Journal: iScience

Article Title: Irradiation combined with PD-L1 −/− and autophagy inhibition enhances the antitumor effect of lung cancer via cGAS-STING-mediated T cell activation

doi: 10.1016/j.isci.2022.104690

Figure Lengend Snippet:

Article Snippet: Rabbit Polyclonal Anti-α-Tubulin , Beyotime , Cat # AF0001.

Techniques: Purification, Recombinant, Red Blood Cell Lysis, Staining, Cell Stimulation, Transfection, cDNA Synthesis, SYBR Green Assay, Protein Extraction, Software, Modification

Journal: PLoS ONE

Article Title: Restriction Endonucleases from Invasive Neisseria gonorrhoeae Cause Double-Strand Breaks and Distort Mitosis in Epithelial Cells during Infection

doi: 10.1371/journal.pone.0114208

Figure Lengend Snippet: A. VK2/E6E7 cells were synchronized and bacteria were allowed to adhere and invade cells for 24 h. Cells were then fixed and stained with DAPI. The nuclear area of 300 infected and 300 uninfected cells were measured. Data were analyzed using a paired 2-tailed Student's t -test. The average nuclear areas observed in 3 independent experiments are shown (* p <0.05). Error bars represent S.E.M. B. Cells were infected with N. gonorrhoeae and observed for 24 h by live-cell microscopy. DIC images were captured every 15 min at randomly selected positions, and times spent transitioning from prophase to cytokinesis were measured for 150 mitotic cells. The graph shows mean ± standard deviation values for the time required for progression from prophase to cytokinesis, measured in 3 independent experiments. C. VK2/E6E7 cells were infected with N. gonorrhoeae for 24 h. RNA from control cells and infected cells was isolated and qPCR was performed. Average mRNA levels of MAD1L1 and MAD2L1 from 3 independent experiments are shown (* p <0.05). Error bars represent S.E.M. Values were normalized against TUBA1A expression and analyzed using a paired 2-tailed Student's t -test. D. Protein expression in infected cells and control cells was analyzed in western blots using antibodies against of MAD1 and MAD2. GAPDH or α-tubulin was used as loading controls. E. Western blot band intensities were quantified and analyzed with a paired 2-tailed Student's t -test, using GAPDH or α-tubulin expression as controls. Mean expression levels of each protein observed in 3 independent western blots are shown (* p <0.05). Error bars represent S.E.M.

Article Snippet: Polyclonal antibodies against α-tubulin (MBS316320, MyBioSource, 1∶1,000) or GAPDH (G-9545, Sigma-Aldrich, 1∶5,000) were used for normalizing total protein loaded in each well.

Techniques: Bacteria, Staining, Infection, Microscopy, Standard Deviation, Control, Isolation, Expressing, Western Blot

Journal: Oncotarget

Article Title: Impact of Polo-like kinase 1 inhibitors on human adipose tissue-derived mesenchymal stem cells

doi: 10.18632/oncotarget.12482

Figure Lengend Snippet: ( A and B ) ASCs were treated with indicated Plk1 inhibitors (50 nM BI 2536, BI 6727 or 50 μM Poloxin) for 48 h and cell cycle analyses were performed for visceral and subcutaneous ASCs. The results are based on three independent experiments, presented as mean ± SEM and statistically analyzed compared to DMSO treated cells. ** p < 0.01. (C and E) ASCs were treated as in (A and B) and stained for α-tubulin, pericentrin, ACA (anti-centromere antibody) and DNA for immunofluorescence microscopy. Mitotic arrested ASCs were evaluated. Representatives are shown for visceral ASCs ( C ) and subcutaneous ASCs ( E ). White arrows indicate mitotic cells; red arrows indicate fragmented cell nuclei. Scale: 25 μm. ( D and F ) Quantification of mitotic cells ( n = 150 analyzed cells for each condition). The results are from three independent experiments and presented as mean ± SEM. ** p < 0.01, *** p < 0.001. ( G and H ) Cellular extracts from treated ASCs were prepared for Western blot analyses with indicated antibodies. β-actin served as loading and DMSO treated cells as vehicle control.

