Journal: Experimental and Therapeutic Medicine

Article Title: Effect of overexpression of GRN on the proliferation and osteogenic capacity of human periodontal cells

doi: 10.3892/etm.2024.12783

Figure Lengend Snippet: pLV-GRN transfection of hPDLCs. (A) Lentiviral infection screening results (magnification, x40). (B) RT-qPCR results of the relative expression of GRN in hPDLCs after transfection ( *** P<0.01). (C) Western blotting results of the relative expression of GRN in hPDLCs following transfection ( ** P<0.05). pLV, lentivirus recombinant plasmid; GRN, granulin precursor gene; hPDLCs, human periodontal ligament cells; RT-qPCR, reverse transcription-quantitative PCR.

Article Snippet: Upon reaching 80% confluence, cell transfection was performed using the jetPRIME Transfection Reagent Kit (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions.

Techniques: Transfection, Infection, Quantitative RT-PCR, Expressing, Western Blot, Recombinant, Plasmid Preparation, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Scientific Reports

Article Title: Nrf2 depletion enhanced curcumin therapy effect in gastric cancer by inducing the excessive accumulation of ROS

doi: 10.1038/s41598-024-81375-1

Figure Lengend Snippet: Cur combined with Nrf2 knockdown effectively inhibited the proliferation, migration and invasion of GC cells. ( A , B ) rt-qPCR validated the efficiency of three siRNA sequences to knock down Nrf2 in AGS and HGC27 cells. ( C , D ) After GC cells were transfected with siNC and siNrf2 for 24 h, they were treated with Cur (0 and 20 µM) for 24 h. Cell viability was detected by the CCK8 assay. ( E – H ) The migratory capacity of GC cells was compared using a wound healing assay and the wound healing area was quantified. ( I – L ) GC cells were transfected with siNC and siNrf2 or treated with Cur (0 and 20 µM) for 24 h. Transwell assay was used to compare the migratory and invasive capacities of GC cells. Scale: 500 μm. Data are presented as the mean ± SD. n = 3. ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: After incubating cells (2 × 10 5 /well) in 6-well plates for 24 h, jetPRIME in vitro siRNA transfection reagent (Yeasen, Shanghai, China) and complete media were added, along with small interfering RNAs (siRNAs) targeting Nrf2 (siNrf2) or negative control (NC) (50 nmol/l).

Techniques: Knockdown, Migration, Quantitative RT-PCR, Transfection, CCK-8 Assay, Wound Healing Assay, Transwell Assay

Journal: Nature Communications

Article Title: Concerted transcriptional regulation of the morphogenesis of hypothalamic neurons by ONECUT3

doi: 10.1038/s41467-024-52762-z

Figure Lengend Snippet: a , a 1 Overexpression of ONECUT3 limited Neuro-2a cell proliferation as shown by a reduction in the mitotic marker pHH3 ( n = 259 cells per condition over 11 fields imaged, p = 7,53E-07). b Cleaved caspase-3 (Cas3) was absent from transfected cells. c Neuro-2a cells three days post-transfection with mock (control; left) or CMV- Onecut3 (OC3) plasmid (right). Note the induction of stable processes (acetylated tubulin + , green) upon ONECUT3 overexpression. d Representative images of Neuro-2a somata (differential interference contrast; DIC) after 4 days in vitro (DIV). Cell body size was measured by tracing the diameter of the somata, and analyzed with an unpaired two-tailed Student’s t -test. ( n = 47/group, p = 8,19508E-06). d 1 , d 2 Live-cell imaging of the cell cluster area ( b 1 ) and neurite length ( b 2 ) over a period of five days. e Dot plot illustrating relative gene expression as measured by sequencing bulk mRNA of Neuro-2a cells four days post-transfection. The top target genes involved in cytoskeletal remodeling were marked in red. Data were assessed with unpaired two-tailed Student’s t -test and expressed as means ± s.e.m. *** p < 0.001 ( a 1 , d ). Source data are provided as a Source Data file. ace. tub. acetylated tubulin, CMV cytomegalovirus promoter, DIV days in vitro, OC3 Onecut3 . Scale bars = 100 µm ( a ), 20 µm ( b – d ).

