Journal: Cells

Article Title: HIF-1-Dependent Induction of β3 Adrenoceptor: Evidence from the Mouse Retina

doi: 10.3390/cells11081271

Figure Lengend Snippet: Effects of oxygen tension on retinal levels of HIF-1α, VEGF and β3-AR from PD7 to PD17. ( A ) Schematic diagram of the OIR model including DMOG administration daily from PD7 to PD12. ( B ) Representative blots showing protein levels of HIF-1α, VEGF and β3-AR as evaluated by Western blot in retinal extracts at different times from normoxic controls or OIR mice without or with DMOG administration. β-actin was used as the loading control. ( C – E ), Relative densitometric analyses of the protein levels of HIF-1α, VEGF and β3-AR. ( F ) Retinal mRNA levels of β3-AR at different times from controls or OIR mice untreated or treated with DMOG. * p < 0.05 vs. normoxic controls. One-way ANOVA followed by Tukey’s multiple comparison post-hoc test. Each histogram represents the mean ± SEM of data from 6 independent samples.

Article Snippet: Blots were blocked in 3% skim milk for 1 h at room temperature and then incubated overnight at 4 °C with the following primary antibodies: rabbit polyclonal against HIF-1α (ab2185; Abcam, Cambridge, UK; 1:500 dilution), rabbit polyclonal against VEGF (ab9570; Abcam; 1:1000 dilution), mouse monoclonal against β3-AR (sc-515763; Santa Cruz Biotechnologies, Santa Cruz, CA, USA; 1:200 dilution), mouse monoclonal against β-actin (A2228; Sigma Aldrich, St. Louis, MO, USA; 1:2500 dilution).

Techniques: Western Blot, Control, Comparison

Journal: Cells

Article Title: HIF-1-Dependent Induction of β3 Adrenoceptor: Evidence from the Mouse Retina

doi: 10.3390/cells11081271

Figure Lengend Snippet: Schematic representation of mouse and human β3-AR genes including their upstream sequences. ( A ) In the mouse gene, 5 exons (E1–E5; solid boxes) and 4 introns (dashed lines) are depicted. They potentially express up to 6 different alternative mRNAs of which 3 codify for the canonical β3-AR protein (yellow mRNAs) while the other 3 for an alternative β3-AR protein with a different C-terminal sequence (purple mRNAs). The putative transcription-start site (TSS) is indicated by the red arrow. The positions of the 6 potential HBSs relative to the TSS are in green. All of them contain the minimal HBS consensus sequence (underlined sequence 5′-ACGTG-3′). ( B ) In the human gene, the positions of the 6 potential HBSs relative to the TSS are in green. All of them contain the minimal consensus sequence (underlined sequence 5′-ACGT-3′). The putative TSS is indicated by the red arrow. The highly conserved nucleotides G −2 and/or C +5 in the mouse and human HBSs sequence are highlighted in red.

Article Snippet: Blots were blocked in 3% skim milk for 1 h at room temperature and then incubated overnight at 4 °C with the following primary antibodies: rabbit polyclonal against HIF-1α (ab2185; Abcam, Cambridge, UK; 1:500 dilution), rabbit polyclonal against VEGF (ab9570; Abcam; 1:1000 dilution), mouse monoclonal against β3-AR (sc-515763; Santa Cruz Biotechnologies, Santa Cruz, CA, USA; 1:200 dilution), mouse monoclonal against β-actin (A2228; Sigma Aldrich, St. Louis, MO, USA; 1:2500 dilution).

Techniques: Sequencing

Journal: Cells

Article Title: HIF-1-Dependent Induction of β3 Adrenoceptor: Evidence from the Mouse Retina

doi: 10.3390/cells11081271

Figure Lengend Snippet: HIF-1α modeling and HIF-1/DNA docking. ( A ) Root-mean-square (RMS) displacement of protein backbone (black arrow indicates the time at which the stabilization of the protein structure occurs). ( B ) RMS fluctuation of aminoacid displacement relative to the starting structure and the principal domains of the HIF-1α protein, accordingly colored in ( C ). ( D ) HIF-1α protein modelized in its dimeric form showing the correct interaction with the DNA fragment. The two monomers are reported in green and orange respectively, while the DNA fragment is highlighted in blue. The binding site generated by dimerization is better shown in the focus section.

Article Snippet: Blots were blocked in 3% skim milk for 1 h at room temperature and then incubated overnight at 4 °C with the following primary antibodies: rabbit polyclonal against HIF-1α (ab2185; Abcam, Cambridge, UK; 1:500 dilution), rabbit polyclonal against VEGF (ab9570; Abcam; 1:1000 dilution), mouse monoclonal against β3-AR (sc-515763; Santa Cruz Biotechnologies, Santa Cruz, CA, USA; 1:200 dilution), mouse monoclonal against β-actin (A2228; Sigma Aldrich, St. Louis, MO, USA; 1:2500 dilution).

