Journal: Cells

Article Title: SOX2 Expression Does Not Guarantee Cancer Stem Cell-like Characteristics in Lung Adenocarcinoma

doi: 10.3390/cells13030216

Figure Lengend Snippet: Inter- and intra-cellular heterogeneity in Lung adenocarcinoma (LUAD) cell lines regarding SOX2 expression. ( A ) SOX2 mRNA expression in LUAD cells. Semi-quantitative RT-PCR was conducted to analyze the mRNA expression of SOX2 and ACTB (β-actin) using total RNA extracted from LUAD cells A549, H23, and H358. NCCIT, an embryonic carcinoma cell line, was used as a positive control. ( B ) SOX2 protein expression in LUAD cells. The antibodies mentioned were employed in Western blotting, with β-actin serving as a loading control. ( C ) Flow cytometry (FACSVerse) was utilized to assess SOX2 protein expression in individual LUAD cells, employing an anti-SOX2 antibody and secondary goat anti-rabbit Alexa 488. A Rabbit IgG was used as a negative control. ( D ) Representative images of immunofluorescence staining with anti-SOX2 antibody in A549 and H358 cells. Nuclei stained with DAPI. Scale bar: 20 μm. ( E ) Quantification of SOX2-positive cells using immunostaining. Cells labeled with SOX2, exhibiting fluorescence intensity surpassing that of the highest controls, were considered as SOX2-positive cells. n = 50 for each sample.

Article Snippet: A rabbit anti-SOX2 monoclonal antibody (Cell Signaling Technology, Danvers, MA, USA) was used followed by incubation with goat anti-rabbit IgG-HRP (Thermo Fisher Scientific).

Techniques: Expressing, Quantitative RT-PCR, Positive Control, Western Blot, Control, Flow Cytometry, Negative Control, Immunofluorescence, Staining, Immunostaining, Labeling, Fluorescence

Journal: Cells

Article Title: SOX2 Expression Does Not Guarantee Cancer Stem Cell-like Characteristics in Lung Adenocarcinoma

doi: 10.3390/cells13030216

Figure Lengend Snippet: SOX2 knockout does not perturb the cell proliferation of LUAD. ( A ) A schematic representation illustrating the g SOX2 target site on SOX2 Exon1 for the generation of SOX2 knockout LUAD cell lines using the CRISPR/Cas9 system. ( B ) Immunoblots showing the SOX2 protein level in A549 and H358 cells stably expressing gMock or gSOX2. ( C ) Immunocytochemical analysis using anti-SOX2 antibody in Mock and SOX2 knockout LUAD cells. Scale bar: 20 μm. ( D ) No impact of SOX2 knockout on the cell proliferation of LUAD cells. Cellular proliferation was measured using sulforhodamine B (SRB) assays, and each value was presented as optical density at 515 nm (OD 515 ). ns, not significant. ( E ) No difference in the ability of a single cell to grow into a colony in LUAD SOX2 knockout cells. Representative images (left) and the quantification (right) of relative colony number in each sample are shown. The crystal violet staining was performed 7 days after seeding of A549 cells and 14 days for H358 cells. Values represent means ± SD from three independent experiments. ns, not significant.

Article Snippet: A rabbit anti-SOX2 monoclonal antibody (Cell Signaling Technology, Danvers, MA, USA) was used followed by incubation with goat anti-rabbit IgG-HRP (Thermo Fisher Scientific).

Techniques: Knock-Out, CRISPR, Western Blot, Stable Transfection, Expressing, Staining

Journal: Cells

Article Title: SOX2 Expression Does Not Guarantee Cancer Stem Cell-like Characteristics in Lung Adenocarcinoma

doi: 10.3390/cells13030216

Figure Lengend Snippet: SOX2 is not required for tumor sphere formation, invasion, and migration of certain LUAD cells. ( A ) No impact of SOX2 knockout on tumor sphere (TS) formation in LUAD cells. TS formation assays were conducted 14 days post-seeding using A549 and H358 cells stably expressing gMock or g SOX2 . Representative images (top) and the quantification of TS number (bottom) are shown. Results from each independent experiment (Experiment 1, 2 and 3) are presented as means ± SD (n = 3). ns, not significant. Scale bar: 100 μm. ( B , C ) No impact of SOX2 knockout on the migration and invasion of LUAD cells. Migration ( B ) and invasion ( C ) assays were carried out using Mock and SOX2 knockout A549 and H358 cells. Representative images (left) are shown, and the total number of migrated and invaded cells was normalized to that of gMock. Mean ± SD from three independent experiments are presented. Scale bar: 100 μm. ns, not significant.