Article Snippet: Cells were transfected with 500 ng pDNA (CMV- Onecut3 or CMV-SERT as CMV control) using a jetPRIME transfection reagent or a Nucleofector (Lonza Biosciences).

Techniques: Over Expression, Marker, Transfection, Control, Plasmid Preparation, In Vitro, Two Tailed Test, Live Cell Imaging, Gene Expression, Sequencing

Journal: Nature Communications

Article Title: Concerted transcriptional regulation of the morphogenesis of hypothalamic neurons by ONECUT3

doi: 10.1038/s41467-024-52762-z

Figure Lengend Snippet: a Reconstruction of a protein-protein interaction network distinguished a pathway from ONECUT3 downstream to NAV2 within the signaling cassette supporting regulation of RhoA-mediated cytoskeletal dynamics. Note that arrows indicate an interaction, but not their directionality per se. b Changes in NAV2 protein levels after ONECUT3 overexpression. NAV2 signal intensity from the somata along neurites until their motile end tips were plotted. Data were acquired by Plot profiling in ImageJ ( n = 68 cells/group). c NAV2 protein localization and distribution in the soma, along the process, and in pseudo-growth cones of Neuro-2a cells after ONECUT3 overexpression. Neuro-2a cells extended their neurites without forming classical synapse-like structures. d Pathway prediction downstream from ONECUT3, including NAV2 and its interacting partners known to trigger cytoskeletal remodeling to promote neuritogenesis. e , e 1 Neurite outgrowth and branching were inhibited by ITX3, a Trio N-terminal RhoGEF domain inhibitor. Experiments were performed in triplicate. Representative DIC images at 3 days post-transfection showed the reduced length of processes upon ITX3 treatment ( e ). Neurite length and branching were measured by IncuCyte live-cell imaging ( e 1 ). A neurite mask (blue) was overlaid on the processes. Note that due to extensive proliferation, the difference in total neurite length was obscured in the first 2 days post-transfection until control cultures reached confluence (the experiment was performed twice). Data were evaluated by unpaired two-tailed Student’s t -test. * p < 0.05. Source data are provided as a Source Data file. ace. tub. acetylated tubulin, CMV cytomegalovirus promoter, DIV days in vitro, gc growth cone, OC3 ONECUT3. Scale bars = 100 µm ( e ), 20 µm ( b , c ).

Article Snippet: Cells were transfected with 500 ng pDNA (CMV- Onecut3 or CMV-SERT as CMV control) using a jetPRIME transfection reagent or a Nucleofector (Lonza Biosciences).

Techniques: Over Expression, Transfection, Live Cell Imaging, Control, Two Tailed Test, In Vitro

Journal: bioRxiv

Article Title: PPM1D truncation-associated overexpression of the stress-related protein NQO1 confers sensitivity to the bioactivatable drug IB-DNQ in diffuse intrinsic pontine glioma

doi: 10.1101/2024.09.05.611476

Figure Lengend Snippet: ( a ) Western blot analyses of PPM1D overexpression in SU-DIPG-VI cell line 6 weeks after stable transfection. EV: empty vector; FL: full length; mut: mutated; Trunc: truncated. ( b ) Western blot of PPM1D and NQO1 in HSJD-DIPG-007 KO cell lines. ( c ) Western blot of PPM1D and NQO1 in HSJD-DIPG-007 and HSJD-DIPG-014 cell lines treated at 100nm during 0; 3; 7 or 14 days. ( d ) NQO1 expression in mouse cell lines derived from IUE tumors analysed by western-blot (left) and real time PCR (right). ( e ) Proteomic and RNA-seq expression of NQO1 in mouse Neural Stem Cells (mNSC) transfected with GFP as control or with PPM1D truncated. For (e) significance was calculated using a moderated t-test. ns: not significant and ** P<0.005.

