Journal: Nucleic Acids Research

Article Title: MRNIP limits ssDNA gaps during replication stress

doi: 10.1093/nar/gkae546

Figure Lengend Snippet: Impaired fork reversal drives MRE11-dependent post-replicative ssDNA gaps in MRNIP KO cells. ( A ) Top: DNA fibre assay schematic. WT and MRNIP KO HeLa cells were labelled with CldU for 20 min, then labelled with IdU for 60 min in the presence of PBS or 150 μM cisplatin. Cells were then treated −/+ S1 nuclease and DNA fibre assays were performed. IdU tract length was measured using ImageJ. ( B ) Top: Schematic of DNA fibre assay. WT and MRNIP KO HeLa cells were treated with 1 μM Olaparib, then nascent DNA was labelled with CldU and IdU −/+S1 nuclease. ( C ) Top: Schematic of DNA fibre assay. WT and MRNIP KO HeLa cells were treated with DMSO, 1 μM Olaparib, 150 μM cisplatin, or 1 μM Olaparib + 150 μM cisplatin, then nascent DNA was labelled with CldU and IdU −/+S1 nuclease. ( D ) Representative examples of DNA fibres from Olaparib + cisplatin-treated WT and MRNIP KO cells. (E, F) WT and MRNIP KO HeLa cells were transfected with a non-targeting control siRNA, or validated siRNAs targeting either SMARCAL1 or ZRANB3. After 48 h, cells were lysed and SMARCAL1 depletion was confirmed by Western blotting ( E ). RNA was isolated and ZRANB3 mRNA levels were quantified by qRT-PCR and normalized to GAPDH ( F ). ( G ) Top: Schematic of DNA fibre assay. WT and MRNIP KO HeLa cells were transfected with a non-targeting control siRNA, or siRNAs targeting SMARCAL1 or ZRANB3. After 48 h, nascent DNA was labelled with CldU, then IdU and cisplatin −/+ S1 nuclease. ( H ) Top: Schematic of DNA fibre assay. WT and MRNIP KO HeLa cells were treated with 1 μM Olaparib and either DMSO or 25 μM PFM39, then nascent DNA was labelled with CldU, followed by IdU labelling in the presence of 100 μM cisplatin −/+ S1 nuclease. Experiments were performed three times independently and were confirmed by at least two members of the laboratory. Results are displayed as dot plots with indicated median values and analysed via one-way ANOVA. ** P < 0.01, **** P < 0.0001

Article Snippet: The following antibodies were used: Western blotting: phospho-CHK1 (133D3, Cell Signalling, 1:1000), CHK1 (Cell Signalling, 2G1D5, 1:1000), MRE11 (Abcam, ab214, 1:1000), PRIMPOL (Proteintech: 29824–1-AP, 1:1000), RAD18 (Cell Signalling, D2B8, 1:1000), UBC13 Antibody (Santa Cruz, F-10, 1:1000), REV1 Antibody (Santa Cruz, A-11, 1:1000), GAPDH Antibody (Santa Cruz, G-9, 1:2000), MRNIP Antibody (Santa Cruz, H-11, 1:500), RPA2 (Abcam 9H8, ab2175, 1:1000), Tubulin (Abcam, ab4074), SMARCAL1 (Santa Cruz Biotechnology, sc-376377), γH2AX (Cell Signaling Technology, 9718), PRIMPOL (gift from Mendez laboratory, 1:1000).