Article Snippet: The following primary antibodies were used: rat polyclonal antibody against α-tubulin (Biozol, Eching), rabbit polyclonal antibodies against pericentrin, human monoclonal antibody against ACA (anti-centromere antibody) (ImmunoVision, Springdale), mouse monoclonal anti-phospho-histone γ-H2AX (Ser139) (Merck Millipore, Darmstadt) and polyclonal rabbit antibodies against 53BP1 (Novus, Cambridge, UK).

Techniques: Staining, Immunofluorescence, Microscopy, Western Blot, Control

Journal: Oncotarget

Article Title: Impact of Polo-like kinase 1 inhibitors on human adipose tissue-derived mesenchymal stem cells

doi: 10.18632/oncotarget.12482

Figure Lengend Snippet: ASCs were subjected to Plk1 compounds (25 nM BI 2536, 25 nM BI 6727 or 25 μM Poloxin) for 4 days and 14 days. Media and compounds were renewed every 3 days. (A and B) To evaluate the induction of DNA damage, treated cells were stained for the DNA damage markers γ-H2AX and 53BP1, α-tubulin and DNA. Representatives are shown. White arrows indicate fragmented cell nuclei and red arrows depict abnormal enlarged cell nuclei. Scale: 25 μm. Insets are a four time magnification of boxed regions in leftist panels. Scale: 6.5 μm. ( C and D ) Quantification of γ-H2AX and 53BP1 double positive cells. The results are based on three independent experiments and presented as mean ± SEM. ** p < 0.01, *** p < 0.001.

Article Snippet: The following primary antibodies were used: rat polyclonal antibody against α-tubulin (Biozol, Eching), rabbit polyclonal antibodies against pericentrin, human monoclonal antibody against ACA (anti-centromere antibody) (ImmunoVision, Springdale), mouse monoclonal anti-phospho-histone γ-H2AX (Ser139) (Merck Millipore, Darmstadt) and polyclonal rabbit antibodies against 53BP1 (Novus, Cambridge, UK).

Techniques: Staining

Journal: Oncotarget

Article Title: Impact of Polo-like kinase 1 inhibitors on human adipose tissue-derived mesenchymal stem cells

doi: 10.18632/oncotarget.12482

Figure Lengend Snippet: ASCs were treated with Plk1 inhibitors (50 nM BI 2536 or 50 nM BI 6727) for 9 h and released into fresh media. Treated ASCs and breast cancer cells were seeded in separated chambers of an culture insert and cultured for 0 h, 8 h and 15 h. Cells were then stained for microtubule marker acetylated α-tubulin, actin marker phalloidin and DNA for analyzing the number of membrane protrusions, including lamellipodia and filopodia, on the migration front of ASCs toward breast cancer cells. ( A ) Evaluation of protrusions in subcutaneous ASCs toward MDA-MB-231 and MCF-7 cells. Each experiment was performed in triplicate, and the results are from three independent experiments and shown as mean ± SEM. ** p < 0.01. ( B ) Representatives are depicted. Left side: subcutaneous ASCs; right side: breast cancer cells. Scale: 25 μm. ( C ) Evaluation of protrusions in visceral ASCs toward MDA-MB-231 and MCF-7 cells. The results are from three independent experiments and shown as mean ± SEM. ( D ) Representatives are shown. Left side: visceral ASCs; right side: breast cancer cells. Scale: 25 μm.

Article Snippet: The following primary antibodies were used: rat polyclonal antibody against α-tubulin (Biozol, Eching), rabbit polyclonal antibodies against pericentrin, human monoclonal antibody against ACA (anti-centromere antibody) (ImmunoVision, Springdale), mouse monoclonal anti-phospho-histone γ-H2AX (Ser139) (Merck Millipore, Darmstadt) and polyclonal rabbit antibodies against 53BP1 (Novus, Cambridge, UK).

