Journal: International Journal of Molecular Sciences

Article Title: Ocular Albinism Type 1 Regulates Melanogenesis in Mouse Melanocytes

doi: 10.3390/ijms17101596

Figure Lengend Snippet: The overexpression of OA1 affects the melanin content in an MITF-dependent fashion. Mice OA1 was PCR amplified and then subcloned into the pMSCV PIG vector with Xho I and EcoR I restriction sites. The pMSCV-OA1-GFP plasmid or empty vector was transfected into the mouse melanocytes. The cells were harvested to extract total RNA and total protein. The expression levels of OA1 were qualitatively and quantitatively analyzed by qRT-PCR and Western blot. ( A ) Melanin contents in melanocytes transfected with OA1 , as well as the control; ( B – D ) transfection efficiency of OA1 in mouse melanocytes; ( E – G ) OA1 overexpression influences microphthalmia-associated transcription factor ( MITF ) mRNA and protein levels in mouse melanocytes. Data are shown as the mean ± standard errors ( n = 3 each), * p < 0.05, ** p < 0.01.

Article Snippet: The mice OA1 gene was PCR amplified and then was cloned into the pMSCV PIG vector (Addgene, Cambridge, MA, USA) with Xho I and EcoR I restriction sites.

Techniques: Over Expression, Amplification, Plasmid Preparation, Transfection, Expressing, Quantitative RT-PCR, Western Blot

Journal: Frontiers in Genetics

Article Title: Quantitative Evaluation of Toxic Polyglycine Biosynthesis and Aggregation in Cell Models Expressing Expanded CGG Repeats

doi: 10.3389/fgene.2018.00216

Figure Lengend Snippet: Fluorescence microscopy and flow cytometry are sensitive and informative tools for quantitative evaluation of RAN translation product, FMRpolyG-GFP. (A) Schematic representation of genetic constructs used in fluorescence microscopy, flow cytometry and FL-SDS-PAGE. Delivery of 5′(CGG exp )- GFP (+1) or ATG(CGG exp )- GFP (+1) leads to biosynthesis of FMRpolyG-GFP as a result of RAN translation from near-cognate ACG or GUG start codons or canonical translation from ACG to AUG mutated start codon, respectively. An additional protein, FMRP-N-X, can be generated from the FMRP native start codon of 5′(CGG exp )- GFP (+1). Administration of 5′(CGG exp )- mCherry (0) leads to biosynthesis of two separate proteins, non-tagged FMRpolyG and FMRP-N-mCherry generated from the FMRP-specific AUG start codon. (B) Fluorescence microscopy of COS7 cells 36, 48, 60, 72, or 84 h following transfection with 5′(CGG exp )- GFP (+1) or ATG(CGG exp )- GFP (+1). Images were taken from the same wells at different time points post transfection. Quantification of the average fluorescence signal is shown in the lower panel ( n = 10). Scale bar, 500 μm. (C) Histograms showing distribution of the FMRpolyG-GFP signal in COS7 cells 24, 36, 48, 72, and 96 h after 5′(CGG exp )- GFP (+1) delivery and flow cytometry. In the lower panel, quantification of the average fluorescence signal after exclusion of dead cells is shown ( n = 3). The threshold for GFP positive cells was set based on the signal obtained for non-transfected cells. For each sample ∼5000 GFP-positive cells were analyzed. Note that the percentage of GFP-positive cells reached maximum 48 h post transfection (39%) while the mean fluorescence signal was highest 72 h post transfection. In the following time points, a decrease in these values was observed as a consequence of cell division and cell death.

Article Snippet: 5′(CGG exp )- mCherry (0) construct was derived from 5′(CGG exp )- GFP (+1). mCherry sequence was amplified from pmCherry_a_tubulin_IRES_puro2 (Addgene #21043) with ( F1 / R1 ) primers with CloneAmp HiFi PCR Premix kit (Takara Bio United States) according to manufacturer’s instructions.

Techniques: Fluorescence, Microscopy, Flow Cytometry, Construct, SDS Page, Generated, Transfection

Journal: Frontiers in Genetics

Article Title: Quantitative Evaluation of Toxic Polyglycine Biosynthesis and Aggregation in Cell Models Expressing Expanded CGG Repeats

doi: 10.3389/fgene.2018.00216

Figure Lengend Snippet: Tagging affects aggregation properties of FMRpolyG. (A) Fluorescence microscopy of COS7 cells 48 h post transfection with ATG(CGG exp )- GFP (+1) or ATG(CGG exp )- mCherry (+1). In contrast to FMRpolyG-GFP, FMRpolyG-mCherry forms only scarce foci (marked with arrows). (B) Visualization of COS7 cells 48 h following delivery of 5′(CGG exp )- mCherry (0) and labeling for FMRpolyG with 8FM antibody. Schematic representations of delivered constructs are shown above the images in (A,B) . Scale bars, 10 μm. (C) FL-SDS-PAGE analyses of soluble fractions of FMRpolyG-mCherry and FMRP-N-mCherry in cell lysates prepared 120 h after transfection with ATG(CGG exp )- mCherry (+1) or 5′(CGG exp )- mCherry (0). Note altered gel migration of the fusion protein in relation to FMRP-N-mCherry. Also, no increase in the FMRpolyG-mCherry signal is observed in gel wells, indicating absence of prominent protein foci. (D) Immunoblotting of COS7 cell lysates for FMRpolyG with 9FM antibody 48 h after administration of 5′(CGG exp )- GFP (+1) or 5′(CGG exp )- mCherry (0). Marked differences in the amounts of FMRpolyG-GFP and FMRpolyG are observed.

Article Snippet: 5′(CGG exp )- mCherry (0) construct was derived from 5′(CGG exp )- GFP (+1). mCherry sequence was amplified from pmCherry_a_tubulin_IRES_puro2 (Addgene #21043) with ( F1 / R1 ) primers with CloneAmp HiFi PCR Premix kit (Takara Bio United States) according to manufacturer’s instructions.