Article Snippet: A rabbit anti-SOX2 monoclonal antibody (Cell Signaling Technology, Danvers, MA, USA) was used followed by incubation with goat anti-rabbit IgG-HRP (Thermo Fisher Scientific).

Techniques: Migration, Knock-Out, Stable Transfection, Expressing

Journal: Cells

Article Title: SOX2 Expression Does Not Guarantee Cancer Stem Cell-like Characteristics in Lung Adenocarcinoma

doi: 10.3390/cells13030216

Figure Lengend Snippet: shRNA−resistant SOX2 is unable to rescue impaired proliferation by sh SOX2 in LUAD. ( A ) A schematic showing the target site for SOX2 knockdown, with DNA sequence substitutions for shRNA-resistant SOX2 . The target site (red line) was used to generate Tet-inducible SOX2 knockdown (sh SOX2 ) in A549 and H358 cells. Red letters indicate three synonymous substitutions, reconstituting shRNA-resistant SOX2 in sh SOX2 cell lines (sh SOX2 /+ SOX2 ). ( B ) SOX2 knockdown and its reconstitution in sh SOX2 LUAD cell lines were assessed. Cells were treated with 1 μg/mL of doxycycline (Dox) for 4 days, and SOX2 protein level was accessed by Western blotting. shMock and shMock + Mock were used as negative controls. ( C , D ) Reduced cell survival caused by sh SOX2 is not restored by SOX2 reconstitution in LUAD cells. Clonogenic assays were conducted using indicated A549 and H358 cell lines. Representative images ( C ) and the quantification ( D ) are shown. The colony number of each sample was normalized to that of shMock without Dox. Mean ± SD from three independent experiments. ***, p < 0.001; ns, not significant. p values were compared with shMock without Dox.

Article Snippet: A rabbit anti-SOX2 monoclonal antibody (Cell Signaling Technology, Danvers, MA, USA) was used followed by incubation with goat anti-rabbit IgG-HRP (Thermo Fisher Scientific).

Techniques: shRNA, Knockdown, Sequencing, Western Blot

Journal: Cells

Article Title: SOX2 Expression Does Not Guarantee Cancer Stem Cell-like Characteristics in Lung Adenocarcinoma

doi: 10.3390/cells13030216

Figure Lengend Snippet: shRNA−resistant SOX2 is unable to rescue suppression of sphere formation, invasion, and migration observed in sh SOX2 LUAD cells. ( A , B ) The impaired sphere formation caused by sh SOX2 is not restored by the reconstitution of SOX2 in LUAD cells. ‘+’ Dox indicates that 1 μg/mL Dox was treated. Representative images (top) and the quantification of TS number (bottom) are shown. TS formation assays were conducted using shMock, sh SOX2 , sh SOX2 /+Mock, and sh SOX2 /+ SOX2 in A549 ( A ) and H358 ( B ) cells. Results from each independent experiment (Experiment 1,2 and 3) are presented as means ± SD (n = 3). ( C , D ) Reconstitution of SOX2 is unable to restore the reduced migration and invasion observed in sh SOX2 LUAD cells. Migration ( C ) and invasion ( D ) assays using indicated A549 with or without Dox treatment. Means ± SD from three independent experiments are presented. ***, p < 0.001; **, p < 0.01 when compared with the shMock without Dox; ns, not significant; TS, tumor sphere. Scale bar: 100 μm.

Article Snippet: A rabbit anti-SOX2 monoclonal antibody (Cell Signaling Technology, Danvers, MA, USA) was used followed by incubation with goat anti-rabbit IgG-HRP (Thermo Fisher Scientific).

