, Expressing, Stable Transfection, Transfection, Western Blot, Quantitation Assay, Activation Assay

Journal: bioRxiv

Article Title: Lysine63-linked ubiquitin chains earmark GPCRs for BBSome-mediated removal from cilia

doi: 10.1101/2020.03.04.977090

Figure Lengend Snippet: A. IMCD3 cells of the indicated genotypes expressing AP SSTR3 NG were treated with somatostatin-14 for 2 h. Cells were fixed and stained for AcTub (magenta) and with antibodies specific for the lysine 63 (UbK63) or lysine 48 (UbK48) Ub chain linkages (yellow). SSTR3 NG (cyan) was imaged through the intrinsic fluorescence of NG. Channels are shifted in the insets to facilitate visualization of overlapping ciliary signals. Scale bar 5μm (main panel), 2μm (inset). B. The fluorescence intensity of the UbK48 and UbK63 channels in the cilium are represented as violin plots. A 14-fold increase in ciliary Ub abundance is detected with the K63Ub linkage-specific antibody. Asterisks indicate ANOVA significance value. ****, p= <0.0001. No ciliary signal is detected with the K48Ub linkage-specific antibody. C. Arl6 -/- IMCD3-[ AP SSTR3; BirA-ER] cells were transfected with the HA-tagged ubiquitin variants WT, noK0 (all seven acceptor lysine residues mutated to arginine) or K63 (where all lysine residues are mutated to arginine except for K63). Biotin was included in the culture medium and cells were treated with sst for 0 or 10 min. Cells were lysed under denaturing conditions and biotinylated SSTR3 was captured on streptavidin resin. Eluates were probed for HA via immunoblotting and for biotin via streptavidin-HRP. Two major biotinylated proteins endogenous to cells are marked by asterisks. Whole cell lysates were probed for Arl6 and, as a loading control, actin. A non-specific band cross-reacting with the anti-Arl6 antibody is marked with a dot. WT IMCD3 cells were processed in parallel as a control. D. Quantitation of SSTR3 ubiquitination. The signals of HA-Ub conjugated to SSTR3 in the streptavidin eluates were measured. The experiment shown in C was repeated four times and for each Ub variant, Ub-SSTR3 signals were normalized to the value at t = 0 of sst stimulation and plotted as grey circles. The horizontal blue lines represent mean values. Asterisks indicate ANOVA significance value. *** p = < 0.001; ** p=<0.01.

Article Snippet: The following monoclonal antibodies were used for immunoblotting: anti-ubiquitin (mouse; clone P4D1; 3936; Cell Signaling; 1:1000), anti-α tubulin (mouse; clone DM1A; MS-581-P1ABX; Thermo Scientific;1:1000), anti-HA (mouse; clone 16B12; 901501; Biolegend;1:500), streptavidin-HRP (Pierce-21140;Thermo Scientific; 1:10000).

Techniques: Expressing, Staining, Fluorescence, Transfection, Ubiquitin Proteomics, Western Blot, Control, Quantitation Assay, Variant Assay

Journal: bioRxiv

Article Title: Lysine63-linked ubiquitin chains earmark GPCRs for BBSome-mediated removal from cilia

