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: Cells were pulsed with 10 µM EdU for 1 hr before harvesting via trypsin digestion and fixation with 4% PFA and later subjected to Click chemistry using Click-iT™ EdU Pacific Blue™ Flow Cytometry Assay Kit according to manufacturer’s instructions (Thermo Scientific C10418).

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, SW1990 and HPAC cells were transfected with short interfering RNA (siRNA) against SGK1, SGK2, or SGK3 and treated with 1µM gemcitabine in NC or CAF-CM and analysed by Comet assay, OTM values were plotted, and two-tailed Mann-Whitney test was used for analysis. Magenta lines represent median values. >250 cells were analysed. b , c SW1990 and HPAC cells were treated with 1 µM gemcitabine, 0.5 µM SN-38, and 100 µM 5-FU in CAF-CM +/- 250 nM SGK1 inhibitor BLU6340, 500nM for HPAC and 24h later subjected to Comet assay. OTM values were analysed using two-tailed Mann-Whitney test. Magenta lines represent median values. >250 cells were analysed. d, SUIT-2 cells were transfected with short interfering RNA (siRNA) against NDRG1 and subjected to immunoblotting to verify the knockdown. The cells were treated with 1 µM gemcitabine, 0.5 µM SN-38, or 100 µM 5-FU in CAF-CM and analysed by Comet assay. Comet assay images were quantified, OTM values were plotted, and two-tailed Mann-Whitney test was used for analysis. >175 cells were analysed, magenta lines represent median values. e , SW1990 NDRG1 KO cells were created using CRISPR/Cas9 system. Data from 3 clonal cells lines are shown, cells used as control (CNTR) are described in Material and methods. f , SW1990 CNTR cells (blue) and NDRG1 KO (magenta) cells were subjected to live image proliferation analysis. Growth curves of cells in 10% FBS are shown over 96h, ANOVA was applied for analysis, error bars represent SEM. g, Fitted dose response curves for gemcitabine and SN-38 in CAF-CM were used to generate IC50 values for SW1990 NDRG1 KO (magenta) and CNTR (blue) cells. Error bars are SEM corresponding to technical replicates. h , NDRG1 was knocked out in AsPC cells using CRISPR/Cas9 and knock-out was verified by Western blotting for NDRG1. i , AsPC CNTR cells (blue) and NDRG1 KO cells (magenta) were subjected to live image proliferation analysis. Growth curves of cells in 10% FBS are shown over 96h, Student’s t-test was applied for analysis, error bars represent SEM. j, Fitted dose response curves for gemcitabine and SN-38 in CAF-CM were used to generate IC50 values for AsPC NDRG1 KO (magenta) and CNTR (blue) cells. Error bars are SEM corresponding to technical replicates. k-l, SW1990 and HPAC cell proliferation was analysed in 10% FBS using live image proliferation analysis in the presence of increasing concentrations of SGK1 inhibitor BLU6340 or vehicle control (DMSO). Error bars are SEM. No significant changes were observed. m, Fitted dose response curves for gemcitabine are shown in the presence of non-conditioned media (NC), CAF-CM, or CAF-CM supplemented with BLU6340 (250nM). Error bars are SEM corresponding to technical replicates. n , SW1990 and AsPC CNTR and NDRG1 KO cells were treated with 1 µM gemcitabine (GEM), or 0.5 µM SN-38. SGK1 inhibitor (250 nM BLU6340) treated CNTR cells were also included in the analysis. 