Techniques: Transfection, Control, Western Blot, Isolation, Quantitative RT-PCR

Journal: Nucleic Acids Research

Article Title: MRNIP limits ssDNA gaps during replication stress

doi: 10.1093/nar/gkae546

Figure Lengend Snippet: Fork reversal and REV1 limit ssDNA gaps and DNA damage markers in MRNIP KO cells. ( A ) Top: Schematic of DNA fibre assay. WT and MRNIP KO HeLa cells were transfected with a non-targeting control siRNA, or siRNAs targeting either SMARCAL1 or ZRANB3. After 48 h, cells were treated with 1 μM JH-RE-06 (REV1 inhibitor), then nascent DNA was labelled with CldU for 20 min, followed by IdU for 60 min. ssDNA was then digested with S1 nuclease. IdU tract length was measured using ImageJ. IdU tract length was measured using ImageJ. (B–D) WT and MRNIP KO cells were transfected with a control siRNA, or an siRNA targeting SMARCAL1. Forty-eight hours later, cells were treated with either DMSO or 1 μM JH-RE-06 for 16 hrs, then fixed and stained with antibodies against γH2AX ( B ) and 53BP1 ( C ), and counterstained with DAPI. Cells with more than 5 foci were counted and results plotted as a percentage of the total number of cells. Representative images are displayed in ( D ). All experiments were performed three times independently and analysed via one-way ANOVA. * P < 0.05, **** P < 0.0001.

Article Snippet: The following antibodies were used: Western blotting: phospho-CHK1 (133D3, Cell Signalling, 1:1000), CHK1 (Cell Signalling, 2G1D5, 1:1000), MRE11 (Abcam, ab214, 1:1000), PRIMPOL (Proteintech: 29824–1-AP, 1:1000), RAD18 (Cell Signalling, D2B8, 1:1000), UBC13 Antibody (Santa Cruz, F-10, 1:1000), REV1 Antibody (Santa Cruz, A-11, 1:1000), GAPDH Antibody (Santa Cruz, G-9, 1:2000), MRNIP Antibody (Santa Cruz, H-11, 1:500), RPA2 (Abcam 9H8, ab2175, 1:1000), Tubulin (Abcam, ab4074), SMARCAL1 (Santa Cruz Biotechnology, sc-376377), γH2AX (Cell Signaling Technology, 9718), PRIMPOL (gift from Mendez laboratory, 1:1000).

Techniques: Transfection, Control, Staining

Journal: Discover Oncology

Article Title: Identifying and validating SMARCAL1 as a prognostic and immunotherapy predictive biomarker for NSCLC

doi: 10.1007/s12672-025-03980-4

Figure Lengend Snippet: SMARCAL1 pathological phases and aberrant expression levels in pan-cancer. A . An examination of SMARCAL1 mRNA expression across 26 different tumor types in comparison to normal tissues. B . Utilizing data from both the TCGA and GTEx databases to ascertain the mRNA expression levels of SMARCAL1. C. CPTAC demonstrated the differences in SMARCAL1 total protein levels across ccRCC, HCC, LUSC, GBM, UCEC, and normal tissues. D . The expression of SMARCAL1 protein in COAD, breast duct carcinoma, PRAD, LUAD, and liver cholangiocarcinoma

Article Snippet: After 20 min of 5% BSA blocking, sections were treated with primary antibodies. (Mouse monoclonal SMARCAL1 antibody, 1:200, Novus, USA) at 4°C overnight.

Techniques: Expressing, Comparison

Journal: Discover Oncology

Article Title: Identifying and validating SMARCAL1 as a prognostic and immunotherapy predictive biomarker for NSCLC

doi: 10.1007/s12672-025-03980-4

Figure Lengend Snippet: Sangerbox analysis of cancer prognosis capacity of SMARCAL1 on TCGA samples. A . Overall survival. B . Disease-specific survival. C . Disease-free interval. D . Progression-free interval

Article Snippet: After 20 min of 5% BSA blocking, sections were treated with primary antibodies. (Mouse monoclonal SMARCAL1 antibody, 1:200, Novus, USA) at 4°C overnight.