doi: 10.1101/2020.03.04.977090

Figure Lengend Snippet: A. IMCD3-[pEF1α Δ - AP SSTR3 NG ] cells of the indicated genotypes were treated with sst for 2h before fixation and staining for acetylated tubulin (acTub, magenta) and ubiquitin (Ub, yellow). SSTR3 NG was visualized through the intrinsic fluorescence of NG (cyan). Channels are shifted in the insets to facilitate visualization of overlapping ciliary signals. SSTR3 exit is blocked in Arl6 -/- cells regardless of the β-Arrestin2 genotype but the ciliary Ub signal is only evident when β-Arrestin2 function is intact. Scale bar: 5μm (main panel), 1μm (inset). B. Violin plots representing the ciliary levels of Ub under the indicated conditions. Asterisks indicate ANOVA significance value. ****, p= <0.0001. C. IMCD3 WT, knockout for Arl6 only and double knockout for Arl6 and the β-Arrestin2 gene Arrb2 were treated with SAG for 2h before fixation and staining for acetylated tubulin (acTub, magenta) and ubiquitin (Ub, yellow). Channels are shifted in the insets to facilitate visualization of overlapping ciliary signals. Scale bar: 5μm (main panel), 1μm (inset). D. Violin plots representing the ciliary Ub levels. Asterisks indicate ANOVA significance value. ****, p= <0.0001; **, p= <0.01. The appearance of a Ub signal in cilia in Arl6 -/- cells depends on β-Arrestin2. E. IMCD3-[ AP SSTR3; BirA-ER] cells of the indicated genotypes stably expressing AP SSTR3 NG and BirA-ER were transfected with HA-Ub, biotin was added to the medium and cells were treated with sst for indicated times. Biotinylated SSTR3 was captured from cell lysates under denaturing conditions on streptavidin resin. Eluates were probed for HA via immunoblotting and for biotin via streptavidin-HRP. Two major biotinylated proteins endogenous to cells are marked by asterisks. Whole cell lysates were probed for Arl6, β-Arrestin2 to verify genotypes and, as a loading control, actin. A non-specific band cross-reacting with the anti-Arl6 antibody is marked with a dot. WT IMCD3 cells were processed in parallel as a control. F. Quantitation of SSTR3 ubiquitination. The signals of HA-Ub conjugated to SSTR3 in the streptavidin eluates were measured. The experiment shown in E was repeated twice and for each experiment, Ub-SSTR3 signals were normalized to the value in Arl6 -/- cells at t = 0 of sst stimulation and plotted as grey circles. The horizontal blue lines represent mean values.

Article Snippet: The following monoclonal antibodies were used for immunoblotting: anti-ubiquitin (mouse; clone P4D1; 3936; Cell Signaling; 1:1000), anti-α tubulin (mouse; clone DM1A; MS-581-P1ABX; Thermo Scientific;1:1000), anti-HA (mouse; clone 16B12; 901501; Biolegend;1:500), streptavidin-HRP (Pierce-21140;Thermo Scientific; 1:10000).

Techniques: Staining, Ubiquitin Proteomics, Fluorescence, Knock-Out, Double Knockout, Stable Transfection, Expressing, Transfection, Western Blot, Control, Quantitation Assay

Journal: Journal of Biological Chemistry

Article Title: Cartilage Oligomeric Matrix Protein Associates with Granulin-Epithelin Precursor (GEP) and Potentiates GEP-stimulated Chondrocyte Proliferation

doi: 10.1074/jbc.m608744200

Figure Lengend Snippet: FIGURE 6. Immunohistochemistry of COMP and GEP in the section of long bone from a 19-day-old mouse embryo. A, low power microphotograph of a section stained with anti-GEP polyclonal antibody (red) and counterstained with Mayer’s hematoxylin (blue). Immunostaining for GEP demonstrates localization of strongly immunopositive chondrocytes in the lower prolifera- tive/upper hypertrophic zones of the growth plate. B, high power micropho- tograph of section in A. C, low power microphotograph of section stained with anti-COMP polyclonal antibody (red) and counterstained with Mayer’s hematoxylin (blue); immunostaining reveals positive staining in chondro- cytes. D, high power microphotograph of section in C. S, resting chondro- cytes; P, proliferating chondrocytes; H, hypertrophic chondrocytes; M, bone metaphysis. Bar, 100 m.

Article Snippet: After rehydration in phosphate-buffered saline and blocking with 20% goat serum in phosphate-buffered saline for 30 min, the cells were incubated with primary antibodies (i.e. mouse monoclonal anti-COMP antibodies (diluted 1:50) and polyclonal goat anti-GEP antibodies (Santa Cruz Biotechnology; diluted 1:50) at room temperature for 1 h.After beingwashedwith phosphate-buffered saline, the coverslips were incubated with secondary antibodies (i.e. goat anti-mouse IgG conjugated with rhodamine (Santa Cruz Biotechnology; diluted 1:100) and chick anti-goat IgG conjugated with fluorescein isothiocyanate (Santa Cruz Biotechnology; diluted 1:400) for 50 min.