Techniques: Cell Culture, Staining, Marker, Membrane, Migration

Journal: PLoS ONE

Article Title: Acetaminophen Induces Apoptosis in Rat Cortical Neurons

doi: 10.1371/journal.pone.0015360

Figure Lengend Snippet: ( A ) Left. Cytochrome c (Cyt c) release from mitochondria to cytosol 24 h after AAP-treatment. Cells were treated with vehicle (DMSO 1‰; V) or AAP at 0.5 (A0.5), 1(A1) or 2 (A2) mM for 24 h and the cytosolic fraction was obtained. α-Tubulin (α-tub) was used as cytosolic protein loading control. Right. Densitometric analysis of Cyt c related to α-tubulin (α-tub.) protein levels detected in cytosolic fraction. Data are expressed as mean ± SEM of 3 independent experiments. ***p<0.001 as compared to vehicle-treated cells. ( B ) Left. Same experiment as in ( A ) , but Cyt c mitochondrial content was determined. COX-IV was used as mitochondrial protein loading control. Right. Densitometric analysis of Cyt c related to COX-IV protein levels detected in mitochondrial fraction. Data are expressed as mean ± SEM of 3 experiments. ***p<0.001 compared to vehicle-treated cells. ( C ) Time-course of caspase 3 activaty induced by AAP. Cortical neurons were incubated with vehicle (DMSO 1‰; •), AAP 1 mM (□) or AAP 2 mM (▪). After different time periods, cell lysates were obtained and caspase 3 activity determined as indicated in Material and Methods. Data represent mean ± SEM of 12 independent experiments. *p<0.05; **p<0.01; *** p<0.001 as compared to vehicle-treated cells. When not shown, SE bars were smaller than the symbol size.

Article Snippet: Membranes were blocked in PBS-Tween 20 (0.1%) containing 5% non-fat dry milk and 0.1% BSA for 1 h at 4°C and incubated with CYP2E1 polyclonal antibody (1∶1,000) (Abcam, Cambrige, UK), anti-cytochrome c polyclonal antibody (1∶1,000) (BD Biosciences, Madrid, Spain), anti-α-tubulin polyclonal antibody (1∶2,000) (Merck Chemicals Ltd., Barcelona, Spain) or anti-OxPhos Complex IV subunit IV (COX-IV) monoclonal antibody (1∶1,000) (Cell Signalling Technology, Barcelona, Spain) overnight at 4°C.

Techniques: Incubation, Activity Assay

Journal: PLoS ONE

Article Title: Acetaminophen Induces Apoptosis in Rat Cortical Neurons

doi: 10.1371/journal.pone.0015360

Figure Lengend Snippet: ( A ) AAP increases CYP2E1 activity in a concentration-dependent manner in rat cortical neurons. Cells were treated with vehicle (DMSO 1‰; V) or AAP 0.5 (A0.5), 1 (A1) and 2 mM (A2)) for 18 h and total lysates were obtained. CYP2E1 activity was quantified as nmol p-nitrophenol transformed per milligram of protein in total lysates. Data represent the mean ± SEM of 9 independent experiments. *** p < 0.001 as compared to vehicle-treated cells (V). ( B ) AAP increases CYP2E1 protein levels in a time- and concentration-dependent manner. Left. CYP2E1 expression detected in total lysates obtained from cells treated with vehicle (DMSO 1‰; V) or AAP at 1 mM (A1) or 2 mM (A2) for 3, 6 and 18 h. α-Tubulin (α-tub) was used as protein loading control. Right. Densitometric analysis of CYP2E1 related to α-tubulin (α-tub.) protein levels detected in total lysates. Data are expressed as mean ± SEM of 3 independent experiments. *p<0.05; **p<0.01; ***p<0.001 compared to vehicle-treated cells. ( C ) Bongkrekic (BA), by inhibiting mitochondrial permeability transition, prevents AAP-induced CYP2E1 induction. Left. CYP2E1 expression detected in total lysates obtained from cells treated with vehicle (DMSO 1‰; V) or AAP 1 mM () and 2 mM in the absence (A1; A2) and the presence of BA 20 µM (A1BA; A2BA) for 18 h. α-Tubulin (α-tub.) was used as protein loading control. Right. Densitometric analysis of CYP2E1 related to α-tubulin (α-tub.) protein levels detected in total lysates. Data are expressed as mean ± SEM of 3 independent experiments. *p<0.05 as compared to A1-treated cells; ## , p < 0.01 as compared to A2-treated cells.

Article Snippet: Membranes were blocked in PBS-Tween 20 (0.1%) containing 5% non-fat dry milk and 0.1% BSA for 1 h at 4°C and incubated with CYP2E1 polyclonal antibody (1∶1,000) (Abcam, Cambrige, UK), anti-cytochrome c polyclonal antibody (1∶1,000) (BD Biosciences, Madrid, Spain), anti-α-tubulin polyclonal antibody (1∶2,000) (Merck Chemicals Ltd., Barcelona, Spain) or anti-OxPhos Complex IV subunit IV (COX-IV) monoclonal antibody (1∶1,000) (Cell Signalling Technology, Barcelona, Spain) overnight at 4°C.