Techniques: Binding Assay, Generated

Journal: Cells

Article Title: HIF-1-Dependent Induction of β3 Adrenoceptor: Evidence from the Mouse Retina

doi: 10.3390/cells11081271

Figure Lengend Snippet: Docking analysis of model 1 and model 3. ( A ) HIF-1/HBS #1 best association complex: full structure and focus on HIF-1-DNA interactions (boxes). ( B ) HIF-1/HBS #3 best association complex: full structure and focus on HIF-1-DNA interactions (boxes).

Article Snippet: Blots were blocked in 3% skim milk for 1 h at room temperature and then incubated overnight at 4 °C with the following primary antibodies: rabbit polyclonal against HIF-1α (ab2185; Abcam, Cambridge, UK; 1:500 dilution), rabbit polyclonal against VEGF (ab9570; Abcam; 1:1000 dilution), mouse monoclonal against β3-AR (sc-515763; Santa Cruz Biotechnologies, Santa Cruz, CA, USA; 1:200 dilution), mouse monoclonal against β-actin (A2228; Sigma Aldrich, St. Louis, MO, USA; 1:2500 dilution).

Techniques:

Journal: Cells

Article Title: HIF-1-Dependent Induction of β3 Adrenoceptor: Evidence from the Mouse Retina

doi: 10.3390/cells11081271

Figure Lengend Snippet: HIF-1α interaction with HBS #1 and corresponding β3-AR gene expression at PD12 (from 0 to 12 h of hypoxia) or at PD17. ( A ) Schematic diagram of the OIR model pointing to the specific times under analysis. ( B ) Data from HIF-1α chromatin immunoprecipitation and HBS #1-specific qPCR (ChIP-qPCR) represented as fold enrichment relative to IgG input. ( C ) Corresponding levels of β3-AR mRNA. White bars represent data from retinas of normoxic controls while grey bars represent data from hypoxic mice. One-way ANOVA followed by Tukey’s multiple comparison post-hoc test. Each histogram represents the mean ± SEM of data from 6 independent samples. * p < 0.05 vs. normoxic controls ( n = 6 samples).

Article Snippet: Blots were blocked in 3% skim milk for 1 h at room temperature and then incubated overnight at 4 °C with the following primary antibodies: rabbit polyclonal against HIF-1α (ab2185; Abcam, Cambridge, UK; 1:500 dilution), rabbit polyclonal against VEGF (ab9570; Abcam; 1:1000 dilution), mouse monoclonal against β3-AR (sc-515763; Santa Cruz Biotechnologies, Santa Cruz, CA, USA; 1:200 dilution), mouse monoclonal against β-actin (A2228; Sigma Aldrich, St. Louis, MO, USA; 1:2500 dilution).

Techniques: Gene Expression, Chromatin Immunoprecipitation, ChIP-qPCR, Comparison

Journal: Journal of Orthopaedic Translation

Article Title: Osteocyte β1 integrin loss causes low bone mass and impairs bone mechanotransduction in mice

doi: 10.1016/j.jot.2022.03.008

Figure Lengend Snippet: Antibodies information.

Article Snippet: β3 Integrin , Cell Signaling , #13166 , 1:1000 , .

Techniques: Western Blot

Journal: Journal of Orthopaedic Translation

Article Title: Osteocyte β1 integrin loss causes low bone mass and impairs bone mechanotransduction in mice

doi: 10.1016/j.jot.2022.03.008

Figure Lengend Snippet: β1 integrin deletion in osteocytes results in significant bone loss in weight-bearing long bones in adult mice ( a – b ) Western blotting results of cortical bone samples from control and cKO mice. Protein extracts were isolated from osteocyte-enriched cortical bone tissues and subjected to western blotting for the expression analyses of β1 integrin. α-actin was used as a loading control. ( c ) Immunohistochemistry (IHC) of the tibial cortical bone sections from control and cKO mice. ( d ) Micro-computerized tomography (μCT) images of the distal trabecular bone and mid-shaft cortical bone of femurs from 4-month-old male control and cKO mice ( e – g ) Quantitative μCT analyses of trabecular separation (Tb.Sp), trabecular number (Tb.N), and cortical thickness (Ct.Th) of femurs. N ​= ​9 for control group and N ​= ​10 for cKO group. ( h ) μCT images of the proximal trabecular bone and mid-shaft cortical bone of tibiae from 4-month-old male control and cKO mice ( i-k ) Quantitative μCT analyses of Tb. Sp, Tb.N, and Ct. Th of tibiae. N ​= ​14 for control group and N ​= ​10–12 for cKO group. ( l ) ​μCT images of the sagittal view of L3-5 ​lumbar spines from 4-month-old male control and cKO mice. ( m ) Cross-section images (at yellow dotted line in l ) and 3D reconstruction images (at red square ROI in l ) of the L4 lumbar spines from control and cKO mice ( n – o ) Quantitative μCT analyses of the bone volume/tissue volume (BV/TV) and arbitrary intensity of spine sections from control and cKO mice. N ​= ​9 for control group and N ​= ​8 for cKO group. Results are expressed as mean ​± ​standard deviation (s.d.). ∗∗ p ​< ​0.01; ∗∗∗ p ​< ​0.001.