Techniques: shRNA, Migration

Journal: Cells

Article Title: SOX2 Expression Does Not Guarantee Cancer Stem Cell-like Characteristics in Lung Adenocarcinoma

doi: 10.3390/cells13030216

Figure Lengend Snippet: SOX2 expression does not significantly affect cancer stem cell-like properties in certain LUAD cell lines. ( A , B ) Cellular response to chemotherapeutic drugs is not altered by SOX2 knockout in LUAD cells. Indicated concentrations of cisplatin ( A ) or paclitaxel ( B ) were treated for 72 h in A549 and H358 cells stably expressing gMock and g SOX2 . Cell viability was measured by SRB assay, and the values are presented as the mean ± SD from three independent experiments. ns, not significant.

Article Snippet: A rabbit anti-SOX2 monoclonal antibody (Cell Signaling Technology, Danvers, MA, USA) was used followed by incubation with goat anti-rabbit IgG-HRP (Thermo Fisher Scientific).

Techniques: Expressing, Knock-Out, Stable Transfection, Sulforhodamine B Assay

Journal: Cell reports

Article Title: Lineage-specific CDK activity dynamics characterize early mammalian development

doi: 10.1016/j.celrep.2025.115558

Figure Lengend Snippet: (A) Confocal images from representative ROSA26 DHB/H2B embryos. Scale bar, 20 μm. (B) Plot showing a quantification of CDK activity in individual SOX2 + or CDX2 + cells from ROSA26 DHB/H2B embryos (dots from the same column) staged based on the number of cells. Percentage of cells (in red) below the arbitrary threshold (−0.25) shown for embryos containing 0–100 cells and above 100 cells. N = 54 embryos. (C) Time-lapse microscopy experiment performed in two representative E3.5 ROSA26 DHB/H2B embryos. Note the embryonic (e)-abembryonic (a) axis. Scale bar, 30 μm. (D) Plots showing CDK activity in individual cells obtained from a representative E3.5 ROSA26 DHB/H2B embryo. The embryo was imaged, fixed, and stained for CDX2 and SOX2 (bottom; scale bar, 30 μm) to determine lineage identity at the endpoint. Highlighted tracks are in cyan and magenta. (E) Plot showing a quantification of CDK activity in individual cells obtained from a pool of E4.5 ROSA26 DHB/H2B embryos. Percentage of cells (in red) below the arbitrary threshold (−0.25). p values are from two-tailed unpaired t tests. **** p < 0.0001; N = 5 embryos. (F) Confocal images from E6.5 ROSA26 DHB/H2B embryos. Dashed line surrounds the EPI. Scale bar, 20 μm.

Article Snippet: Rabbit Anti Sox2 , Cell Signaling , 23064S, RRID:AB_2714146.

Techniques: Activity Assay, Time-lapse Microscopy, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: P53 toxicity is a hurdle to CRISPR/CAS9 screening and engineering in human pluripotent stem cells

doi: 10.1101/168443

Figure Lengend Snippet: (A) Detailed depiction of all-in-one dox inducible (pAAVS1-iCas9), inducible enhanced Cas9 (pAAVS1-ieCas9) and Shield1 inducible (pPB-ddCas9 iNgn2) Cas9 constructs. Although not utilized for this manuscript the ddCas9 transgene has an all-in-one dox inducible Ngn2 that can be used for rapid generation of cortical excitatory neurons from hPSCs. TRE3G and pTRE-tight, tetracycline response element promoter, ins, insulator CAG, constitutive promoter, Tet-On 3G and rtTA16, tetracycline transactivator protein, T2A, self-cleaving peptide, neo R , neomycin resistance gene, DD, destabilizing domain, PB, piggyBac repeats, LA, left homology arm, RA, right homology arm, HSVtk, herpes simplex virus (HSV) thymidine kinase promoter, ieCas9, dox inducible - enhanced Streptococcus Pyogenes Cas9 1.1 (eSpyCas9(1.1). (B) Karyotype analysis for the clones used in the study revealed no chromosomal abnormalities when the lines were first banked. 8402-iPSCs expressing ieCas9 are described by (C) Induction of Cas9 protein by addition of dox or Shield1 increase the amount of Cas9 protein detected by immunofluorescence using an antibody to detect FLAG tagged Cas9. (D) qPCR to detect cas9 mRNA reveals that cas9 expression is only induced in the presence of dox. Relative expression was calculated in comparison to untreated control. Bars represent mean expression. n=3 samples per condition. (E) Targeting of the iCas9 construct to the pAAVS1 safe harbor locus. Using a primer pair to span the AAVS1 knock-in site only amplifies in controls and indicates that iCas9 clone used in this study is homozygous. Junction PCR was used to detect both 5’ and 3’ specific junctions only in iCas9 transgenic cells. H1-ieCas9 is properly targeted homozygous as described by . (F) iCas9 cells infected with MAPT targeting sgRNAs retain pluripotency marker expression. Control sgRNAs targeting OCT4 and SOX2 loose TRA-1-60 expression and either OCT4 or SOX2 expression respectively. Cells were fixed and stained after 8 days of dox treatment when on-target editing is near completion.