Techniques: Immunohistochemistry, Staining, Immunostaining

Journal: Antioxidants

Article Title: The Effect of Sea Buckthorn ( Hippophae rhamnoides L.) Seed Oil on UV-Induced Changes in Lipid Metabolism of Human Skin Cells

doi: 10.3390/antiox7090110

Figure Lengend Snippet: The level of endocannabinoids (AEA, 2-AG) and endocannabinoid receptors (CB1, CB2) in keratinocytes and fibroblasts after exposure to UVA (30 J/cm 2 and 20 J/cm 2 ) and UVB radiation (60 mJ/cm 2 and 200 mJ/cm 2 , respectively) and sea buckthorn seeds oil (500 ng/mL) treatment. Mean values ± SD of five independent experiments are presented. x statistically significant differences vs. control group, p < 0.05; y statistically significant differences between UVA/UVB and oil treated groups vs. oil treated control group, p < 0.05; a statistically significant differences between UVA and oil treated group vs. UVA treated group, p < 0.05; b statistically significant differences between UVB and oil treated group vs. UVB treated group, p < 0.05.

Article Snippet: Primary monoclonal antibodies against CB1 and CB2 (Santa Cruse Biotechnology, Santa Cruz, CA, USA) were used at a concentration of 1:1000.

Techniques: Control

Journal: Nature

Article Title: Hippo signalling maintains ER expression and ER + breast cancer growth.

doi: 10.1038/s41586-020-03131-5

Figure Lengend Snippet: Fig. 1 | LATS is essential to maintaining ER expression. a, Deletion of LATS1/2 causes an enrichment of YAP target-gene signature and depletion of ERα target-gene signature. NES, normalized enrichment score. b, c, LATS1/2 double knockout (dKO) downregulates ESR1 mRNA (b) and ERα protein (c) in MCF-7 cells. Two independent LATS1/2 double-knockout MCF-7 clones are shown (labelled no. 1 and no. 2). pYAP (S127), YAP phosphorylated at S127. WT, wild type. d, LATS1/2 double knockout inhibits target genes of ERα, with (+E2) or without (−E2) treatment with oestradiol (E2) (1 nM) for 45 min. e, LATS1/2 deficiency downregulates ERα. T47D (left) and ZR-75-1 (right) cells with lentivirus-mediated CRISPR deletion of LATS1/2 (single-guide RNA against LATS1/2 (sgLATS1/2)) were subjected to immunoblot analysis with indicated antibodies. No. 1 and no. 2 denote two independent clones of each cell line. f–h, Deletion of Lats1/2 activates YAP and TAZ, and downregulates Esr1 expression, in mouse mammary organoids. Organoids derived from the mammary tissues of Lats1+/+Lats2+/+ and Lats1fl/flLats2fl/fl mice were infected with Cre-encoding adenovirus and subjected to immunohistochemistry (f), immunoblot (g) and qPCR analysis (h). Scale bar, 50 μm. i, LATS1

Article Snippet: Those for ERα (mouse) (no. sc-542), YAP and TAZ (no. sc-101199) and GAPDH (no. sc-25778) were obtained from Santa Cruz Biotechnology.

Techniques: Expressing, Double Knockout, Clone Assay, CRISPR, Western Blot, Derivative Assay, Infection, Immunohistochemistry

Journal: Journal of Clinical Investigation

Article Title: The retinoblastoma tumor suppressor controls androgen signaling and human prostate cancer progression

doi: 10.1172/jci44239

Figure Lengend Snippet: Figure 6 The RB/E2F1 axis regulates the AR locus, and the kinetics of E2F1 and AR expression are cell cycle dependent. (A) Schematic of the AR regulatory locus, with the TSS and translational start site (TLS) shown. Results from ChIP analyses of E2F1 binding are shown, including semiquantitative and real-time PCR results, with occupancy in shCon1 cells set to 1. (B) ChIP analyses of RB binding, Sin3B, and histone H4 acetylation at sites enriched for E2F1 after RB depletion, and promoters of CCNA2 and ALB. (C) qPCR analyses of E2F1 and AR from LNCaP cells arrested in G0/G1 (CDT), G1 (ROS), early S-phase (APH), or mid–S-phase (HU). Transcript levels in CDT condition were set to 1. Data represent 3 replicates (mean ± SD); similar results were obtained in at least 3 independent analyses. See also Supplemental Figure 6.