Techniques:

Journal: Discover Oncology

Article Title: Identifying and validating SMARCAL1 as a prognostic and immunotherapy predictive biomarker for NSCLC

doi: 10.1007/s12672-025-03980-4

Figure Lengend Snippet: Enrichment analysis SMARCAL1-associated genes. A Protein–Protein Interaction network of the top 20 co-expressed genes of SMARCAL1 in the GEPIA database. B KEGG pathway analysis. C GO analysis of SMARCAL1

Article Snippet: After 20 min of 5% BSA blocking, sections were treated with primary antibodies. (Mouse monoclonal SMARCAL1 antibody, 1:200, Novus, USA) at 4°C overnight.

Techniques:

Journal: Discover Oncology

Article Title: Identifying and validating SMARCAL1 as a prognostic and immunotherapy predictive biomarker for NSCLC

doi: 10.1007/s12672-025-03980-4

Figure Lengend Snippet: Analysis of SMARCAL1 with the correlation of heterogeneity, genomic instability, mutation profiles, and methylation levels. A – D . Lollipop picture of the correlation between SMARCAL1 expression and TMB, MSI, MATH, and purity

Article Snippet: After 20 min of 5% BSA blocking, sections were treated with primary antibodies. (Mouse monoclonal SMARCAL1 antibody, 1:200, Novus, USA) at 4°C overnight.

Techniques: Mutagenesis, Methylation, Expressing

Journal: Discover Oncology

Article Title: Identifying and validating SMARCAL1 as a prognostic and immunotherapy predictive biomarker for NSCLC

doi: 10.1007/s12672-025-03980-4

Figure Lengend Snippet: Relevance analysis between two immune cell types and SMARCAL1. A Macrophage M2. B Regulatory T cells

Article Snippet: After 20 min of 5% BSA blocking, sections were treated with primary antibodies. (Mouse monoclonal SMARCAL1 antibody, 1:200, Novus, USA) at 4°C overnight.

Techniques:

Journal: Discover Oncology

Article Title: Identifying and validating SMARCAL1 as a prognostic and immunotherapy predictive biomarker for NSCLC

doi: 10.1007/s12672-025-03980-4

Figure Lengend Snippet: Analysis of the relationship between antitumor immunity and SMARCAL1 A Using GBM, SKCM, and KIRC cohorts, Kaplan–Meier plots show how OS differs between SMARCAL1 high and low expression groups following ICB treatment. B Comparison of SMARCAL1’s predictive power for ICB therapy with that of standardized biomarkers. C Correlation between SMARCAL1 and CTL. D The effect of SMARCAL1 expression levels on overall survival in cancer patients is demonstrated using a Kaplan–Meier survival study. E – G . Kaplan–Meier survival analysis illustrating the effect of SMARCAL1 expression levels on overall survival in cancer patients undergoing anti-PD-1, anti-PD-L1, and anti-CTLA-4 therapy, respectively

Article Snippet: After 20 min of 5% BSA blocking, sections were treated with primary antibodies. (Mouse monoclonal SMARCAL1 antibody, 1:200, Novus, USA) at 4°C overnight.

Techniques: Expressing, Comparison

Journal: Discover Oncology

Article Title: Identifying and validating SMARCAL1 as a prognostic and immunotherapy predictive biomarker for NSCLC

doi: 10.1007/s12672-025-03980-4

Figure Lengend Snippet: An investigation into the responsiveness of SMARCAL1 to chemotherapeutic agents and its predictive value for drug sensitivity. A The left panel’s box plot illustrates the association between SMARCAL1 expression and chemotherapy response, whereas the ROC curve on the right, sourced from the online ROC plotter resource, indicates the predictive accuracy of SMARCAL1 levels for chemotherapy responsiveness. B The study reveals that SMARCAL1 expression correlates with the chemosensitivity to fludarabine and methylprednisolone

Article Snippet: After 20 min of 5% BSA blocking, sections were treated with primary antibodies. (Mouse monoclonal SMARCAL1 antibody, 1:200, Novus, USA) at 4°C overnight.