Article Snippet: β3 Integrin , Cell Signaling , #13166 , 1:1000 , .

Techniques: Western Blot, Control, Isolation, Expressing, Immunohistochemistry, Tomography

Journal: Journal of Orthopaedic Translation

Article Title: Osteocyte β1 integrin loss causes low bone mass and impairs bone mechanotransduction in mice

doi: 10.1016/j.jot.2022.03.008

Figure Lengend Snippet: β1 integrin loss impairs bone mechanical properties and alters collagen fiber integrity. ( a ) Representative load–displacement curve from three-point-bending (3 ​PB) test in 4-month-old male control and cKO mice ( b – d ) Quantitative analyses of maximum load force (Max-load), stiffness and work-to-fracture in 3 ​PB test. N ​= ​14 for control group and N ​= ​12 for cKO group ( e – f ) Quantitative analyses of Young's modulus and hardness for 4-month-old male control and cKO mice in nanoindentation test. N ​= ​8 for each group ( g – i ) Sirius red/fast green dye staining of tibial sections from 4-month-old male control and cKO mice ( j ) Semi-quantitative measurement of the collagen content on the sagittal tibial sections from control and cKO mice. N ​= ​6 for each group ( k ) Representative images of collagen fibers under two-photon microscopy from 4-month-old male control and cKO tibial sections ( l – m ) Quantitative analyses of collagen fiber intensity and length for control and cKO mice. N ​= ​6 for each group. Results are expressed as mean ​± ​standard deviation (s.d.). n. s. p ​> ​0.05; ∗ p ​< ​0.05; ∗∗ p ​< ​0.01; ∗∗∗ p ​< ​0.001.

Article Snippet: β3 Integrin , Cell Signaling , #13166 , 1:1000 , .

Techniques: Control, Staining, Microscopy

Journal: Journal of Orthopaedic Translation

Article Title: Osteocyte β1 integrin loss causes low bone mass and impairs bone mechanotransduction in mice

doi: 10.1016/j.jot.2022.03.008

Figure Lengend Snippet: β1 integrin loss alters lacunar-canalicular (LCS) size, cellular morphology and nuclear orientation in osteocytes ( a , c ) H/E staining results of trabecular and cortical sections from 4-month-old male control and cKO tibiae ( b , d ) Quantitative analyses of trabecular and cortical osteocyte numbers per 100 ​μm 2 area in tibial sections. ( e ) Representative LCS images from the cortical tibial sections of 4-month-old male control and cKO mice ( f – g ) Quantitative analyses of size and roundness of LCS in control and cKO mice. ( h ) Representative actin cytoskeleton images from Rhodamine-Phalloidin staining of the cortical tibial sections from 4-month-old male control and cKO mice. Zoom-in images of #1 and #2 represent control and cKO osteocyte actin cytoskeleton, respectively ( i – j ) Quantitative analyses of cortical osteocyte process length and number for control and cKO mice ( k , l ) Western blotting results and quantifications for β1 integrin, β3 integrin, Kindlin-2 and Gapdh expression from osteocyte-enriched cortical bone samples of control and cKO mice ( m ) Representative images for osteocyte nuclei from the cortical tibial sections of 4-month-old male control and cKO mice. Zoom-in images of #1 and #2 represent the angle α and β, respectively, i.e., angles between longitudinal direction of tibia sections and major axis of control and cKO nuclei ( n – p ) Quantitative analyses of nuclear size, nuclear roundness, and angle α/β from control and cKO cortical osteocytes. Results were collected from three biological replicates ( N ​= ​3) for each group. Results are expressed as mean ​± ​standard deviation (s.d.). n. s. p ​> ​0.05; ∗ p ​< ​0.05; ∗∗ p ​< ​0.01; ∗∗∗ p ​< ​0.001.

Article Snippet: β3 Integrin , Cell Signaling , #13166 , 1:1000 , .

Techniques: Staining, Control, Western Blot, Expressing

Journal: Journal of Orthopaedic Translation

Article Title: Osteocyte β1 integrin loss causes low bone mass and impairs bone mechanotransduction in mice

doi: 10.1016/j.jot.2022.03.008

Figure Lengend Snippet: β1 integrin loss in osteocytes reduces bone formation without affecting osteoclast activities in bone. ( a ) Representative images of tartrate-resistant acid phosphatase (TRAP) staining from tibial sections of 4-month-old male control and cKO mice ( b – c ) Quantitative analyses of osteoclast surface and osteoclast numbers in tibial sections from TRAP staining. ( d ) Serum levels of collagen type I cross-linked C-telopeptide (CTX-1) from ELISA detection of 4-month-old male control and cKO mice. ( e ) In vivo double calcein labeling for bone formation detection in the trabecular (Tb) and cortical bone (Cb) of ulna sections of 4-month-old male control and cKO mice ( f – g ) Quantitative analyses of MAR, MS/BS for double calcein labeling results. ( h ) Serum levels of procollagen type 1 ​N-terminal propeptide (P1NP) from ELISA detection of 4-month-old male control and cKO mice. ( i ) Alkaline phosphatase (ALP) activity detection from BMSC osteogenic differentiation experiments ( j ) Quantitative analysis of ALP-positive regions in BMSC osteogenic differentiation experiments ( k ) The transcription of Collα1 , Runx2 , Sp7 and Bglap were detected through quantitative PCR analysis after BMSC osteogenic differentiation. Results were collected from three biological replicates ( N ​= ​3) for each group. Results are expressed as mean ​± ​standard deviation (s.d.). n. s. p ​> ​0.05; ∗ p ​< ​0.05; ∗∗ p ​< ​0.01.