Article Snippet: Antibodies were used against RB (for immunoblot, 554136, BD Biosciences; for ChIP, Ab-1 [1F8], Thermo Scientific), Lamin B (sc-6217, Santa Cruz Biotechnology Inc.), AR (N-20, Santa Cruz Biotechnology Inc.), E2F1 (sc-193, Santa Cruz Biotechnology Inc.), E2F2 (sc-9967, Santa Cruz Biotechnology Inc.), E2F3 (sc-878, Santa Cruz Biotechnology Inc.), cdk-4 (sc-601, Santa Cruz Biotechnology Inc.), AcH4 (06-866, Upstate), and Sin3B (sc-768, Santa Cruz Biotechnology Inc.).

Techniques: Expressing, Binding Assay, Real-time Polymerase Chain Reaction

Journal: Journal of Clinical Investigation

Article Title: The retinoblastoma tumor suppressor controls androgen signaling and human prostate cancer progression

doi: 10.1172/jci44239

Figure Lengend Snippet: Figure 7 Selective restoration of RB activity at sites of E2F1 action suppresses AR expression; AR regulation is E2F selective; and E2F1-induced AR deregulation is required for RB loss–mediated castration resistance. (A) Schematic of the E2F1-AB chimera. Also shown is RT-PCR for CCNA2 and AR mRNA from cells transfected with plasmids encoding E2F1, E2F1-AB, or control (pcDNA). (B) qPCR analyses of data in A, with AR levels in shCon1/control–transfected (pcDNA) cells set to 1. Data represent triplicate analyses of at least 2–3 independent biological replicates. (C) Immunoblot analyses for transduced E2F1, E2F2, E2F3, and lamin B (loading control) in LNCaP. (D) Parallel semiquantitative RT-PCR and qPCR analysis of AR mRNA in cells from C. Results are plotted relative to control (Ad-GFP–transduced LNCaP) from triplicate replicates (mean ± SD); similar results were obtained in at least 3 independent analyses. (E) AR immunoblot in control and E2F1-transduced cells from C and D. (F) Immunoblot for E2F1 and AR after siRNA-mediated knockdown of E2F1; verification of AR knockdown is also shown. Impact of E2F1 and AR knockdown on castration-resistant proliferation in RB-depleted cells was determined by trypan blue exclusion and cell counting. Data reflect duplicate experiments, each with 3 independent biologic replicates. *P < 0.05, Student’s t test.

Article Snippet: Antibodies were used against RB (for immunoblot, 554136, BD Biosciences; for ChIP, Ab-1 [1F8], Thermo Scientific), Lamin B (sc-6217, Santa Cruz Biotechnology Inc.), AR (N-20, Santa Cruz Biotechnology Inc.), E2F1 (sc-193, Santa Cruz Biotechnology Inc.), E2F2 (sc-9967, Santa Cruz Biotechnology Inc.), E2F3 (sc-878, Santa Cruz Biotechnology Inc.), cdk-4 (sc-601, Santa Cruz Biotechnology Inc.), AcH4 (06-866, Upstate), and Sin3B (sc-768, Santa Cruz Biotechnology Inc.).

Techniques: Activity Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Transfection, Control, Western Blot, Knockdown, Cell Counting

Journal: Journal of Clinical Investigation

Article Title: The retinoblastoma tumor suppressor controls androgen signaling and human prostate cancer progression

doi: 10.1172/jci44239

Figure Lengend Snippet: Figure 8 AR levels correlate inversely with RB levels and directly with E2F1 levels in CRPC specimens. (A) Log2 scaled expression ratios of AR versus RB1 and AR versus E2F1 from 39 human CRPC soft tissue metastases. (B) Heatmap depicting AR, RB1, E2F1, and E2F3 expression levels in 39 CRPC tumors. Pearson correlations are provided. See also Table 2 and Supplemental Figure 7.

Article Snippet: Antibodies were used against RB (for immunoblot, 554136, BD Biosciences; for ChIP, Ab-1 [1F8], Thermo Scientific), Lamin B (sc-6217, Santa Cruz Biotechnology Inc.), AR (N-20, Santa Cruz Biotechnology Inc.), E2F1 (sc-193, Santa Cruz Biotechnology Inc.), E2F2 (sc-9967, Santa Cruz Biotechnology Inc.), E2F3 (sc-878, Santa Cruz Biotechnology Inc.), cdk-4 (sc-601, Santa Cruz Biotechnology Inc.), AcH4 (06-866, Upstate), and Sin3B (sc-768, Santa Cruz Biotechnology Inc.).

Techniques: Expressing

Journal: Journal of Clinical Investigation

Article Title: The retinoblastoma tumor suppressor controls androgen signaling and human prostate cancer progression

doi: 10.1172/jci44239

Figure Lengend Snippet: Figure 9 Model for RB loss in controlling tumor progression through nuclear receptor expression and output. The present data suggest what we believe to be a new model for RB tumor suppressor function in tumor progression. While RB is actively engaged in response to hormone therapy, RB loss induces E2F1-mediated deregulation of the AR locus. This event is sufficient to induce unchecked AR activity and progres- sion to CRPC. This model strongly suggests that RB plays a significant role in tumor progression that is independent of genes directly associ- ated with cell cycle, manifest by controlling nuclear receptor expres- sion, function, and output.

Article Snippet: Antibodies were used against RB (for immunoblot, 554136, BD Biosciences; for ChIP, Ab-1 [1F8], Thermo Scientific), Lamin B (sc-6217, Santa Cruz Biotechnology Inc.), AR (N-20, Santa Cruz Biotechnology Inc.), E2F1 (sc-193, Santa Cruz Biotechnology Inc.), E2F2 (sc-9967, Santa Cruz Biotechnology Inc.), E2F3 (sc-878, Santa Cruz Biotechnology Inc.), cdk-4 (sc-601, Santa Cruz Biotechnology Inc.), AcH4 (06-866, Upstate), and Sin3B (sc-768, Santa Cruz Biotechnology Inc.).

Techniques: Expressing, Activity Assay

Journal: bioRxiv

Article Title: Integrin-TGFβ axis induces partial EMT in basal-like cells to lead collective invasion

doi: 10.1101/2025.04.04.647177

Figure Lengend Snippet: (A) Confocal imaging of ITGα2, Vimentin, and K8 in an IDC patient tissue section. Green contour: cluster of cancer cells with protrusive morphology; green arrowheads: basal-like cells (K8-low) at the tumor–stroma interface with high ITGα2 expression. White contour: cluster of cancer cells lacking basal-like cells (K8-high), with low ITGα2 expression; magenta arrowheads. White arrowheads: fibroblast-like cells (elongated, spindle-shaped). (B) Quantification of mean gray values for ITGα2 and Vimentin in basal-like (n = 33), luminal-like (n = 32), and fibroblast-like (n = 32) cells from one IDC patient tissue section. (C) Representative brightfield images of MMTV-PyMT organoids (ITGα2-WT or ITGα2-KO, gRNA1 and gRNA2) cultured in 3D Collagen I. Black arrowheads: invasive strands. (D) Percentage of organoids exhibiting one or more invasive strands in ITGα2-WT and ITGα2-KO (clones 1 and 2 from gRNA1) MMTV-PyMT organoids. (E) qPCR analysis of classical TGF-β and EMT target genes in ITGα2-WT and ITGα2-KO MMTV-PyMT organoids cultured in 3D Collagen I for three days. Values represent mean normalized mRNA expression (relative to housekeeping genes), shown for KO organoids relative to WT controls (dashed line). Data are presented as mean ± SD from three independent experiments. (F) Confocal imaging of Col ¾ and F-actin in ITGα2-WT and ITGα2-KO MMTV-PyMT organoids after one day in 3D Collagen I. (G, H) qPCR analysis of Vimentin and Slug mRNA expression in ITGα2-KO versus ITGα2-WT MMTV-PyMT organoids treated with Activin A (20 ng/μl) or vehicle control (0.1% BSA) for three days. Bar graphs represent mean normalized expression values ± SD from four independent experiments. (I) Kaplan–Meier analysis correlating high vs. low mRNA expression of INHBA, ITGA2, ITGB1, and their combinations (ITGA2 + ITGB1, or INHBA + ITGA2 + ITGB1) with distant metastasis-free survival (DMFS) in patients with grade 3 breast cancer. Scale bars: 100 μm (A, C), 50 μm (A, zoom-in), 50 μm (F), 10 μm (F, zoom-in). P values: two-sided unpaired Mann–Whitney test (E), two-sided Kruskal-Wallis test with Dunn’s multiple comparisons (G, H), Log-rank test (I).