Techniques: Fluorescence, Microscopy, Transfection, Labeling, Construct, SDS Page, Migration, Western Blot

Journal: Frontiers in Genetics

Article Title: Quantitative Evaluation of Toxic Polyglycine Biosynthesis and Aggregation in Cell Models Expressing Expanded CGG Repeats

doi: 10.3389/fgene.2018.00216

Figure Lengend Snippet: FL-SDS-PAGE and fluorescence microscopy allow for precise quantification of soluble and aggregated in foci forms of FMRpolyG-GFP. (A) FL-SDS-PAGE analyses of soluble fractions of FMRpolyG-GFP and FMRP-N-mCherry in cell lysates prepared 24, 36, 48, 72, and 96 h after co-transfection with 5′(CGG exp )- GFP (+1) and 5′(CGG exp )- mCherry (0). Fluorescence detected in gel wells is characteristic for FMRpolyG-GFP positive samples (invisible in case of other GFP-fusion proteins or GFP alone; not shown) and comes from a fraction of undissolved protein aggregates. The FMRpolyG-GFP fluorescence signal shown on the graph was normalized to FMRP-N-mCherry signal ( n = 4). Note that after 36 h solubility of FMRpolyG-GFP decreases significantly in favor of the insoluble form of this protein. The FMRpolyG-GFP band was additionally identified based on western blotting with anti-FMRpolyG antibodies (see Supplementary Figure ). (B) Fluorescence microscopy of COS7 cells 24, 36, 48, 72, and 96 h following transfection with 5′(CGG exp )- GFP (+1) construct and Hoechst 33342 staining. Insoluble fraction of FMRpolyG-GFP was calculated as the number of foci per 100 cells ( n = 7). Note that up to 72 h post transfection the number of FMRpolyG-GFP foci per cell increases significantly reaching ∼10 per 100 cell, and then decreases due to cell division and cell death. Scale bar, 50 μm.

Article Snippet: 5′(CGG exp )- mCherry (0) construct was derived from 5′(CGG exp )- GFP (+1). mCherry sequence was amplified from pmCherry_a_tubulin_IRES_puro2 (Addgene #21043) with ( F1 / R1 ) primers with CloneAmp HiFi PCR Premix kit (Takara Bio United States) according to manufacturer’s instructions.

Techniques: SDS Page, Fluorescence, Microscopy, Cotransfection, Solubility, Western Blot, Transfection, Construct, Staining

Journal: Frontiers in Genetics

Article Title: Quantitative Evaluation of Toxic Polyglycine Biosynthesis and Aggregation in Cell Models Expressing Expanded CGG Repeats

doi: 10.3389/fgene.2018.00216

Figure Lengend Snippet: FMRpolyG fluorescence-based system can be used for efficient monitoring of therapeutic potential of molecules. (A) RT-qPCR analyses showing FMR1 expression levels in FXTAS patient-derived fibroblasts after 48 h treatment with 10 nM control (ASO-Ctrl) or directed against FMR1 antisense oligonucleotides (ASO- FMR1 ). Significant downregulation was observed in either control fibroblasts (CGG norm /-) or two fibroblast lines obtained from FXTAS patients: CGG norm /CGG exp (mutant and normal allele), CGG exp /CGG exp (two mutant alleles). All values are normalized to the level of GAPDH mRNA ( n = 3). (B) Representative histograms showing distribution of FMRpolyG-GFP signals in COS7 cells 48 h post transfection with 5′(CGG exp )- GFP (+1) and early delivery of ASO-Ctrl or ASO- FMR1 as well as quantification of the signals obtained from the flow cytometry experiments (lower panel). Note that ASO- FMR1 , but not ASO-Ctrl, induced reduction of the number of cells showing high fluorescence and mean fluorescence signals in concentration dependent manner ( n = 3 for each condition). (C) Quantitative FL-SDS-PAGE analysis of COS7 cells 48 h after co-transfection with 5′(CGG exp )- GFP (+1) and 5′(CGG exp )- mCherry (0) and early administration of either 5 or 25 nM ASO-Ctrl and ASO- FMR1 . Delivery of ASO- FMR1 at 25 nM concentration significantly decreased the amount of soluble FMRpolyG-GFP and FMRP-N-mCherry by ∼90%. Results were normalized to the reference mCherry signals derived from the control mCherry construct ( n = 3). (D) Diagram showing quantification of foci numbers per 100 cells 48 h following 5′(CGG exp )- GFP (+1) administration and early ASO delivery ( n = 6). Cells treated with 25 nM ASO- FMR1 revealed a ∼60% decrease of FMRpolyG-GFP foci. (E) Quantification of the average fluorescence FMRpolyG-GFP signal in COS7 cells 72 h after transfection with 5′(CGG exp )- GFP (+1) and late delivery of 10 nM ASO-Ctrl or ASO- FMR1 ( n = 3). Note that 10 nM ASO- FMR1 only slightly reduced the total FMRpolyG-GFP signal.

Article Snippet: 5′(CGG exp )- mCherry (0) construct was derived from 5′(CGG exp )- GFP (+1). mCherry sequence was amplified from pmCherry_a_tubulin_IRES_puro2 (Addgene #21043) with ( F1 / R1 ) primers with CloneAmp HiFi PCR Premix kit (Takara Bio United States) according to manufacturer’s instructions.

Techniques: Fluorescence, Quantitative RT-PCR, Expressing, Derivative Assay, Control, Mutagenesis, Transfection, Flow Cytometry, Concentration Assay, SDS Page, Cotransfection, Construct