Techniques: Activity Assay, Concentration Assay, Transformation Assay, Expressing, Permeability

Journal: Molecular Systems Biology

Article Title: Proteome-wide copy-number estimation from transcriptomics

doi: 10.1038/s44320-024-00064-3

Figure Lengend Snippet: ( A ) Gene ontology (GO) enrichments for M genes. The largest non-redundant GO term is shown with the fold enrichment (FE) and false discovery rate-corrected p value ( q ). The complete list of GO enrichments for each relationship class is available in Dataset . ( B ) HL outperforms competing mRNA-to-protein relationships. Models encoding linear, hyperbolic, three-parameter logistic, and HL relationships were built for all genes and compared by Bayesian Information Criterion (BIC). Results are shown as the smoothed density of BIC differences (∆BIC) relative to the best model for that gene (∆BIC = 0). Distributions of BIC weights (Wagenmakers and Farrell, ) are shown in Fig. . ( C , D ) HL captures different empirical classes of mRNA-to-protein relationships. Log-concave genes ( C ) saturate at high mRNA abundance, whereas log-convex genes ( D ) plateau at low mRNA abundance. The remaining genes exhibited characteristics of both fits or linear relationships to varying degrees (Fig. ). ( E , F ) Ratio compression of a log-convex gene. The indicated cell lines were immunoblotted for SERPINB6 along with vinculin and tubulin as loading controls ( E ), and loading-normalized SERPINB6 abundance was quantified relative to the median copy number for these cell lines in the meta-assembly. Observations were compared to the meta-assembly ( F ) assuming direct proportionality (blue). ( G ) Feature weights of HL + LASSO genes are biologically sensible. Smoothed densities of LASSO feature weights (indicating strength and direction of modulation for an HL fit) among mRNAs encoding subunits of the proteasome (blue) and the ribosome (red) are shown. ( H ) HL + LASSO features are highly enriched for STRING interactions. For each HL + LASSO gene, LASSO-selected features were replaced with random genes to build a null distribution for finding binary interactions in STRING (Szklarczyk et al, ). The actual number of STRING interactions among HL + LASSO genes of Pinferna is indicated. Data information: For ( B ), n = 4366 genes. For ( F ), n = 3 biological replicates. For ( G ), n = 127 feature weights (blue) or 397 feature weights (red). For ( H ), n = 10,000 randomizations.

Article Snippet: α-Tubulin Chicken Polyclonal Ab , LSBio , Cat # LS-C108486.

Techniques:

Journal: Molecular Systems Biology

Article Title: Proteome-wide copy-number estimation from transcriptomics

doi: 10.1038/s44320-024-00064-3

Figure Lengend Snippet: ( A , B ) Knockdown of NUP37 increases CDK4 abundance in B2B1 breast epithelial cells (Wang et al, ). ( C , D ) Overexpression of inducible NUP37 (iNUP37) decreases CDK4 abundance in B2B1 breast epithelial cells. ( E , F ) Overexpression of iNUP37 decreases CDK4 abundance in MCF7 luminal breast cancer cells. ( G , H ) Residual DNM1L protein after knockdown correlates with CDK4 abundance in T47D ( G ) and ZR751 ( H ) luminal breast cancer cells. Actin was used as a housekeeping control not used for loading normalization (“Methods”). Quantities are scaled to shScramble control samples. Data information: For ( A , C , E ), representative immunoblots for NUP37 and CDK4 are shown with vinculin and tubulin ( A , C ) or p38 ( E ) as loading controls and FLAG ( C , E ) to confirm ectopic expression of iNUP37 or luciferase (luc) control. For ( B , D , F ), immunoblot results are summarized as the mean total NUP37 (endogenous ( B , D , F ) and induced ( D , F )) or CDK4 ± s.e.m. of n = 6 biological replicates. Differences were assessed by one-sided t test. For ( G , H ), representative immunoblots for DNM1L and CDK4 are shown with vinculin and p38 as loading controls and actin as a housekeeping control. Data are summaries from n = 6 biological replicates.

Article Snippet: α-Tubulin Chicken Polyclonal Ab , LSBio , Cat # LS-C108486.

Techniques: Knockdown, Over Expression, Control, Western Blot, Expressing, Luciferase