Article Snippet: The following antibodies were used: rabbit anti-human Vimentin (Cat#ab92547, Abcam), chicken anti-human Vimentin (Cat#PA1-16759, Invitrogen) rabbit anti-human Keratin 14 (Cat# 905301, Biolegend), rat anti-mouse Keratin 8 (Cat# 531826, DSHB), rabbit anti-rat Collagen I cleavage site (Col ¾, Cat#0217-025, immunoGlobe), rabbit anti-human integrin α2 (Cat#ab181548, Abcam), rabbit anti-human Inhba (Cat#10651-1-AP, Proteintech), Mouse IgG1 isotype control (MAB002, R&D Systems), anti-Activin A antibody (Cat#AF338, R&D Systems).

Techniques: Imaging, Expressing, Cell Culture, Clone Assay, Control, MANN-WHITNEY

Journal: bioRxiv

Article Title: Integrin-TGFβ axis induces partial EMT in basal-like cells to lead collective invasion

doi: 10.1101/2025.04.04.647177

Figure Lengend Snippet: (A) DNA sequence of the Itgα2 gene to confirm gene knockout by Crispr-Cas9 gene editing. Red regions indicate the insertion of one base pair compared to the wildtype Itgα2 sequence. (B) Western blot analysis showing Itgα2 and GAPDH expression from whole cell lysates of MMTV-PyMT organoids WT or KO (gRNA1 or gRNA2). (C) Confocal imaging of Itgα2 and K8 in MMTV-PyMT organoids with Itgα2 WT or knockout (KO) grown in Collagen I for 3 days. White arrowheads: invading strands in Itgα2 WT organoids led by basal-like cells (low K8, high Itgα2, insets), White arrows: non-invading basal-like cells at the ECM interface in Itgα2 KO organoids (low K8, low Itgα2, insets). (D) Percentage of invasive organoids in MMTV-PyMT organoids Itgα2-WT versus Itgα2-KO (gRNA2). (E) Mean gray value of Col ¾ relative to Collagen I reflection in MMTV-PyMT organoids with Itgα2-WT and Itgα2-KO. Median: red lines, from n = 11 organoids per group from two independent experiments. (F) Single confocal slice showing INHBA and K8 expression in Itgα2-WT and Itgα2-KO MMTV-PyMT organoids cultured in 3D Collagen I for three days. Insets show basal-like cells (K8 low) guiding invasive strands (Itgα2-WT) or remaining at the organoid rim (Itgα2 KO, non-invasive). (G) Mean-gray value of Inhba in basal-like cells (K8-low) located at the rim of Itgα2 WT vs. KO MMTV-PyMT organoids (3D Collagen I, day 1). Median: red lines, n =11 cells from 6 Itgα2-WT organoids, and n = 12 cells from 7 Itgα2-KO organoids from one experiment. (H) qPCR analysis showing relative mRNA expression of CTGF in MMTV-PyMT Itgα2-KO organoids compared to Itgα2-WT organoids treated with Activin A ligand (20 ng/μl) or vehicle control (0.1% BSA) for three days. Bar graph represents mean normalized expression values (relative to housekeeping genes) ± SD from four independent experiments. (I) Kaplan-Meier plot correlating ITGB1 gene expression with DMFS in grade 3 breast cancer patients. Scale bars: 50 μm (C, F), 25 μm (C, F, Zoom in). P values, two-sided unpaired Mann–Whitney test (E, G), two-sided Kruskal-Wallis test (Dunn’s multiple comparison) (H), Logrank test (I).

Article Snippet: The following antibodies were used: rabbit anti-human Vimentin (Cat#ab92547, Abcam), chicken anti-human Vimentin (Cat#PA1-16759, Invitrogen) rabbit anti-human Keratin 14 (Cat# 905301, Biolegend), rat anti-mouse Keratin 8 (Cat# 531826, DSHB), rabbit anti-rat Collagen I cleavage site (Col ¾, Cat#0217-025, immunoGlobe), rabbit anti-human integrin α2 (Cat#ab181548, Abcam), rabbit anti-human Inhba (Cat#10651-1-AP, Proteintech), Mouse IgG1 isotype control (MAB002, R&D Systems), anti-Activin A antibody (Cat#AF338, R&D Systems).

Techniques: Sequencing, Gene Knockout, CRISPR, Western Blot, Expressing, Imaging, Knock-Out, Cell Culture, Control, Gene Expression, MANN-WHITNEY, Comparison