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: International immunopharmacology

Article Title: Inhibiting the SARM1-NAD + axis reduces oxidative stress-induced damage to retinal and nerve cells.

doi: 10.1016/j.intimp.2024.112193

Figure Lengend Snippet: Fig. 6. DSRM ameliorated oxidative stress-induced neurite injury in N2a cells. (A-C) GOx inhibited N2a cell proliferation and neurite growth in the RA-induced N2a cell differentiation model (magnification 400 × ); (D-E) Immunocytochemistry demonstrated that 10 μM DSRM pre-treatment for 2 h enhanced β3-tubulin expression induced by 7 mU/mL GOx for 12 h in N2a cells (scale bar: 50 μm). N ≥3, **p < 0.01, ****p < 0.0001.

Article Snippet: After culturing and treating the cells in 24-well plates and fixing and permeabilizing them with methanol at -20 ◦C for 15 min, the cells were then washed 3 times with PBS, and then incubated in containment buffer (5 % donkey serum for containment) for 20 min, and then incubated with anti-GSDMD primary antibody (1:500, Abclonal, Wuhan, China, Cat#A20197), anti-β3-tubulin primary antibody (1:1000, CST, USA, Cat#5568S) was incubated at 4 ◦C overnight.

Techniques: Cell Differentiation, Immunocytochemistry, 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 , 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: PathoGenetics

Article Title: Regulation of TGF-β signalling by Fbxo11 , the gene mutated in the Jeff otitis media mouse mutant

doi: 10.1186/1755-8417-2-5

Figure Lengend Snippet: Immunolocalization in palate tissue . a . Coronal sections through the palate of E15.5 wild-type (WT) and homozygote ( Jf/Jf ) embryos, immunohistochemically stained with antibodies against Fbox11, TGF-β3, TGFβR-I, Smad2, and Smad4. Scale bar 50 μm. Medial edge epithelium (mee), nasal palatal epithelium (ne) and oral palatal epithelium (oe). Graph : comparison of the percentage of epithelial cells positive for Smad2 in E15.5 wild-type and homozygote palates. b . Coronal sections through the palate of E14.5 (before fusion) and E15.5 (after fusion) wild-type (WT) and homozygote ( Jf/Jf ) embryos, immunohistochemically stained with pSmad2 antibody. Scale bar 50 μm. Medial edge epithelium (mee). Graph : comparison of the percentage of epithelial cells positive for nuclear and cytoplasmic localization of pSmad2 in E15.5 wild-type and homozygote palates. c . Coronal sections through the palate of E15.5 wild-type (WT) and homozygote ( Jf/Jf ) embryos, immunohistochemically stained with antibodies against Ki67 and cleaved caspase-3. Scale bar 50 μm. Graph : comparison of the percentage of epithelial cells positive for cleaved caspase-3 in E15.5 wild-type and homozygote palates. P -values were determined using two-tailed T -test.

Article Snippet: Rabbit ABC Staining system (sc-2018 Santa Cruz Biotechnology) and goat ABC staining system (sc-2023 Santa Cruz Biotechnology) were used to develop the specific signals with all the antibodies except for TGF-β3.

Techniques: Staining, Two Tailed Test

Journal: PathoGenetics

Article Title: Regulation of TGF-β signalling by Fbxo11 , the gene mutated in the Jeff otitis media mouse mutant

doi: 10.1186/1755-8417-2-5

Figure Lengend Snippet: Immunolocalization in eyelid tissue . a . Coronal sections through the eye of E16 wild-type (WT) and homozygote ( Jf/Jf ) embryos immunohistochemically stained with antibodies against Fbox11, TGF-β3, TGFβR-I, Smad3, Smad2 and Smad4. Scale bar 200 μm. Graph : comparison of the percentage of epithelial cells positive for Smad2 in E16 upper and lower eyelids. b . Coronal sections through the eyes of E15.5 (before fusion) and E16 (after fusion) in wild-type (WT) and homozygote ( Jf/Jf ) embryos stained with pSmad2 antibody (scale bar 200 μm). Epidermis (ep), basal cells (bc) and dermis (d). Graph : comparison of the percentage of epithelial cells with positive nuclear and cytoplasmic localization of pSmad2 in E16 upper and lower eyelids. c . Coronal sections through the eyes of E16 in wild-type (WT) and homozygote ( Jf/Jf ) embryos stained with Ki67 and cleaved caspase-3 antibodies. Scale bar 200 μm. Graph : comparison of the percentage of epithelial cell positive for Ki67 and cleaved caspase-3 in E16 upper and lower eyelids. P -values were determined using two-tailed T -test comparing each homozygote eyelid with the wild type.

Article Snippet: Rabbit ABC Staining system (sc-2018 Santa Cruz Biotechnology) and goat ABC staining system (sc-2023 Santa Cruz Biotechnology) were used to develop the specific signals with all the antibodies except for TGF-β3.

Techniques: Staining, Two Tailed Test