Article Snippet: β3 Integrin , Cell Signaling , #13166 , 1:1000 , .

Techniques: Staining, Control, Enzyme-linked Immunosorbent Assay, In Vivo, Labeling, Activity Assay, Real-time Polymerase Chain Reaction

Journal: Journal of Orthopaedic Translation

Article Title: Osteocyte β1 integrin loss causes low bone mass and impairs bone mechanotransduction in mice

doi: 10.1016/j.jot.2022.03.008

Figure Lengend Snippet: β1 integrin loss in osteocytes enhances BMSC adipogenic differentiation in vitro and in bone. ( a ) Oil red O staining results of adipogenic differentiation from cultured primary BMSC derived from control and cKO mice. ( b ) Quantitative analyses of numbers of oil droplets per frame in Oil red O staining results. ( c ) The transcription of Cebpα , Cebpβ , Ap2 , Pref-1 , Adipo , and Pparγ were detected through quantitative PCR analysis after BMSC adipogenic differentiation ( d , g ) H/E staining of trabecular and cortical bone and bone marrow of tibial sections from control and cKO mice ( e , h ) Merged immunofluorescence images of nuclei (DAPI in blue) and oil droplets (marked with Perilipin in green) in the trabecular and cortical bone sections from control and cKO tibiae ( f , i ) Quantitative analyses of numbers of oil droplets per 1 ​mm 2 in the trabecular and cortical sections from control and cKO tibiae. Results were collected from three biological replicates ( N ​= ​3) for each group. Results are expressed as mean ​± ​standard deviation (s.d.). n. s. p ​> ​0.05; ∗ p ​< ​0.05; ∗∗ p ​< ​0.01; ∗∗∗ p ​< ​0.001.

Article Snippet: β3 Integrin , Cell Signaling , #13166 , 1:1000 , .

Techniques: In Vitro, Staining, Cell Culture, Derivative Assay, Control, Real-time Polymerase Chain Reaction, Immunofluorescence

Journal: Journal of Orthopaedic Translation

Article Title: Osteocyte β1 integrin loss causes low bone mass and impairs bone mechanotransduction in mice

doi: 10.1016/j.jot.2022.03.008

Figure Lengend Snippet: β1 integrin loss in osteocytes impairs load-induced bone formation and collagen fiber integrity. ( a ) In vivo μCT images on Day 1 (D1, before loading) and Day 14 (D14, after loading) of experimental control and cKO mice. Yellow asterisks indicate the trabecular bones; red arrows point to the cortical bones. ( b ) Representative double calcein labeling images for the trabecular bones from unload and load tibiae of control and cKO mice ( c – e ) Quantitative analyses of relative ratio between load and unload tibia in MAR, MS/BS and BFR/BS. Results were collected from three biological replicates ( N ​= ​3) for each group. ( f ) Relative cortical thickness changes after load compared to unload conditions. N ​= ​5 for control group and N ​= ​7 for cKO group ( g – h ) Collagen I fiber images from two-photon microscopy of unload and load cortical and trabecular bones of control and cKO tibiae. Results are expressed as mean ​± ​standard deviation (s.d.). n. s. p ​> ​0.05; ∗ p ​< ​0.05; ∗∗ p ​< ​0.01; ∗∗∗ p ​< ​0.001.

Article Snippet: β3 Integrin , Cell Signaling , #13166 , 1:1000 , .

Techniques: In Vivo, Control, Labeling, Microscopy

Journal: bioRxiv

Article Title: A novel DNA repair protein, N-Myc downstream regulated gene 1 (NDRG1), links stromal tumour microenvironment to chemoresistance