Article Snippet: 1:50 FITC conjugated anti-TRA-1-60 antibody (FCMAB115F – Millipore), 1:50 647 conjugated anti-OCT4 (C30A3) antibody (5263-CST), 1:50 647 conjugated anti-Sox2 (D6D9) antibody (5067-CST).

Techniques: Construct, Virus, Clone Assay, Expressing, Immunofluorescence, Comparison, Control, Knock-In, Transgenic Assay, Infection, Marker, Staining

Journal: Theranostics

Article Title: Pharmacological inhibition of MYC to mitigate chemoresistance in preclinical models of squamous cell carcinoma

doi: 10.7150/thno.88759

Figure Lengend Snippet: MYCi975 suppresses cancer stemness in HNSCC cells. (A) Western blot analysis showing the inhibition of MYC, BMI1, SOX2, and ALDH1 after MYCi975 treatment. GAPDH was used as the internal control. (B-C) Representative FACS plots of ALDH high cells in HN6 and SCC15 cells after MYCi975 treatment. Means ± SD are shown. ** P < 0.01 using an unpaired Student's t test. A specific inhibitor of ALDH, diethylaminobenzaldehyde (DEAB), was used to control for background fluorescence. (D-G) Images and numbers of tumorspheres formed by ALDH high and ALDH low HN6 and SCC15 cells, with or without MYCi975 treatment. Scale bar: 100 μm. Means ± SD are shown. ** P < 0.01 using an unpaired Student's t test. (H-I) Extreme limiting dilution analysis (ELDA) of ALDH high CSC‑like cells from HN6 cells treated with or without MYCi975 in vivo (n = 5). The frequency of allograft formation is displayed for each cell dose injected. ELDA software was used to analyze the data. Scale bar: 1 cm.

Article Snippet: The primary antibodies used in this study were: anti-MYC (Cell Signaling Technology; Cat# 18583; 1:1000), anti-B lymphoma Mo-MLV insertion region 1 homolog (BMI1) (Cell Signaling Technology; Cat# 6964; 1:1000), anti-SRY-box transcription factor 2 (SOX2) (Cell Signaling Technology; Cat# 14962; 1:1000), anti‐ALDH1 (Cell Signaling Technology; Cat# 54135; 1:1000), anti-phospho-Histone H2A.X (Cell Signaling Technology; Cat# 9718; 1:1000), anti-p-IRF3 (Cell Signaling Technology; Cat# 37829; 1:1000), anti-GAPDH (ZSGB-BIO; Cat# TA-08; 1:1000), and anti-Histone H3 (ABclonal, Wuhan, China; Cat# A2348; 1:1000).