Journal: Cell Host & Microbe

Article Title: SARS-CoV-2 spike L452R variant evades cellular immunity and increases infectivity

doi: 10.1016/j.chom.2021.06.006

Figure Lengend Snippet: Increase in the binding affinity to ACE2, viral infectivity, viral fusogenicity, and viral replication capacity by the L452 mutation (A) Location of the NF9 peptide (residues 448–456) in the cocrystal structure of the SARS-CoV-2 S and human ACE2 proteins (PDB: 6M17 ) ( Yan et al., 2020 ). An overview (left), an enlarged view of the boxed area in the left panel (middle), and a view of the middle panel rotated 180° on the y axis (right) are shown. Residues 448–456 of SARS-CoV-2 S (corresponding to the NF9 peptide) are shown in black. (B–D) Binding affinity of SARS-CoV-2 S RBD to ACE2 by yeast surface display. The percentage of the binding of the SARS-CoV-2 S RBD expressed on yeast to soluble ACE2 (B) and the K D values (C) are shown. Assays were performed in quadruplicate. (D) The level of stable expression of the SARS-CoV-2 RBD on yeast (x axis) and the binding affinity toward ACE2 (y axis) compared to the parental RBD. In (B), the fitting curve of parental RBD is shown as black lines in all panels. (E and F) Pseudovirus assay. The HIV-1-based reporter virus pseudotyped with the parental SARS-CoV-2 S or its derivatives (E, L452R, Y453F, and N501Y; F, D614G, B.1.429 [S13I/W152C/L452R/D614G], and B.1.1.298 [HV69-70del/Y453F/D614G]) was inoculated into HEK293 cells transiently expressing human ACE2 and TMPRSS2 at four different doses (1, 3, 5, and 10 ng p24 antigens). Percentages of infectivity compared to the virus pseudotyped with parental S (10 ng p24 antigen) are shown. (G) Gain of electrostatic complementarity by the L452R substitution. Left: the surface structure of SARS-CoV-2 S and ACE2 (PDB: 6M17 ) ( Yan et al., 2020 ). Residue 452 of SARS-CoV-2 S and the negatively charged patch on ACE2 (residues E35, E37, and D38) are indicated in black and red. The boxed area is enlarged in the upper right panel. Right: Coulombic surface coloring of the structures of SARS-CoV-2 S and ACE2 (PDB: 6M17 ) ( Yan et al., 2020 ) (top) and a model of the L452R substitution (bottom). The black line indicates the border between SARS-CoV-2 S and ACE2. (H) Chromatograms of the mutated regions of SARS-CoV-2 viruses artificially generated by reverse genetics. Chromatograms of nucleotide positions 22,913–22,924 (left) and 23,060–23,068 (right) of parental SARS-CoV-2 (strain WK-521; GISAID ID: EPI_ISL_408667) and the L452R (T22917G in nucleotide), Y453F (A22920T in nucleotide), and N501Y (A23063T in nucleotide) mutants are shown. (I–L) Growth kinetics of parental SARS-CoV-2 and SARS-CoV-2 mutants. Parental SARS-CoV-2 and the L452R, Y453F, and N501Y mutants (100 plaque-forming units [PFU]) were inoculated into HEK293-ACE2 cells (I and J), A549-ACE2 cells (K), and VeroE6/TMPRSS2 cells (L), and the copy number of viral RNA in the culture supernatant was quantified by real-time PCR. (I) Representative bright-field images of HEK293-ACE2 cells uninfected or infected with the viruses indicated at 24, 48, or 72 h post-infection are also shown. Bars, 200 μm. (J–L) Left: the growth curve of the viruses inoculated. The result for the parental virus is shown in all panels as a black line. Right: the amount of viral RNA in the culture supernatant at 72 h post-infection. Assays were performed in triplicate (J) or quadruplicate (K and L). (M) Competition assay. Parental virus and the L452R mutant were mixed at a 1:1 ratio based on PFU, and the mixture was inoculated into HEK293-ACE2 cells. The percentage of L452R mutant at each time point was analyzed as described in . The data are shown as the average of four biological replicates. (N) SARS-CoV-2 S-based fusion assay. Effector cells (S-expressing cells) and target cells (ACE2-expressing cells) were prepared, and the fusion activity was measured as described in . Assays were performed in quadruplicate, and fusion activity (arbitrary unit) is shown. In (C), statistically significant differences ( ∗ p < 0.05) versus parental S are determined by the Mann-Whitney U test. In (E and F), statistically significant differences ( ∗ p < 0.05) versus parental S (E) and the D614G mutant (F) at the same dose were determined by Student’s t test. In (J–L and N), statistically significant differences ( ∗ p < 0.05) versus parental virus (J–L) or parental S (N) were determined by Student’s t test. In (M), values at respective time points were compared with those at the last time point using a two-tailed, paired Student’s t test, and an asterisk denotes familywise error rate <0.05 using the Holm test. See also Figure S1 .

Article Snippet: To construct the expression plasmid for human ACE2 (GenBank: NM_021804.3 ) (pLV-EF1a-human ACE2-IRES-Puro), the MluI-SmaI fragment pTargeT-human ACE2 (kindly provided by Shuetsu Fukushi) ( ) was inserted into the MluI-HpaI site of pLV-EF1a-IRES-Puro (Addgene #85132).

Techniques: Binding Assay, Infection, Mutagenesis, Expressing, Virus, Residue, Generated, Real-time Polymerase Chain Reaction, Competitive Binding Assay, Single Vesicle Fusion Assay, Activity Assay, MANN-WHITNEY, Two Tailed Test

Journal: Cell Host & Microbe

Article Title: SARS-CoV-2 spike L452R variant evades cellular immunity and increases infectivity

doi: 10.1016/j.chom.2021.06.006

Figure Lengend Snippet:

Article Snippet: To construct the expression plasmid for human ACE2 (GenBank: NM_021804.3 ) (pLV-EF1a-human ACE2-IRES-Puro), the MluI-SmaI fragment pTargeT-human ACE2 (kindly provided by Shuetsu Fukushi) ( ) was inserted into the MluI-HpaI site of pLV-EF1a-IRES-Puro (Addgene #85132).

Techniques: Virus, Recombinant, Modification, Expressing, Reverse Transcription, Transfection, Luciferase, Mutagenesis, Gel Extraction, Cloning, Plasmid Preparation, Quantitative RT-PCR, Competitive Binding Assay, Sequencing, Random Hexamer, Software, Pore Size, Membrane

Journal: Nature Communications

Article Title: Ufbp1 promotes plasma cell development and ER expansion by modulating distinct branches of UPR

doi: 10.1038/s41467-019-08908-5

Figure Lengend Snippet: Defective differentiation of Ufbp1-deficient B cells into plasmablasts. a , c Sorted naive B cells from lymph nodes of indicated mice were cultured with lipopolysaccharide (LPS; 1 µg/ml) + interleukin (IL)-4 (10 ng/ml) + IL-5 (5 ng/ml). Four days later cells were analyzed for expression of BLIMP1, CD138, IRF4, and Pax5 by flow cytometry. Left panels in a and c show control IgG2a, IgG1, and IG2a isotype staining for BLIMP1, IRF4, and Pax5 respectively. Right panels in a and c show staining for BLIMP1 and CD138, and IRF4 and Pax5 respectively. Numbers represent the percent positive cells in the adjacent/corresponding area. b Enumeration of the frequency of BLIMP1 + CD138 high cells in a ( n = 3 mice/genotype). d Enumeration of the frequency of IRF4 + Pax5 − cells in c ( n = 3 mice/genotype). e Naive B cells from Ufbp1 F/F and Ufbp1 F/F CD19 cre mice were cultured with LPS, IL-4, and IL-5 as above, and transduced with retrovirus containing empty vector (MSCV-PIG), expressing human Ufbp1 (MSCV-PIG-hUfbp1) or Ufbp1K267R (MSCV-PIG-hUfbp1K267R). Transduction of cells with retroviruses was monitored by expression of green fuorescent protein (GFP). Two days after transduction, cells were analyzed for GFP and CD138. Numbers represent percent CD138 + cells within GFP + cells. f Enumeration of the frequency of CD138 + cells among GFP + cells in e ( n = 3 mice/genotype). Error bars represent mean ± standard error. ** P < 0.01, *** P < 0.001. Unpaired Student’s two-tailed t -test was used. A representative of at least two experiments is shown

Article Snippet: cDNA from mouse XBP1u, XBP1s, and human Ufbp1, and its mutant Ufbp1K267R were cloned into retrovirus vector MSCV-PIG (Addgene, cat# #18751) and/or MSCV-Thy1.1 (Addgene, cat# 17442).

Techniques: Cell Culture, Expressing, Flow Cytometry, Control, Staining, Transduction, Plasmid Preparation, Two Tailed Test

Journal: Nature Communications

Article Title: Ufbp1 promotes plasma cell development and ER expansion by modulating distinct branches of UPR

doi: 10.1038/s41467-019-08908-5

Figure Lengend Snippet: Ufbp1 downstream to IRE1/XBP1 induces endoplasmice reticulum (ER) expansion. a Naive B cells and plasma cells were sorted from the spleens and bone marrows respectively of Ire1α F/F and Ire1α F/F CD19 cre mice. Expression of the indicated molecules normalized against β-actin was quantified by quantitative real-time PCR (qRT-PCR). b Total cell lysates from naive B cells (untreated) and treated with lipopolysaccharide (LPS) for 3 days from indicated mice were immunoblotted with antibodies against indicated molecules. c Naive B cells from Ire1α F/F and Ire1α F/F CD19 cre mice were treated with LPS. Three days later cells were sorted into CD138 − and CD138 + populations. Expression of Uba5, Ufc1, Ufl1, Ufbp1, Ufm1, and IRE1α mRNA normalized against β-actin was quantified by qRT-PCR. d Naive B cells from Ire1α F/F and Ire1α F/F CD19 cre mice were stimulated with LPS and transduced with retroviruses expressing empty vector (MSCV-PIG), unspliced XBP1 (MSCV-XBP1u), or spliced XBP1 (MSCV-XBP1s) on day 2. On day 4, CD138 + cells transduced with viruses were sorted and expression of Uba5, Ufc1, Ufl1, Ufbp1, and Ufm1 mRNA normalized against β-actin was quantified by qRT-PCR. Error bars represent standard error of mean. e LPS-stimulated B cells from wild-type and Ire1α F/F CD19 cre mice were transduced with empty retrovirus (MSCV-Thy1.1), retrovirus expressing Ufbp1 or Ufbp1K267R. Cells were stained with anti-Thy1.1, CD138, and ER-tracker dye, and analyzed by flow cytometry. Shown is the mean fluorescent intensity (MFI) of the ER-tracker staining on the CD138 + cells among cells transduced with retrovirus (Thy1.1 + cells). Error bars represent mean ± standard error. * P < 0.05, ** P < 0.01, *** P < 0.001, ns nonsignificant. Unpaired Student’s two-tailed t -test was used. A representative of at least two experiments is shown

Article Snippet: cDNA from mouse XBP1u, XBP1s, and human Ufbp1, and its mutant Ufbp1K267R were cloned into retrovirus vector MSCV-PIG (Addgene, cat# #18751) and/or MSCV-Thy1.1 (Addgene, cat# 17442).

Techniques: Clinical Proteomics, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Transduction, Plasmid Preparation, Staining, Flow Cytometry, Two Tailed Test

Journal: Cell communication and signaling : CCS

Article Title: HMGB3 promotes the malignant phenotypes and stemness of epithelial ovarian cancer through the MAPK/ERK signaling pathway.

doi: 10.1186/s12964-023-01172-7

Figure Lengend Snippet: Fig. 1 HMGB3 promotes ovarian cancer proliferation. A pLKO.1, HMGB3 shRNA-1 (shHMGB3-1), HMGB3 shRNA-2 (shHMGB3-2), pCMV, and pCMV HMGB3 plasmids were stably transfected into A2780 and SKOV3 cells. HMGB3 protein levels were determined by western blot. B Cells were seeded into 96-well plates and cultured for 1, 2, 3, 4, and 5 days, and an MTT assay was performed to assess cell viability. C A clonogenic assay was used to assess the colony formation efficiency of A2780 and SKOV3 cells with HMGB3 knocked down or overexpressed. D Quantification of the number of clones in C. E Proliferation of A2780 and SKOV3 cells with HMGB3 knocked down or overexpressed detected by EdU assay. Nuclei were stained using DAPI. Scale bar, 10 μm. F Quantification of the ratio of EdU positive cells in (E). Data are presented as the mean ± SEM, *p < 0.05, **p < 0.01, n = 3

Article Snippet: The pCMV-HMGB3 plasmid was generated by cloning the human HMGB3 open reading frame into the pCMV vector (PS100069, OriGene, Rockville, MD, USA).

Techniques: shRNA, Stable Transfection, Transfection, Western Blot, Cell Culture, MTT Assay, Clonogenic Assay, Clone Assay, EdU Assay, Staining

Journal: Cell communication and signaling : CCS

Article Title: HMGB3 promotes the malignant phenotypes and stemness of epithelial ovarian cancer through the MAPK/ERK signaling pathway.

doi: 10.1186/s12964-023-01172-7

Figure Lengend Snippet: Fig. 3 RNA sequencing analysis of signaling pathways involved in HMGB3 function. A2780 cells were transfected with HMGB3 siRNA (siHMGB3) or negative control siRNA (Ctr) for 48 h. High-throughput RNA sequencing analysis was used to compare mRNA expression profiles of the siHMGB3 and Ctr groups. A Volcano plot showing differentially expressed genes (DEGs) between the siHMGB3 and Ctr groups. In total, 91 genes were up-regulated (Up) and 679 genes were down-regulated (Down); other genes expression levels were not significantly altered (no-DEGs) (B) Kyoto Encyclopedia of Genes and Genomes enrichment analysis of genes down-regulated in the siHMGB3 group relative to the Ctr group. C Heatmap showing genes involved in stem cell pluripotency and the MAPK signaling pathway down-regulated in the siHMGB3 group relative to the Ctr group. D A2780 cells were transfected with HMGB3 siRNA (siHMGB3) or negative control siRNA (Ctr) for 48 h, and qRT-PCR used to verify down-regulation of representative genes in the siHMGB3 group relative to the Ctr group. Data are presented as the mean ± SEM, *p < 0.05, **p < 0.01, n = 3

Article Snippet: The pCMV-HMGB3 plasmid was generated by cloning the human HMGB3 open reading frame into the pCMV vector (PS100069, OriGene, Rockville, MD, USA).

Techniques: RNA Sequencing, Protein-Protein interactions, Transfection, Negative Control, High Throughput Screening Assay, Expressing, Quantitative RT-PCR

Journal: Cell communication and signaling : CCS

Article Title: HMGB3 promotes the malignant phenotypes and stemness of epithelial ovarian cancer through the MAPK/ERK signaling pathway.

doi: 10.1186/s12964-023-01172-7

Figure Lengend Snippet: Fig. 4 HMGB3 activates the MAPK/ERK signaling pathway in ovarian cancer cells. A p-MEK1/2, MEK1/2, p-ERK1/2, ERK1/2, ETS-1, CCND1, c-Myc, HMGB3, and β-actin protein levels of in A2780 and SKOV3 cells with HMGB3 knocked down or overexpressed detected by western blot. B Quantification of the protein levels in (A). C Protein levels in ovarian cancer cells transfected with pLKO.1, HMGB3 shRNA-1 (shHMGB3-1) and/or pCMV HMGB3 detected by western blot. (D) Quantification of the protein levels in (C). Data are presented as the mean ± SEM, *p < 0.05, **p < 0.01, n = 3

Article Snippet: The pCMV-HMGB3 plasmid was generated by cloning the human HMGB3 open reading frame into the pCMV vector (PS100069, OriGene, Rockville, MD, USA).

Techniques: Western Blot, Transfection, shRNA

Journal: Cell communication and signaling : CCS

Article Title: HMGB3 promotes the malignant phenotypes and stemness of epithelial ovarian cancer through the MAPK/ERK signaling pathway.

doi: 10.1186/s12964-023-01172-7

Figure Lengend Snippet: Fig. 6 HMGB3 promotes the malignant phenotypes of ovarian cancer via the MAPK/ERK signaling pathway. A A2780 and SKOV3 cells with HMGB3 overexpression were seeded into 6 cm dishes and then treated with or without AZD6244 (5 µM)/PD0325901 (10 µM) for 24 h. p-ERK1/2, ERK1/2, HMGB3, and β-actin protein levels detected by western blot. B A2780 and SKOV3 cells with HMGB3 overexpression were seeded into 96-well plates and then treated with or without AZD6244 (5 µM)/PD0325901 (10 µM) for 72 h. Cell viability was detected by MTT assay. C A2780 and SKOV3 cells with HMGB3 overexpression were treated with or without AZD6244 (1 µM)/PD0325901 (2 µM) for 1–2 weeks. Colony formation efficiency was assessed by clonogenic assay. D Quantification of the number of clones in (C). E A2780 and SKOV3 cells with HMGB3 overexpression were seeded into transwell plates and then treated with or without AZD6244 (5 µM)/PD0325901 (10 µM) for 12–24 h. Cell migration and invasion were evaluated by transwell assay. Scale bar, 50 μm. F Quantification of the number of cells in (E). Data are presented as the mean ± SEM, **p < 0.01, n = 3

Article Snippet: The pCMV-HMGB3 plasmid was generated by cloning the human HMGB3 open reading frame into the pCMV vector (PS100069, OriGene, Rockville, MD, USA).

Techniques: Over Expression, Western Blot, MTT Assay, Clonogenic Assay, Clone Assay, Migration, Transwell Assay

Journal: Cell communication and signaling : CCS

Article Title: HMGB3 promotes the malignant phenotypes and stemness of epithelial ovarian cancer through the MAPK/ERK signaling pathway.

doi: 10.1186/s12964-023-01172-7

Figure Lengend Snippet: Fig. 7 HMGB3 promotes ovarian cancer cell proliferation in vivo through the MAPK/ERK signaling pathway. A2780 cells (5 × 106) transfected with pLKO.1, HMGB3 shRNA-1 (shHMGB3-1), or HMGB3 shRNA-2 (shHMGB3-2) were subcutaneously injected into nude mice. Mice were divided into three groups: pLKO.1 (Ctr), shHMGB3-1, and shHMGB3-2 (n = 6 per group). Two weeks post-injection, mice were euthanized and the xenograft tumors were removed. A Tumors from each group are shown. B Tumor volumes of each group. C Body weight of each group. D p-MEK1/2, MEK1/2, p-ERK1/2, ERK1/2, HMGB3, and β-actin protein levels in tumor tissues detected by western blot. E Quantification of the protein levels in (D). A2780 cells (5 × 106) transfected with pCMV or pCMV HMGB3 were subcutaneously injected into nude mice. Mice were divided into three groups: pCMV (Ctr), pCMV HMGB3, and pCMV HMGB3 + AZD6244 (n = 6 per group). One group of mice received an intraperitoneal injection of AZD6244 (25 mg/ kg) once a day. Two weeks post-injection, mice were euthanized and xenograft tumors were removed. F Tumors from each group are shown. G Tumor volumes of each group. H Body weight of each group. I p-MEK1/2, MEK1/2, p-ERK1/2, ERK1/2, HMGB3, and β-actin protein levels in tumor tissues detected by western blot. J Quantification of the protein levels in (I). Data are presented as mean ± SEM, #p > 0.05, **p < 0.01, n = 6

Article Snippet: The pCMV-HMGB3 plasmid was generated by cloning the human HMGB3 open reading frame into the pCMV vector (PS100069, OriGene, Rockville, MD, USA).

Techniques: In Vivo, Transfection, shRNA, Injection, Western Blot

Journal: Cell communication and signaling : CCS

Article Title: HMGB3 promotes the malignant phenotypes and stemness of epithelial ovarian cancer through the MAPK/ERK signaling pathway.

doi: 10.1186/s12964-023-01172-7

Figure Lengend Snippet: Fig. 8 Schematic summary of the study findings. HMGB3 overexpression activates the MAPK/ERK signaling pathway, thereby promoting ovarian cancer proliferation, metastasis, and stemness. Inhibition of the MAPK/ERK signaling pathway using specific inhibitors counteracts the effects of HMGB3

Article Snippet: The pCMV-HMGB3 plasmid was generated by cloning the human HMGB3 open reading frame into the pCMV vector (PS100069, OriGene, Rockville, MD, USA).

Techniques: Over Expression, Inhibition

Journal: Molecular Cancer

Article Title: Tumour-intrinsic PDL1 signals regulate the Chk2 DNA damage response in cancer cells and mediate resistance to Chk1 inhibitors

doi: 10.1186/s12943-024-02147-z

Figure Lengend Snippet: Tumour PDL1 promotes Chk2 content. a . Immunoblot from whole cell lysates of CTRL and PDL1 KO B16 cells. Vinculin (VINC), loading control. γH2AX Ser139 blot is shown as γH2AX. b . Immunoblot from whole cell lysates of CTRL and PDL1 KO ES2 cells. VINC, loading control. c . Immunoblot of CTRL and PDL1 KO ES2 cells transfected with empty vector (EV, gray) and/or PDL1 plasmid (black) at depicted μg. d . Immunoblot of CTRL versus PDL1 KO T24 cells after 72-h incubation with vehicle or gemcitabine (5 ng/mL). e . Quantification of relative intracellular PDL1 and Chk2 expression of 12 human high-grade serous ovarian tumours at upfront treatment. Statistical analysis by linear regression

Article Snippet: Transient induction of differential PDL1 expression into PDL1 KO ES2 cells by was by transfecting empty vector (Addgene 72299) or human Flag-PDL1 (Addgene 121446) expression plasmids.

Techniques: Western Blot, Control, Transfection, Plasmid Preparation, Incubation, Expressing

Journal: Molecular Cancer

Article Title: Tumour-intrinsic PDL1 signals regulate the Chk2 DNA damage response in cancer cells and mediate resistance to Chk1 inhibitors

doi: 10.1186/s12943-024-02147-z

Figure Lengend Snippet: Tumour PDL1 deficiency promotes Chk2-dependent synthetic lethality to Chk1i and ATRi in vitro. a . In vitro cell viability of CTRL versus PDL1 KO B16, T24, and ID8agg cells or CTRL versus PDL1 lo 4T1 and MDA-MB-231 cells incubated with the selective Chk1i rabusertib for 5 days at indicated doses (mean ± SD, n = 3 independent replicates, P values by two-way ANOVA). Viability normalised to vehicle controls. b . Flow cytometry analysis of Annexin/PI-stained CTRL versus PDL1 KO T24 cells and c . CTRL versus PDL1 lo 4T1 cells after treatment with vehicle (DMSO) or 1 µM rabusertib. Q1 (% dead cells) quantified. P, two-way ANOVA. d . Clonogenic crystal violet stain of PDL1 KO T24 or PDL1 lo 4T1 and respective CTRL cells treated with indicated rabusertib (Chk1i) concentrations for 5 days. e . Cell viability of indicated CTRL versus PDL1-deficient cell lines after ATRi (AZD6738) or f . ATMi (AZD0156) incubation. P , two-way ANOVA. g . IC 50 for the Chk1i prexasertib and h . ATMi AZD0156 in 44 human SCLC-A cell lines segregated by low (lo) or high (hi) PDL1 expression. P, two-sided t -test. Grubb’s test was used to identify outliers. i . Immunoblot after Chk2 re-expression plasmids (Chk2 RE ) or empty vector controls were transiently introduced into CTRL or PDL1 lo 4T1 cells and treated with rabusertib (Chk1i, 2.5 µM) or vehicle (DMSO) for 72 h. j . PDL1 lo 4T1 viability with transient Chk2 RE as in i , treated with 2.5 µM rabusertib for 5 days in vitro versus CTRL and PDL1 lo with empty vector. Data are mean ± SD normalised to vehicle. P, unpaired t -test

Article Snippet: Transient induction of differential PDL1 expression into PDL1 KO ES2 cells by was by transfecting empty vector (Addgene 72299) or human Flag-PDL1 (Addgene 121446) expression plasmids.

Techniques: In Vitro, Incubation, Flow Cytometry, Staining, Expressing, Western Blot, Plasmid Preparation

Journal: Molecular Cancer

Article Title: Tumour-intrinsic PDL1 signals regulate the Chk2 DNA damage response in cancer cells and mediate resistance to Chk1 inhibitors

doi: 10.1186/s12943-024-02147-z

Figure Lengend Snippet: Tumour PDL1 depletion promotes Chk1i synthetic lethality in vivo that requires adaptive immunity in distinct models. Growth in mammary fat pad of a . CTRL and b . PDL1 lo 4T1 tumours in immune deficient NSG mice treated with vehicle or rabusertib (Chk1i). Data represented as mean ± SEM. P by two-way ANOVA. c. Tumour end-point weights from a and b . P, unpaired t -test. d-e . Tumour growth curves of NSG mice challenged with d . CTRL and e . PDL1 KO B16 cells and treated with rabusertib daily. P , two-way ANOVA. f . NSG mice bearing B16 tumours as in d-e were sacrificed day 21 post-challenge and tumours were weighed. P , unpaired t -test. g - h . Growth of subcutaneous g . CTRL versus h . PDL1 KO B16 tumours in WT mice treated with vehicle or rabusertib. Data represented as mean ± SEM. P, two-way ANOVA. i . Tumour end-point weights from g and h . P, unpaired t- test. j - l . Flow cytometry immune analyses of indicated populations in CTRL versus PDL1 KO B16 tumours treated with vehicle or rabusertib in vivo. Mice were sacrificed day 17 post-challenge. P, unpaired t -test. m . Tumour growth curves of Rag2 KO mice bearing PDL1 KO B16 tumours treated with vehicle or rabusertib daily. P , two-way ANOVA. n . Rag2 KO mice as in m were sacrificed day 20 post-challenge. P , unpaired t -test

Article Snippet: Transient induction of differential PDL1 expression into PDL1 KO ES2 cells by was by transfecting empty vector (Addgene 72299) or human Flag-PDL1 (Addgene 121446) expression plasmids.

Techniques: In Vivo, Flow Cytometry

Journal: Molecular Cancer

Article Title: Tumour-intrinsic PDL1 signals regulate the Chk2 DNA damage response in cancer cells and mediate resistance to Chk1 inhibitors

doi: 10.1186/s12943-024-02147-z

Figure Lengend Snippet: Autochthonous PDL1-deficient tumour cells phenocopy genetic PDL1 silencing by inducing Chk2 deficiency and Chk1i synthetic lethality in vitro and in vivo. a . Immunoblot validation of PDL1 expression in NRAS Q61R -mutant melanoma cells derived from CTRL TN 61R (NCH1; PDL1 + ) or PDL1 − TN 61R (NFH1; PDL1 − ) mice. Cells were treated in vitro with 0.1 ng/mL IFNγ for 48 h. b . Immunoblot of NCH1 (PDL1 + ) or NFH1 (PDL1 − ) cells treated in vitro with the Chk1i rabusertib or vehicle (veh) for indicated proteins. c-d . Cell viability of PDL1 + NCH1 versus PDL1 − NFH1 melanoma cells treated with the Chk1i c . rabusertib or d . prexasertib for 72 h in vitro. Viability normalised to vehicle. P, two-way ANOVA. NSG mice were challenged with e. PDL1 + NCH1 cells or f . PDL1 − NFH1 cells and treated with rabusertib daily. P , two-way ANOVA. g . Tumour-bearing NSG mice were sacrificed day 18 post-challenge. Tumours were excised and weighed. P , unpaired t -test. WT mice were challenged with h . NCH1 or i . NFH1 cells and treated with vehicle or rabusertib daily. P , two-way ANOVA. j . Tumour-bearing WT mice were sacrificed, and tumours were excised and weighed. P , unpaired t -test. k - m . WT mice bearing NCH1 or NFH1 tumours were sacrificed day 28 post-challenge and profiled for indicated immune populations by flow cytometry. MFI, mean fluorescence intensity. P , unpaired t -test

Article Snippet: Transient induction of differential PDL1 expression into PDL1 KO ES2 cells by was by transfecting empty vector (Addgene 72299) or human Flag-PDL1 (Addgene 121446) expression plasmids.

Techniques: In Vitro, In Vivo, Western Blot, Expressing, Mutagenesis, Derivative Assay, Flow Cytometry, Fluorescence

Journal: Molecular Cancer

Article Title: Tumour-intrinsic PDL1 signals regulate the Chk2 DNA damage response in cancer cells and mediate resistance to Chk1 inhibitors

doi: 10.1186/s12943-024-02147-z

Figure Lengend Snippet: Tumour PDL1 control of Chk2 is through intracellular PDL1 and independent of PD1 and immunity. a . Immunoblots of PDL1 KO and PD1 KO B16 cells versus CTRL B16 cells for indicated proteins. Vinculin (VINC) loading control. b . In vitro MTT cell viability of CTRL T24 cells treated with the αPD1 antibodies pembrolizumab (50 µg/mL), balstilimab (50 µg/mL), or genetic shRNA PD1 knockdown (PD1 lo ) with increasing doses of the Chk1i rabusertib for 5 days at indicated doses. P by two-way ANOVA. Viability shown as relative values following normalisation to respective isotype or vehicle-treated controls. c . Schematic of membrane (mem)-localising versus intracellular (cyto)-localising PDL1 expression constructs (see methods). d . RNA-seq analyses of PDL1 mem versus PDL1 cyto B16 cells using gene set enrichment analysis. Top 10 altered hallmark pathways ( p < 0.01) are shown by PDL1 subcellular location. e . Immunoblot comparing ATM, Chk2, γH2AX, and vimentin expression of indicated B16 cells. Vinculin (VINC) as loading control. f . MTT viability assay of PDL1 mem versus PDL1 cyto cells after 96-h of in vitro treatment with the Chk1i rabusertib at indicated concentrations. P, two-way ANOVA. g . Viability analysis of CTRL versus PDL1 KO T24 cells treated with indicated rabusertib concentrations ± 1 ng/mL IFNγ, 10 ng/mL TNFα, or vehicle. P, two-way ANOVA. h . Immunoblot of cell lysates of membrane and cytosolic fractions of CTRL T24 cells treated with vehicle (PBS), IFNγ, or TNFα as in g . Na + /K + pump as membrane loading control and GAPDH as cytosolic loading control.

Article Snippet: Transient induction of differential PDL1 expression into PDL1 KO ES2 cells by was by transfecting empty vector (Addgene 72299) or human Flag-PDL1 (Addgene 121446) expression plasmids.