doi: 10.1101/2025.01.22.634323

Figure Lengend Snippet: a , To characterize the PDAC CAF lines used in this study, three lines were stained and imaged by IF for α-SMA, b-tubulin and DAPI. Scale bar: 50µm. b , The same CAF lines as above, and indicated epithelial cell lines were probed for α-SMA and GAPDH by Western blot. c , Deposition of Collagen 1, pan-Laminin, and Fibronectin by starved CAFs was visualized by immunofluorescence at Day 9, α-SMA was used as a marker for CAF activation, scale bar: 20µm. d , Additional CAF lines were kept in 10% serum, or starved similarly as in , and media were harvested at indicated timepoints, TCA precipitated and probed for Collagen 1 (Col1), Fibronectin (FN) and pan-Laminin (Lam). The whole cell lysates (WCL) were probed for Vinculin. The TCA precipitates were loaded according to protein concentrations in the WCL. e , CAF-CM effect on SW1990 cell proliferation was analysed using automated live cell imager over 36h. Three CAF lines were used and NC media was used as a control. Data are represented as the confluency fold change normalized to day 0. Error bars represent SEM. Data were analysed by Mann-Whitney test. f , CAF-CM effect on SW1990 cell cycle was analysed using flow cytometry with cells labelled with Click-EdU (S-phase). CAF-CM increases PDAC cells in S-phase. Data are shown as mean with SD. Student’s t-test test was used for significance. g , SUIT-2 tumour cells’ IC50 responses towards chemotherapies were compared in CAF-CM (magenta) and in non-conditioned media (NC, blue). Fitted dose response curves to gemcitabine, 5-fluorouracil, and oxaliplatin were used to generate IC50 values for each treatment condition. Error bars are SEM corresponding to technical replicates. h-j , SW1990, BxPC-3 and AsPC tumour cell IC50 responses towards chemotherapies were compared as above ( g ) in CAF-CM (magenta) and in NC media (blue). Fitted dose response curves to gemcitabine, SN-38, 5-fluorouracil, and oxaliplatin were used to generate IC50 values for each treatment condition. k , SW1990 cells were treated with 1µM gemcitabine, 0.5 µM SN-38, 100 µM 5-FU, or 50 µM oxaliplatin for 24h in NC or CAF-CM (obtained from three patient-derived CAF lines). yH2AX foci analysis was used to assess DNA damage. Cells were fixed and immunostained for yH2AX followed by image acquisition and quantification by CellProfiler, data are shown as foci counts per single nucleus. >200 nuclei were analysed per condition. Mann-Whitney test was applied, magenta bars represent median. l-o , Similarly, as above in ( k ), HPAC, BxPC3, SUIT-2 and AsPC cells were analysed for yH2AX foci in three different CAF-CM vs. NC media, treated with 1µM gemcitabine, 0.5 µM SN-38, 100 µM 5-FU, or 50 µM oxaliplatin. p , CAF-CM and NC media were either boiled, or CAF-CM was filtered using 3kDa cut-off spin filters. PDAC cells (SW1990, HPAC) were treated with indicated medias (NC, CAF-CM, CAF-CM <3kDa, CAF-CM >3kDa, NC boiled, CAF-CM boiled) for 24h in the presence of 1 µM (SW1990) or 2 µM (HPAC) gemcitabine and cell numbers were counted. Student’s t-test was used to measure significance, data are shown as mean with SD. q , SUIT-2 were analysed for DNA damage by the alkaline comet assay. Proteins were filtered from CAF-CM using 3kDa cut-off spin filters. Cells were treated with 1µM gemcitabine or 0.5µM SN-38 for 24h in NC, CAF-CM, filtered CAF-CM (<3kDa proteins) and the top fraction (>3kDa proteins). Afterwards cells were plated on Comet assay slides and DNA was run under alkaline conditions and stained with SYBR-gold. Images were acquired and quantified using Comet Score software. Olive Tail Moment (OTM) were plotted in Graphpad Prism8 and Mann-Whitney test was used to establish significance. Magenta lines are median, >200 cells were analysed for each treatment condition. r-s, SW1990 and SUIT-2 cells were analysed for yH2AX foci in 1 µM gemcitabine or 0.5 µM SN-38-treatment after incubation for 24h in the different fractionated media: NC, CAF-CM, CAF-CM <3kDa, or CAF-CM >3kDa. yH2AX foci were measured and quantified as above ( k ). t , SW1990 cells were starved overnight after which NC, CAF-CM, purified matrix proteins (collagen 1, fibronectin, laminin) or RGD peptides were added to the cells along with 0.25 µM SN-38 for 24h. yH2AX foci were quantified as above ( k ). Mann-Whitney was used to measure statistical significance, and magenta lines represent median. u , SUIT-2 cells were treated as above with 0.5 µM gemcitabine or 0.25 µM SN-38 with indicated media or purified matrix proteins, stained for yH2AX (green) and DAPI (blue) and imaged. Representative images are shown. Scale bar: 20 µm. v , yH2AX foci analysis of SUIT-2 cells treated with NC, CAF-CM, or purified matrix proteins in the presence of 0.5 µM gemcitabine (GEM) or 0.25 µM SN-38 for 24h. yH2AX foci were quantified as above ( k ). Mann-Whitney was used to measure statistical significance, magenta lines represent median. w, Integrin profiling of PDAC lines. SW1990, HPAC, SUIT-2, PANC-1 and PA-TU-8988T cells were grown in 10% serum and probed for indicated integrins by Western blotting. Vinculin/actin was used as a loading control. x , CAF-CM changes integrin expression profile. HPAC and SW1990 cells were grown in NC or CAF-CM and probed for indicated integrins by Western blot. Actin was used as a loading control.