Techniques: Western Blot, Inhibition, Control, Fluorescence, In Vivo, Injection, Software

Journal: Nature Communications

Article Title: The transcribed pseudogene RPSAP52 enhances the oncofetal HMGA2-IGF2BP2-RAS axis through LIN28B-dependent and independent let-7 inhibition

doi: 10.1038/s41467-019-11910-6

Figure Lengend Snippet: RPSAP52 displays oncogenic features in breast cancer cells. a Viability/cytotoxicity assays in MCF10A, Hs578T and HCC1143 clones. The experiment was performed three times and one representative graph is shown for each cell line. Values are mean ±SD of n ≥ 6 measurements. One-way ANOVA was used (*** P < 0.001, **** P < 0.0001). b Effect of RPSAP52 silencing on colony formation ability. Representative plates are shown. Colonies were counted from three replicate plates and two independent experiments. Values are mean ±SD. Two-tailed unpaired t -test were used (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001). c Migration capacity of RPSAP52 -depleted clones was monitored over 24 h ( n = 5 replicates per condition), with higher cell index indicating higher migration. Values are mean ±SD. A two-tailed Mann–Whitney U test of data at end point was used (** P < 0.01). Inset: migration was also assessed with transwells. Scale bar = 100 µm. d Western blot analysis of NANOG, OCT4, and SOX2 in RPSAP52 -depleted cells. e The clonogenic ability was assessed with at least n = 12 replicates per condition. Values are mean ±SD. A two-tailed Mann–Whitney U test was used (*** P < 0.001). f Growth-inhibitory effect of RPSAP52 knockdown in MCF10A mice xenografts. Upper graph: tumor volume ( n = 10) was monitored over time. Mean values are shown ±SEM. Lower graph: tumors were excised and weighed at 77 days (*** P < 0.001, two-tailed Mann–Whitney U test). Western blot was carried out from sh4 tumors since no material could be recovered from sh1 tumors, and the levels of RAS and IGF2BP2 proteins were analyzed. The photograph shows the relative size of all tumors extracted. Scale bar = 10 mm. g Growth-inhibitory effect of RPSAP52 knockdown in Hs578T mice xenografts. Upper graph: tumor volume ( n = 9 for scr and n = 10 for sh4 clones) was monitored over time. Mean values are shown ±SEM. Lower graph: tumors were excised and weighed at 28 days (* P < 0.05, *** P < 0.001, two-tailed Mann–Whitney U test). Western blot was carried out from tumors at end point and the levels of IGF2BP2 protein were analyzed. The photograph shows the relative size of all tumors extracted. Scale bar = 10 mm. Source data are provided as a Source Data file

Article Snippet: The detected proteins were IGF2BP2 (H00010644-M01, Abnova, 1:500), RAS (ab55391, Abcam, 1:500, which recognizes all RAS proteins), LIN28B (ab71415, Abcam, 1:1000), IGF1R (#3027, Cell Signaling, 1:1000), ERK (#4695, Cell Signaling, 1:1000), p-ERK (#9101, Cell Signaling, 1:1000), LAMIN B1 (ab16048, Abcam, 1:4000), LIN28A (#8641, Cell Signaling, 1:750), α-TUBULIN HRP (ab40742, Abcam, 1:5000), HNRNPQ (ab184946, Abcam, 1:10,000), β-ACTIN HRP (a3854, Sigma, 1:20,000), NANOG (#4903, Cell Signaling, 1:2000), OCT4 (#2750, Cell Signaling, 1:1000), SOX2 (#4195, Cell Signaling, 1:1000), NUCLEOLIN (#8031, Santa Cruz, 1:2000), HMGA2 (ab97276, Abcam, 1:1000), HISTONE H3 (ab1791, Abcam, 1:5000), RPSA (ab133645, Abcam, 1:1000), and RPL5 (A303-933A, Company Bethyl, 1:1000).

Techniques: Clone Assay, Two Tailed Test, Migration, MANN-WHITNEY, Western Blot, Knockdown

Journal: Frontiers in Molecular Neuroscience

Article Title: The cAMP Response Element- Binding Protein/Brain-Derived Neurotrophic Factor Pathway in Anterior Cingulate Cortex Regulates Neuropathic Pain and Anxiodepression Like Behaviors in Rats

doi: 10.3389/fnmol.2022.831151

Figure Lengend Snippet: The primary antibodies for western blotting.

Article Snippet: CaMKII α , Cat no. 5049S, Cell Signaling Technology, Inc., United States , 1:1,000.

Techniques: Western Blot