Techniques: Control, Western Blot, In Vitro, shRNA, Knockdown, Membrane, Expressing, Construct, RNA Sequencing Assay, MTT Viability Assay

Journal: Molecular Cancer

Article Title: Tumour-intrinsic PDL1 signals regulate the Chk2 DNA damage response in cancer cells and mediate resistance to Chk1 inhibitors

doi: 10.1186/s12943-024-02147-z

Figure Lengend Snippet: PDL1 protects Chk2 from ubiquitination in distinct tumours. a . Volcano plot of RNA-seq data depicting PDL1 KO effect on select mRNA levels versus CTRL B16. b . qRT-PCR of Chek2 mRNA in CTRL versus PDL1 KO B16 cells treated with DMSO or rabusertib. P , unpaired t -test. c . Immunoblot of CTRL versus PDL1 KO B16 cells treated with cycloheximide (CHX). Summary graph depicts normalised total Chk2 protein levels quantified using ImageJ. d . Immunoblot of CTRL versus PDL1 KO B16 and e . ES2 cells treated with MG132 (2 µM) for 12-h. f. Immunoblot of immunoprecipitated Chk2 from CTRL, PDL1 KO , and stable PDL1 knock-in (PDL1 KI ) B16 cells and g . CTRL and PDL1 KO ES2 cells after MG132 treatment. h . CTRL and PDL1 KO T24 cells treated with MG132 for 16-h plus vehicle (H 2 O) or 10 ng/mL gemcitabine (gem). Endogenous Chk2 was immunoprecipitated and Chk2 and ubiquitination (Ub) were detected by immunoblot

Article Snippet: Transient induction of differential PDL1 expression into PDL1 KO ES2 cells by was by transfecting empty vector (Addgene 72299) or human Flag-PDL1 (Addgene 121446) expression plasmids.

Techniques: RNA Sequencing Assay, Quantitative RT-PCR, Western Blot, Immunoprecipitation, Knock-In

Journal: Molecular Cancer

Article Title: Tumour-intrinsic PDL1 signals regulate the Chk2 DNA damage response in cancer cells and mediate resistance to Chk1 inhibitors

doi: 10.1186/s12943-024-02147-z

Figure Lengend Snippet: Tumour PDL1 protects Chk2 from PIRH2 E3 ligase-mediated degradation in distinct tumours through its immunoglobulin-like domains. a . Non-targeting or PIRH2-specific siRNA vectors transfected into CTRL or PDL1 KO B16 cells or b . PDL1 + NCH1 cells and PDL1 − NFH1 cells. c . Interaction among PDL1, Chk2 and PIRH2 in B16 cells stably expressing vector control or turbo-GFP (tGFP)-tagged PDL1 (PDL1 tGFP ) treated with MG132 and co-immunoprecipitated with tGFP trap beads followed by immunoblotting. d . Endogenous PIRH2 co-immunoprecipitated in CTRL, PDL1 KO , and PDL1 KI B16 cells. e . Left: Flag-tagged human PDL1 and Streptavidin-tagged human Chk2 were co-expressed in Tni cells and affinity pull-downs of lysates with Flag resin and eluate fractions (1,2,3,4) followed by SDS-PAGE and Coomassie blue staining. Right: eluates were pooled for reciprocal Chk2 affinity pulldown using Strep-tactin resin. L, lysates; FT, flow-through; I, input. f . Viability of PDL1 truncation mutant-expressing B16 cells 72-h post rabusertib (2.5 μM) treatment. P, unpaired t -test. g . Immunoblot of domain-expressing B16 cells immunoprecipitated using anti-mCherry beads

Article Snippet: Transient induction of differential PDL1 expression into PDL1 KO ES2 cells by was by transfecting empty vector (Addgene 72299) or human Flag-PDL1 (Addgene 121446) expression plasmids.

Techniques: Transfection, Stable Transfection, Expressing, Plasmid Preparation, Control, Immunoprecipitation, Western Blot, SDS Page, Staining, Mutagenesis

Journal: Molecular Cancer

Article Title: Tumour-intrinsic PDL1 signals regulate the Chk2 DNA damage response in cancer cells and mediate resistance to Chk1 inhibitors

doi: 10.1186/s12943-024-02147-z

Figure Lengend Snippet: Pharmacologic tumour PDL1 depletion replicates genetic tumour PDL1 KO by depleting Chk2 and inducing Chk1i synthetic lethality. a . Immunoblot of CTRL T24 cells for indicated proteins after 72 h of treatment with vehicle (DMSO), 1 µM rabusertib (Chk1i), 80 μM cefepime, or combination. b . Immunoblot of CTRL, PDL1 KO , and stable PDL1 KI B16 cells treated with 80 μM cefepime for indicated time points. c-e . Cell viability of c. CTRL MDA-MB-231, d . CTRL T24, and e. PDL1 KO T24 cells treated with indicated concentrations of rabusertib ± 80 μM cefepime or vehicle (DMSO) for 96 h. P, two-way ANOVA. f . Immunoblot of CTRL T24 cells treated with vehicle, 1 μM rabusertib, 80 μM ceftazidime, or both for 72 h. g . Immunoblot of CTRL, PDL1 KO and stable PDL1 KI B16 cells treated with 80 μM ceftazidime for indicated time points. h - j . Cell viability of h . CTRL MDA-MB-231, i . CTRL T24, and j . PDL1 KO T24 cells treated with indicated concentrations of rabusertib ± 80 μM ceftazidime or vehicle for 96 h. P, two-way ANOVA. k . MTT viability analysis of CTRL T24 cells treated for 96 h with 80 μM of indicated β-lactams with rabusertib. P, two-way ANOVA. l . Growth curves of PDL1 + NCH1 tumours implanted subcutaneously in WT mice treated with vehicle, ceftazidime, rabusertib, or both. P, two-way ANOVA. m . Tumour weights of NCH1 tumours from l . at endpoint (day 18). P, unpaired t -test. Vehicle (veh), ceftazidime (ctz), rabusertib (rab), and ceftazidime + rabusertib (combo) groups are indicated

Article Snippet: Transient induction of differential PDL1 expression into PDL1 KO ES2 cells by was by transfecting empty vector (Addgene 72299) or human Flag-PDL1 (Addgene 121446) expression plasmids.

Techniques: Western Blot