Article Snippet: The membranes were incubated with the following antibodies: Collagen 1 (Abcam #ab34710), Fibronectin 1 (Abcam #ab6328, Invitrogen #MA5-11981), pan-Laminin (Dako #Z-0097), EGFR (#sc-03G, Santa Cruz), GAPDH (#5174S, CST), phospho-NDRG1 T346 (#5482S, CST), NDRG1 (#9485S, CST), phospho-FAK Y397 (#3283S, CST), phospho-Src Y418 (#44-660G, Invitrogen), phospho-AKT S473 (9271, CST), V5-tag (R96025, Invitrogen), PCNA (#2586S, CST), Histone H3 (#4499S, CST), pH2AX S129 (yH2AX) (#9718S, CST and 05-636 Millipore Sigma), α-tubulin (B-5-1-2) (T5168, Sigma-Aldrich), β-actin (A5316, Sigma-Aldrich), vinculin (#13901, CST), integrin ⍺5 (#4705, CST), integrin ⍺V (#4711, CST), integrin β4 (#14803, CST, Abcam #ab110167), integrin ⍺4 (#8440, CST), integrin β1 (#9699, CST, BD clone 9EG7 #553715), integrin β1 active (Millipore clone Huts-4 #MAB2079Z), integrin β3 (#13166, CST), integrin β5 (#3629, CST), integrin ⍺6 (Millipore #MAB1378).

Techniques: Staining, Western Blot, Immunofluorescence, Marker, Activation Assay, Control, MANN-WHITNEY, Flow Cytometry, Derivative Assay, Alkaline Single Cell Gel Electrophoresis, Single Cell Gel Electrophoresis, Software, Incubation, Purification, Expressing

Journal: bioRxiv

Article Title: A novel DNA repair protein, N-Myc downstream regulated gene 1 (NDRG1), links stromal tumour microenvironment to chemoresistance

doi: 10.1101/2025.01.22.634323

Figure Lengend Snippet: a, Reverse Phase Protein Array (RPPA) was performed on three pancreatic cancer cell lines (HPAC, SW1990, BxPC-3), probing for 304 phospho- and total proteins. The cells were treated with 1 µM gemcitabine (GEM) for 72h in NC or CAF-CM. The data are shown as CAF-CM media normalized over NC media. Only significantly (p<0.05) changed proteins are shown. Yellow indicates proteins that were most significantly up-regulated in the CAF-CM and blue indicates down-regulated proteins (heat-map: log2 fold change). b , Western blot validation of the RPPA. Cells were treated with NC media or CAF-CM +/-1 µM GEM for 72h, media were changed daily, and cells were harvested 1h after the last media change. Gels were run at least thrice with similar results and probed for p-NDRG1 (T346), total NDRG1, EGFR, p-FAK (Y397), and actin. c , Purified ECM proteins (Laminin-5, Fibronectin, or Collagen 1) were added to serum starved HPAC or SW1990 cells for 3h, NC and CAF-CM media were used as controls. Cells were lysed and analysed for p-NDRG1, total NDRG1, and tubulin. d , General scheme of ECM-triggered signalling events, and pharmacological inhibition approaches used in experiments e-k . e-g , SW1990 and HPAC cells were treated with Integrin β1 or Integrin β4 function blocking antibodies or control Mouse IgG1 in CAF-CM. NC media were used as a control. g, Cells were immuno-stained for p-NDRG1 T346 (white). Scale bar: 100 µm. e-f, Mean fluorescence intensities of p-NDRG1 signal were quantified in Fiji, plotted, and analysed using Mann-Whitney test. >150 cells were analysed per condition. Magenta lines represent medians. h-i , Src-family inhibitor Saracatinib (2 µM) or FAK inhibitor Defactinib (2 µM) were added to serum starved cells (SW1990, HPAC) 1h before addition of CAF-CM, the cells were lysed 3h later and immunoblotted for p-NDRG1 T346, NDRG1, p-FAK Y397, p-Src Y418, and tubulin. j-k , SW1990 and HPAC cells were treated with vehicle control (DMSO) or inhibitors targeting AKT (MK-2206 250 nM) or SGK1 (BLU6340 250 nM) and CAF-conditioned media was added for indicated times. Cells were lysed and probed for p-NDRG1, NDRG1, p-AKT S473 and vinculin.

Article Snippet: The membranes were incubated with the following antibodies: Collagen 1 (Abcam #ab34710), Fibronectin 1 (Abcam #ab6328, Invitrogen #MA5-11981), pan-Laminin (Dako #Z-0097), EGFR (#sc-03G, Santa Cruz), GAPDH (#5174S, CST), phospho-NDRG1 T346 (#5482S, CST), NDRG1 (#9485S, CST), phospho-FAK Y397 (#3283S, CST), phospho-Src Y418 (#44-660G, Invitrogen), phospho-AKT S473 (9271, CST), V5-tag (R96025, Invitrogen), PCNA (#2586S, CST), Histone H3 (#4499S, CST), pH2AX S129 (yH2AX) (#9718S, CST and 05-636 Millipore Sigma), α-tubulin (B-5-1-2) (T5168, Sigma-Aldrich), β-actin (A5316, Sigma-Aldrich), vinculin (#13901, CST), integrin ⍺5 (#4705, CST), integrin ⍺V (#4711, CST), integrin β4 (#14803, CST, Abcam #ab110167), integrin ⍺4 (#8440, CST), integrin β1 (#9699, CST, BD clone 9EG7 #553715), integrin β1 active (Millipore clone Huts-4 #MAB2079Z), integrin β3 (#13166, CST), integrin β5 (#3629, CST), integrin ⍺6 (Millipore #MAB1378).

Techniques: Protein Array, Western Blot, Biomarker Discovery, Purification, Inhibition, Blocking Assay, Control, Staining, Fluorescence, MANN-WHITNEY

Journal: bioRxiv

Article Title: A novel DNA repair protein, N-Myc downstream regulated gene 1 (NDRG1), links stromal tumour microenvironment to chemoresistance

doi: 10.1101/2025.01.22.634323

Figure Lengend Snippet: a, Schematic for the sister fork symmetry analysis. Asymmetric sister forks show a ratio of a shorter IdU track to longer track <1, while symmetric forks show ratio ≈ 1. SW1990 CNTR, NDRG1 deficient and cells treated with 250nM BLU6340 were analysed for sister fork symmetry. 89 bidirectional forks were analysed for CNTR cells, 44 for NDRG1 deficient and 49 for BLU6340-treated cells, magenta lines represent median, Mann-Whitney test was used for significance. b, Schematic of the RNase H1 constructs used in the assays, WT-RNase H1 removes R-loops whereas the catalytically dead D210N RNase H1 is unable to do so. Replication fork progression was analysed using DNA fiber assays. SW1990 cells were treated with vehicle control (DMSO) or 250nM BLU6340 and co-transfected with constructs encoding WT-RNase H1 (RNH1) or the catalytically dead (dRNH1). Cells were pulsed with IdU (20min) followed by CldU (40min). Median IdU track lengths (µm) of >300 double-labelled fibers are shown in magenta. Mann–Whitney test was applied to test significance. c, Fork dynamics were analysed using DNA fiber assays. SW1990 CNTR, NDRG1 KO, and H194A addback cells were used and transfected with either WT- RNH1 , or dead RNH1 (dRNH1). Median IdU track lengths (µm) of >150 double-labelled fibers are shown in magenta. Mann–Whitney test was applied to test significance. d, Schematic of the recombinant RNase H1 construct used for R-loop visualisation. The bacterial purified GFP-labelled catalytic dead (D210N) RNase H1 was used to probe for RNA-DNA hybrids in fixed cells. e , Confocal images of SW1990 cells stained with GFP-dRNH1 (green) in CNTR, NDRG1 KO and rescue (WT, H194A) SW1990 cells. Scale bar: 20µm. f, Analysis of the fluorescence intensity of nuclear GFP-dRNase H1 in SW1990 cells from ( e ). At least 1500 nuclei were analysed per condition, magenta lines represent median, Mann-Whitney test was used for significance. g, GFP-dRNH1 nuclear intensity was analysed in SW1990 CNTR cells, cells with NDRG1 knockdown (siNDRG1), or after BLU6340 250nM 24h. Knockdown of Senataxin (siSETX) was used a positive control to induce R-loops. At least 1600 nuclei were analysed per condition, magenta lines represent median, Mann-Whitney test was used for significance. h, To assess whether NDRG1 KO increases R-loop content in the presence of chemotherapies SW1990 CNTR and KO cells were treated with 0.5µM gemcitabine (GEM) or 0.5 µM SN-38 for 24h and analysed for RNA-DNA hybrid formation by GFP-dRNH1 staining. At least 2100 nuclei were analysed per condition, magenta lines represent median, Mann-Whitney test was used for significance. i, Confocal images of the transcription-replication conflicts (TRC) in SW1990 cells. Proximity ligation assay (PLA) between PCNA and RNA PolII (RNAPII) was performed in CNTR, NDRG1 KO and rescue (WT, H194A) cells. PLA foci between PCNA and RNAPII are shown in white. Cytokeratin (green) was used to visualize cell shape, Scale bar: 10µm. j, SW1990 CNTR, NDRG1 KO, and rescue (WT, H194A) cells were synchronized by double thymidine block at G1/S border and TRC formation was analysed at different time-points post dT release. TRC accumulation was also analysed in non-synchronized cells. TRCs were assessed by PLAs between PCNA and RNA PollI, imaged and TRC foci counts analysed by CellProfiler. At least 210 nuclei per timepoint were analysed, magenta lines represent median, Mann-Whitney test was used for significance. At 6h all the lines had significant TRC load which were resolved in the CNTR and WT add-back cells at 10h timepoint. k, DNA fiber assay in SW1990 CNTR, NDRG1 KO and H194A add-back cells in the presence of transcription inhibitors α-amanitin (10mg/ml) and flavopiridol (10µM). Transcription inhibitors reduce TRC conflicts, thus preventing R-loops accumulation and rescuing replication fork dynamics in NDRG1-deficient or H194A NDRG1 expressing cells. Cells were pulsed with CldU (20min) and IdU (40min). Median IdU track lengths (µm) of >275 double-labelled fibers are shown in magenta. Mann–Whitney test was applied to test significance. l, Schematic of the proposed mechanism by which ECM proteins stimulate DNA repair and reduce R-loop formation via integrin-FAK-SRC-SGK1-NDRG1 pathway.

Article Snippet: The membranes were incubated with the following antibodies: Collagen 1 (Abcam #ab34710), Fibronectin 1 (Abcam #ab6328, Invitrogen #MA5-11981), pan-Laminin (Dako #Z-0097), EGFR (#sc-03G, Santa Cruz), GAPDH (#5174S, CST), phospho-NDRG1 T346 (#5482S, CST), NDRG1 (#9485S, CST), phospho-FAK Y397 (#3283S, CST), phospho-Src Y418 (#44-660G, Invitrogen), phospho-AKT S473 (9271, CST), V5-tag (R96025, Invitrogen), PCNA (#2586S, CST), Histone H3 (#4499S, CST), pH2AX S129 (yH2AX) (#9718S, CST and 05-636 Millipore Sigma), α-tubulin (B-5-1-2) (T5168, Sigma-Aldrich), β-actin (A5316, Sigma-Aldrich), vinculin (#13901, CST), integrin ⍺5 (#4705, CST), integrin ⍺V (#4711, CST), integrin β4 (#14803, CST, Abcam #ab110167), integrin ⍺4 (#8440, CST), integrin β1 (#9699, CST, BD clone 9EG7 #553715), integrin β1 active (Millipore clone Huts-4 #MAB2079Z), integrin β3 (#13166, CST), integrin β5 (#3629, CST), integrin ⍺6 (Millipore #MAB1378).

Techniques: MANN-WHITNEY, Construct, Control, Transfection, Recombinant, Purification, Staining, Fluorescence, Knockdown, Positive Control, Proximity Ligation Assay, Blocking Assay, Expressing

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: Association of the α 2 δ 1 Subunit with Ca v 3.2 Enhances Membrane Expression and Regulates Mechanically Induced ATP Release in MLO-Y4 Osteocytes

doi: 10.1002/jbmr.437

Figure Lengend Snippet: RT-PCR primers used for detection of VSCC subunits and osteocyte markers

Article Snippet: The rabbit anti-β 3 antibody was purchased from Alomone Research Laboratories (Jerusalem, Israel).

Techniques: Sequencing

Journal: Frontiers in Molecular Neuroscience

Article Title: LRRK2 Regulates Voltage-Gated Calcium Channel Function

doi: 10.3389/fnmol.2016.00035

Figure Lengend Snippet: Biophysical properties of HEK293 cells recorded in different experimental conditions .

Article Snippet: The primary antibodies used were: rabbit anti-LRRK2 at 1:500 (MJFF2, c41-2 Abcam), rabbit anti-calcium channel β3 subunit 1:200 (Sigma), rabbit anti-calcium channel α1a subunit 1:200 (Sigma), mouse anti-actin 1:2000, mouse anti-myc 1:1000 (Sigma), rabbit anti-S6 ribosomal protein (S6RP) 1:1000 (Cell Signalling) applied in blocking buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 0.1% Tween 20) and 5% nonfat dry milk, overnight at 4°C.

Techniques: Transfection

Journal: Frontiers in Molecular Neuroscience

Article Title: LRRK2 Regulates Voltage-Gated Calcium Channel Function

doi: 10.3389/fnmol.2016.00035

Figure Lengend Snippet: LRRK2 interacts with the Ca V 2.1 channel. (A) Mouse forebrain specimens obtained from littermate wt and heterozygous BAC hG2019S (GS) mice were cross-linked and then processed for immunoprecipitation with rat anti LRRK2 antibodies. Eluted proteins were resolved by SDS-PAGE and detected with anti LRRK2, anti Ca V 2.1 β3 subunit antibodies, anti Ca V 2.1 α1a and anti S6 ribosomal protein (S6RP). (B,C) The graphs report the yield of protein recovered upon LRRK2 immunoprecipitation expressed as a percentage of relative input (B) and as amount of pulled Ca V 2.1 β3 subunit relative to the amount of pulled LRRK2 (C) . Bars represent mean ± SEM ( n = 4) where * indicates p < 0.05; unpaired t -test.

Article Snippet: The primary antibodies used were: rabbit anti-LRRK2 at 1:500 (MJFF2, c41-2 Abcam), rabbit anti-calcium channel β3 subunit 1:200 (Sigma), rabbit anti-calcium channel α1a subunit 1:200 (Sigma), mouse anti-actin 1:2000, mouse anti-myc 1:1000 (Sigma), rabbit anti-S6 ribosomal protein (S6RP) 1:1000 (Cell Signalling) applied in blocking buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 0.1% Tween 20) and 5% nonfat dry milk, overnight at 4°C.

Techniques: Immunoprecipitation, SDS Page