24h later the cells were fixed and stained for yH2AX followed by image acquisition and quantification by CellProfiler, data are shown as foci counts per single nucleus. >220 nuclei were analysed per condition. Mann-Whitney test was applied, magenta bars represent median. o-r, Cell cycle analyses of SW1990 and AsPC cells. o, q SW1990 and AsPC NDRG1 KO and CNTR cells were subjected to double thymidine block and released into 10% DMEM. Time points were collected with unsynchronised (non-sync) cells, cells were fixed and processed for flow cytometry using PI for total DNA content, EdU-click coupled to Pacific Blue 450 azide as a marker for cells in S phase, phosphorylated histone H3 Ser10 (H3-pS10) conjugated to Phycoerythrin (PE) as a marker for mitosis. Due to partial emission spectra overlap between PI and PE it is possible to present total DNA content and mitotic population on the same axis. Representative cell cycle scatter plots showing propidium iodide (PI), phospho-histone H3 (pH3) (x-axis) and EdU-positive cells (y-axis) staining intensities. Plots were generated in Flowjo after gating on single cells only. Cells stained for each of the fluorophores were used as gating controls. Time point 0h shows synchronization of the cells at G1/S border. p,r , SW1990 and AsPC cells were subjected to double thymidine block, pre-treated with BLU6340 250nM for 16h and released into 10% DMEM supplemented with BLU6340 250nM when indicated. Time points were collected with unsynchronised (non-sync) cells, cells were fixed and processed for flow cytometry using PI for total DNA content, EdU-click coupled to Pacific Blue 450 azide as a marker for cells in S phase, phosphorylated histone H3 Ser10 (H3-pS10) conjugated to Phycoerythrin (PE) as a marker for mitosis. Representative cell cycle scatter plots showing propidium iodide (PI), phospho-histone H3 (pH3) (x-axis) and EdU-positive cells (y-axis) staining intensities. Plots were generated in Flowjo after gating on single cells only. Cells stained for each of the fluorophores were used as gating controls. Time point 0h shows synchronization of the cells at G1/S border. s, 2×10 SW1990 CNTR and a mix of NDRG1 KO clonal cell lines (KO mix) were injected into flanks of male athymic nude mice (10 animals/group). Representative images of tumours of CNTR versus NDRG1 KO cells are shown; NDRG1 expression was evaluated by IHC, scale bar: 1mm (whole size tumour images), and 100 μm for magnified areas. t, Growth dynamics of SW1990 tumour xenografts based on tumour volume, error bars are SEM, some error bars are not visible due to small variation in sample group. Student’s t-test was applied. u, Tumour weights between CNTR and NDRG1 KO groups at end point were plotted, student’s t-test was applied for significance, error bars are SEM. v, NDRG1 was knocked down in AsPC cells using CRISPR/Cas9 and after selection, batch KD population and CNTR cells were pooled and injected to nude mice. Tumours were allowed to grow until palpable and treated with 25mg/kg gemcitabine 2× week for 13 days. Tumours were harvested and imaged. w, AsPC tumour volumes were measured at end point and plotted. Student’s t-test was applied for significance, error bars are SEM. x, AsPC NDRG1 KD and CNTR IHC sections of the tumours were stained for NDRG1 expression and imaged. Scale bar: 2 mm. y , AsPC tumour weights were measured and plotted. Student’s t-test was applied for significance, error bars are SEM. z, AsPC xenograft tumours sections were stained for pan-cytokeratin to define cancer cells within the tumour, for Ki67 to define proliferative cells, and the nuclei were counterstained by DAPI. Whole tumour image analysis was performed in QuPath by applying appropriate thresholds and thus defining Ki-67 positivity. Percent of the Ki67 positive cells in relation to pan-cytokeratin positive cells within each tumour is plotted. Student’s t-test was applied for significance. Error bars are SEM.

Article Snippet: Cells were pulsed with 10 µM EdU for 1 hr before harvesting via trypsin digestion and fixation with 4% PFA and later subjected to Click chemistry using Click-iT™ EdU Pacific Blue™ Flow Cytometry Assay Kit according to manufacturer’s instructions (Thermo Scientific C10418).

Techniques: Transfection, Small Interfering RNA, Single Cell Gel Electrophoresis, Two Tailed Test, MANN-WHITNEY, Western Blot, Knockdown, CRISPR, Control, Knock-Out, Staining, Blocking Assay, Flow Cytometry, Marker, Generated, Injection, Expressing, Selection

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 of the Tus/ Ter replication fork barrier system allowing induction of site-specific replication fork stalling and measurement of homologous recombination (HR) at a defined locus in mouse embryonic stem cells. HR may be measured following either expression of the I-SceI nuclease and induction of a conventional double-strand break (DSB), or after site-specific replication fork stalling upon expression of the bacterial protein Tus. Tus binding to the 6× Ter array imposes a physical barrier to replication fork progression, stalling approaching replication forks, and triggering HR. (Orange triangle: 6×Ter element array. Blue hexagon: Tus protein, yellow rectangle: I-Sce-I endonuclease cut site). HR outcomes are quantified by flow cytometry: the reporter contains two non-functional heteroalleles of GFP (grey boxes) flanking synthetic exons encoding RFP in a tail to head orientation (A and B: 5’ and 3’ artificial RFP exons). HR may reconstitute a functional GFP allele and thus GFP+ cells - the 6× Ter -I-SceI GFP allele may undergo gene conversion upon copying a short region of the 5’-truncated GFP allele off the intact sister-chromatid, producing a wild-type GFP allele. These short-tract gene conversion (STGC) events represent conservative HR outcomes. A distinct minority of error-prone HR outcomes, termed long-tract gene conversion (LTGC), results in functional GFP with the duplication of the interior of the cassette harboring synthetic RFP exons. LTGC events produce cells that are both GFP+ and RFP+. b-c , Quantification of repair outcomes induced by fork stalling or I- Sce- I incision. Reporter cells were co-transfected with either empty vector, Tus or I- Sce -I endonuclease vectors and with siRNAs targeting either NDRG1 or control Luc. Transfected cells were analysed after 72 hours. Normalized STGC and LTGC repair frequencies were calculated and corrected for transfection efficiency. Triplicate samples and mean values from six independent experiments and Student’s t-test are shown. NDRG1 knockdown has a specific effect on HR at stalled forks (A); loss of NDRG1 inhibits total HR and error-free STGC at stalled forks (top panel), whereas it did not influence conventional DSB repair (I- Sce -I, lower panel). P-value: *<0.05, **,<0.01. ns: not significant. d , Quantification of yH2AX formation in AsPC CNTR, NDRG1 KO, and rescue (WT, H194A) cells after 1µM gemcitabine or 0.5µM SN-38 treatment (24h). yH2AX foci quantification was performed using CellProfiler, foci numbers per single nucleus were plotted and Mann-Whitney test was used for significance. Magenta lines represent median values. >200 cells were quantified per experiment. e , Replication fork dynamics in AsPC CNTR, NDRG1 KO, and rescue cells (WT and H194A) were analysed by DNA Fiber Assay. Left panel: Cells were labelled with CldU followed by IdU as indicated. Right panel: Fork stalling was investigated by including HU (2mM, 1h) in between the CldU and the IdU pulse. Median IdU track lengths (µm) of >200 double-labelled fibers were analysed and shown in magenta. Mann–Whitney test was applied to test significance. f , To analyse the effect of short-term and long-term SGK1 inhibition on replication fork dynamics AsPC cells were treated with either vehicle control (DMSO), 250nM BLU6340 (0.5h) or pre-treated with BLU6340 for 24h, then labelled with CldU (20min) followed by IdU (40min). Median IdU track lengths (µm) of >200 double-labelled fibers are shown in magenta. Mann–Whitney test was applied to test significance. g , To assess the effect of CAF-CM on replication fork progression SW1990 cells (CNTR: siLUC, siNDRG1, and 250nM BLU6340 treated cells) were labelled with CldU (20min) followed by IdU (40min) in the presence of different CAF-CM media harvested from three patient lines supplemented with 2%FBS. Median IdU track lengths (µm) of >200 double-labelled DNA fibers are shown in magenta. h, To assess the effect of CAF-CM on fork stalling in SW1990 cells, experiment described in (g) was repeated with the inclusion of 2mM HU (1h) to stall the replication fork in between the CldU and IdU pulses. IdU pulse was 60 min. Median IdU track lengths (µm) of >200 double-labelled DNA fibers indicating fork restart are shown in magenta. Mann–Whitney test was applied to test significance.

Article Snippet: Cells were pulsed with 10 µM EdU for 1 hr before harvesting via trypsin digestion and fixation with 4% PFA and later subjected to Click chemistry using Click-iT™ EdU Pacific Blue™ Flow Cytometry Assay Kit according to manufacturer’s instructions (Thermo Scientific C10418).

Techniques: Homologous Recombination, Expressing, Binding Assay, Flow Cytometry, Functional Assay, Transfection, Plasmid Preparation, Control, Knockdown, MANN-WHITNEY, Inhibition

Journal: Communications Biology

Article Title: Context dependent effects of ascorbic acid treatment in TET2 mutant myeloid neoplasia

doi: 10.1038/s42003-020-01220-9

Figure Lengend Snippet: a Natural TET2 -mutant HEL and TET2 wild-type MEG-01 and CMK cells were stably transfected with either an empty pOZ (Vector) or a pOZ-TET2-overexpression vector ( TET2 OE ). Levels of 5hmC and 5mC were assessed by dot blot assay. b – d Cell lines (Vector and TET2 OE of HEL, MEG-01, and CMK) were treated for 6 days with either PBS or 250 µM AA. Surviving cells were assessed by trypan blue exclusion assay on Vi-CELL XR cell viability analyzer (Beckman Coulter) and the cell output was plotted. b MEG-01. c CMK. d HEL. e – h Human K562 and MOLM13 cells were transduced with scrambled shRNA (scr) or shRNA targeting TET2 ( shTET2 ) and treated with 250 µM AA. e – f 5hmC and 5mC were assessed after 24 h by 2D-UPLC-MS/MS. g – h Cellular proliferation was accessed by the number of viable cells at different time. i – j Primary human cord blood CD34 + cells and proliferation was assessed after 14 days by colony forming assay. a – j Data are shown as mean ± SEM ( n = 3) and are representative of two independent experiments. k TET2 WT ( n = 8) and TET2 MT ( n = 11) human BM levels of 5hmC/5mC was measured by 2D-UPLC-MS/MS. Data are shown as mean with SEM. l TET2 WT and TET2 MT human BM cells were treated for 48 h with 250 µM AA or left untreated. Base oxidation levels were assessed by 2D-UPLC-MS/MS. Data from the same cell are connected with arrow, TET2 WT , n = 3; TET2 MT , n = 4. TET2 MT MN patient information are provided in Supplementary Data . Paired t test was used in ( l ), p values are indicated, ns: not significant.

Article Snippet: Human cord blood was acquired from Cleveland Cord Blood Center, Cleveland, Ohio, and CD34 + cells were isolated by human CD34 MicroBead Kit (Miltenyi Biotec) according to the manufacturer’s protocol.

Techniques: Mutagenesis, Stable Transfection, Transfection, Plasmid Preparation, Over Expression, Dot Blot, Trypan Blue Exclusion Assay, Transduction, shRNA, Tandem Mass Spectroscopy

Journal: International Journal of Nanomedicine

Article Title: Enrichment of breast cancer stem-like cells by growth on electrospun polycaprolactone-chitosan nanofiber scaffolds

doi: 10.2147/IJN.S55720

Figure Lengend Snippet: Culture on polycaprolactone-chitosan nanofibers results in increased resistance to chemotherapy. Notes: MCF-7 cells (top panel) and T47D cells (lower panel) were cultured on polycaprolactone-chitosan nanofibers or in polystyrene dishes for 72 hours and then exposed to chemotherapy for 48 hours. Data are expressed as percentage of untreated cells as determined by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay. *Significant difference ( P ≤0.05).

Article Snippet: Cells were treated with docetaxel or doxorubicin at the indicated concentrations and assayed for survival 48 hours later using the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) cell viability kit assay (Biotium Inc, Hayward, CA, USA).

Techniques: Cell Culture, MTT Assay