Article Snippet: Lentiviruses containing this construct were produced by cotransfecting HEK-293T cells with the recombinant plasmid, with packaging vectors and jetPRIME® Transfection Reagent (Polyplus Transfection) following the supplier instructions.

Techniques: Western Blot, Over Expression, Stable Transfection, Plasmid Preparation, Expressing, Derivative Assay, Real-time Polymerase Chain Reaction, RNA Sequencing Assay, Transfection, Control

Journal: bioRxiv

Article Title: PPM1D truncation-associated overexpression of the stress-related protein NQO1 confers sensitivity to the bioactivatable drug IB-DNQ in diffuse intrinsic pontine glioma

doi: 10.1101/2024.09.05.611476

Figure Lengend Snippet: ( a ) Stable transfection in HSJD-DIPG-011 of MDM2 S395A validated by western blot leads to NQO1 increased expression analysed by western blot (left), but do not modulate the transcript expression of NQO1 analysed by real time PCR (right). For real time PCR, significance was calculated using an unpaired t-test. ns: not significant and ****P<0.0001.

Article Snippet: Lentiviruses containing this construct were produced by cotransfecting HEK-293T cells with the recombinant plasmid, with packaging vectors and jetPRIME® Transfection Reagent (Polyplus Transfection) following the supplier instructions.

Techniques: Stable Transfection, Western Blot, Expressing, Real-time Polymerase Chain Reaction

Journal: bioRxiv

Article Title: PPM1D truncation-associated overexpression of the stress-related protein NQO1 confers sensitivity to the bioactivatable drug IB-DNQ in diffuse intrinsic pontine glioma

doi: 10.1101/2024.09.05.611476

Figure Lengend Snippet: ( a ) IC50 determination using the MTT assay in the DIPG cell lines. DIPG cells harbouring NQO1 overexpression (HSJD-DIPG-007; -008 and -014) show higher sensitivity to IB-DNQ in comparison to NQO1 normal expression cells (HSJD-DIPG-011; -012 and SU-DIPG-VI). ( b ) DIPG cellular models of NQO1 depletion by short hairpin (sh) RNA validated by real time PCR (left panel) and western blots (middle) for the cell line HSJD-DIPG-007. NQO1 depletion leads to IB-DNQ resistance shown by IC50 determination (right panel). ( c ) NQO1 overexpression model by stable transfection in HSJD-DIPG-011 cell line validated by real time PCR (left panel) and western-blots (middle) that leads to IB-DNQ sensitivity (right panel). ( d ) PPM1D overexpression by stable transfection in HSJD-DIPG-011 cell line leads to IB-DNQ sensitivity. ( e ) Cell lines derived from IUE mouse models of PPM1D are more sensitive to IB-DNQ. ( f ) MDM2 and mainly MDM2 S395A stable transfection in HSJD-DIPG-011 lead to IB-DNQ sensitivity. ( g ) Kaplan-Meier analysis of survival according treatment conditions (IB-DNQ treated vs mock group) in a mouse model with implanted tumors derived from the NQO1 overexpressing DIPG cell line DIPG 7. Mice was treated at days 20; 22; 24; 26 and 28 (red vertical lines). Significance of the log-rank test is shown. Results of the univariate Cox regression analysis are represented by the Hazards Ratio (HR) and 95% Confidence Interval (CI). Significance was calculated using a paired t test for IC50s and an unpaired t-test for real time PCR. *P<0.05, **P<0.005, ***P<0.0005 and ****P<0.0001.

Article Snippet: Lentiviruses containing this construct were produced by cotransfecting HEK-293T cells with the recombinant plasmid, with packaging vectors and jetPRIME® Transfection Reagent (Polyplus Transfection) following the supplier instructions.

Techniques: MTT Assay, Over Expression, Comparison, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Stable Transfection, Derivative Assay