Techniques: Expressing

Journal: Discover Oncology

Article Title: Identifying and validating SMARCAL1 as a prognostic and immunotherapy predictive biomarker for NSCLC

doi: 10.1007/s12672-025-03980-4

Figure Lengend Snippet: Expression of SMARCAL1 mRNA and protein in non-small cell lung cancer (NSCLC). A Relative expression of SMARCAL1 in Normal and LUAD tissues. B qRT-PCR examination of the relative expression of SMARCAL1 mRNA in normal and NSCLC cell lines. C The protein level of SMARCAL1, as determined by Western blot. D – E . Immunohistochemical analysis detected the presence of SMARCAL1 protein in both corresponding normal and lung adenocarcinoma (LUAD) samples. **** p < 0.001

Article Snippet: After 20 min of 5% BSA blocking, sections were treated with primary antibodies. (Mouse monoclonal SMARCAL1 antibody, 1:200, Novus, USA) at 4°C overnight.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Immunohistochemical staining

Journal: Discover Oncology

Article Title: Identifying and validating SMARCAL1 as a prognostic and immunotherapy predictive biomarker for NSCLC

doi: 10.1007/s12672-025-03980-4

Figure Lengend Snippet: Expression of SMARCAL1 mRNA and protein in LUAD, NSCLC. A SMARCAL1 expression levels were obtained from the GEO database for normal, LUAD, and NSCLC tissues. B The ROC curve of SMARCAL1 in three databases. C – E . GSEA enrichment analysis of SMARCAL1 in NSCLC based on GSE31547 , GSE40791 , and GSE19804

Article Snippet: After 20 min of 5% BSA blocking, sections were treated with primary antibodies. (Mouse monoclonal SMARCAL1 antibody, 1:200, Novus, USA) at 4°C overnight.

Techniques: Expressing

Journal: Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc

Article Title: SMARCAL1 loss and alternative lengthening of telomeres (ALT) are enriched in giant cell glioblastoma.

doi: 10.1038/s41379-021-00841-7

Figure Lengend Snippet: Fig. 2 Immunohistochemistry to detect SMARCAL1. IHC for SMARCAL1 was performed on U87 cell line variants, either SMARCAL1 wild-type (A) or SMARCAL1 knockout (B). C Example of giant cell GBM lacking SMARCAL1 protein expression in the cancer cells as revealed by IHC. For all panels, original magnification ×400.

Article Snippet: For SMARCAL1, slides were steamed in EDTA buffer (Cat# AM9849, Invitrogen, Carlsbad, CA) for 45 min, blocked twice (Cat# S2003 and X0909, Dako, Denmark), then were incubated for 2 h at room temperature in primary antibody against SMARCAL1 (1:100 dilution, Cat# HPA020337, Atlas Antibodies, Bromma, Sweden).

Techniques: Immunohistochemistry, Knock-Out, Expressing

Journal: Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc

Article Title: SMARCAL1 loss and alternative lengthening of telomeres (ALT) are enriched in giant cell glioblastoma.

doi: 10.1038/s41379-021-00841-7

Figure Lengend Snippet: Fig. 4 Association between overall survival in classic giant cell glio- blastomas and glioblastomas with giant cell features based on mole- cular features. Kaplan–Meier curves and log rank tests demonstrate that there is not significant difference in overall survival based on A ATRX status (p = 0.08) or B SMARCAL1 status (p = 0.09); however, C there was a significantly better overall survival in patients with ALT-positive tumors compared to patients with ALT-negative tumors (p < 0.03).

Article Snippet: For SMARCAL1, slides were steamed in EDTA buffer (Cat# AM9849, Invitrogen, Carlsbad, CA) for 45 min, blocked twice (Cat# S2003 and X0909, Dako, Denmark), then were incubated for 2 h at room temperature in primary antibody against SMARCAL1 (1:100 dilution, Cat# HPA020337, Atlas Antibodies, Bromma, Sweden).

Techniques: