Journal: International Journal of Molecular Sciences

Article Title: The Novel HSF1 Inhibitor NXP800 Exhibits Robust Antitumor Activity in Hepatocellular Carcinoma

doi: 10.3390/ijms27062781

Figure Lengend Snippet: Cytotoxicity assessment of the HSF1 inhibitor NXP800 in human hepatocellular carcinoma (HCC) cell lines. ( A ) The levels of the HSF1 protein were assessed in Hep3B, HLE, HLF, Huh7, and SNU449 cell lines by Western blot analysis. GAPDH was used as a loading control. ( B ) Cell viability was measured via MTT assay. The cell viability in percentage to DMSO control, expressed as non-linear regression curve fit over log10 drug concentrations, is depicted. The mean ± SD (n = 3) of at least three independent experiments each performed in triplicate is shown. Horizontal dotted line: 50% viable cells. IC 50 values per cell line are shown in each plot.

Article Snippet: The human hepatocellular carcinoma cell lines HLE (XenoTech, Kansas City, KS, USA; Cat. No. JCRB0404), HLF (Tebubio GmbH, Le Perray en Yvelines, France; Cat. No. JCRB0405), PLC/PRF/5 (CLS Cell Lines Service GmbH, Eppelheim, Germany; Cat. No. 300315), Huh7 (Cytion GmbH, Heidelberg, Germany; Cat. No. 300156), and SNU449 (LGC Standards GmbH, Teddington, Middlesex, UK; Cat. No. CRL-2234) were purchased as indicated and cultured according to standard procedures.

Techniques: Western Blot, Control, MTT Assay

Journal: Scientific Reports

Article Title: Osthol ameliorates obesity-associated lipid metabolic disorders by inhibiting ADRA1D-dependent Th17 cell differentiation

doi: 10.1038/s41598-025-20719-x

Figure Lengend Snippet: OST Regulates Adipocyte Lipid Metabolism via ADRA1D. ( A ) Oil red O staining was performed to assess lipid droplet accumulation in 3T3-L1 adipocytes treated with OST. ( B ) Intracellular triglyceride (TG) content was quantified to evaluate lipid accumulation. ( C , D ) RT-qPCR and western blotting were used to measure the mRNA and protein expression levels of FASN, PPARγ, and ADRA1D. (E) Western blot was used to detect the phosphorylation levels of ERK1/2 and PI3K. n = 3. ** P < 0.01, **** P < 0.0001. One-way ANOVA followed by Tukey’s post hoc test was used for multiple group comparisons.

Article Snippet: 3T3-L1 preadipocytes (400,107, Cytion, Eppelheim, Germany) were cultured in Dulbecco’s modified Eagle medium (DMEM; 11,885,084, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; A5670401, Thermo Fisher Scientific) at 37 °C until reaching 100% confluence.

Techniques: Staining, Quantitative RT-PCR, Western Blot, Expressing, Phospho-proteomics

Journal: BMC Microbiology

Article Title: First human cell-based cultivation system for the syphilis spirochete Treponema pallidum

doi: 10.1186/s12866-026-04856-5

Figure Lengend Snippet: Human foreskin fibroblast cell lines support the growth of T. pallidum in vitro . A Human foreskin fibroblast cell lines (MoNa, HFF1, and HFFC) support the growth of T. pallidum in long-term, continuous, in vitro cultivation. While cultivation of strain SS14 was discontinued after 10 weeks, in vitro cultures of the DAL-1 strain have been ongoing for over one year. During this period, scheme of subcultures was optimized (up to 1:20). A booster passage (*), extending the standard 7-day subculture to a 14-day period with a cultivation medium exchange on day 7, was employed to enrich the treponemal culture during critical declines. In vitro cultivation was performed in triplicate (i.e., in three cultivation wells) and the mean values for each subculture are presented in graph. Source data from individual wells are shown in Supplementary Table . B Parallel cultivation for seven days ( n = 15) with defined DAL-1 inoculum (10 6 treponemes) validated the differences in growth support of individual foreskin cell lines. Treponemal growth on human foreskin fibroblast cells was slower compared to growth on rabbit epithelial Sf1Ep cells. Red bar, mean. C Human foreskin-based cultivation system supports the growth of various T. pallidum strains ( n = 8), from the Nichols-like as well as the SS14-like cluster. Note that human foreskin fibroblast cells were prepared as an equal mixture of three tested cell lines. Each T. pallidum strain was cultivated in a single in vitro well, representing a sole experimental replicate used for data acquisition

Article Snippet: Human foreskin fibroblasts HFF1 (SCRC-1041; ATCC) and HFFC (300715; Cytion) were purchased, while the third cell line (MoNa) was kindly provided by Dr. Vladimir Rotrekl (Masaryk University), and was originally obtained from the National Tissue Centre (Czech Republic).

Techniques: In Vitro

Journal: Biomedicines

Article Title: Reference Gene Validation for Quantitative PCR Analysis in 2D and 3D AML12 Hepatocyte Models

doi: 10.3390/biomedicines14010150

Figure Lengend Snippet: Representative morphology of AML12 cells cultured under 2D and 3D conditions at day 7 (20× magnification). Cells were maintained in four culture setups: ( a ) 2D standard tissue culture plate (2D-TC); ( b ) 2D thin-layer gelysate-peptone coating (2D-GelyP); ( c ) 2D thin-layer collagen type I coating (2D-Coll); ( d ) 3D collagen type I sandwich culture (3D-Coll). Distinct differences in cell morphology, spatial organization, and cell–cell interactions are observed depending on culture dimensionality and extracellular matrix context.

Article Snippet: The hepatic parenchymal cell line AML12 (300643, Cytion, Heidelberg, Germany) was maintained under standard culture conditions in a 1:1 mixture of Dulbecco’s modified Eagle’s medium (DMEM) and Ham’s F12 medium (Cytion, Heidelberg, Germany) supplemented with 10% fetal bovine serum (FBS), 1% insulin–transferrin–selenium (ITS, I3146, 100×), 1% antibiotic–antimycotic solution (penicillin, streptomycin, amphotericin B), and dexamethasone (D4902, 40 ng/mL) (all from Sigma-Aldrich Chemie GmbH, Merck KGaA, Darmstadt, Germany) to support hepatocyte-like characteristics.

Techniques: Cell Culture

Journal: Biomedicines

Article Title: Reference Gene Validation for Quantitative PCR Analysis in 2D and 3D AML12 Hepatocyte Models

doi: 10.3390/biomedicines14010150

Figure Lengend Snippet: Stability ranking of candidate housekeeping genes in AML12 cells based on ( a ) NormFinder and ( b ) geNorm algorithms. The y -axis represents the stability value (NormFinder) or stability measure M (geNorm), where lower values indicate higher expression stability. Analysis was performed using efficiency-corrected RT-qPCR data, with amplification efficiencies ranging from 97% to 103%. Gene abbreviations are defined in .

Article Snippet: The hepatic parenchymal cell line AML12 (300643, Cytion, Heidelberg, Germany) was maintained under standard culture conditions in a 1:1 mixture of Dulbecco’s modified Eagle’s medium (DMEM) and Ham’s F12 medium (Cytion, Heidelberg, Germany) supplemented with 10% fetal bovine serum (FBS), 1% insulin–transferrin–selenium (ITS, I3146, 100×), 1% antibiotic–antimycotic solution (penicillin, streptomycin, amphotericin B), and dexamethasone (D4902, 40 ng/mL) (all from Sigma-Aldrich Chemie GmbH, Merck KGaA, Darmstadt, Germany) to support hepatocyte-like characteristics.

Techniques: Expressing, Quantitative RT-PCR, Amplification

Journal: Biomedicines

Article Title: Reference Gene Validation for Quantitative PCR Analysis in 2D and 3D AML12 Hepatocyte Models

doi: 10.3390/biomedicines14010150

Figure Lengend Snippet: Comprehensive ranking of candidate reference genes in AML12 cells generated by RefFinder based on Ct values. The y -axis represents the overall ranking score derived from the integration of multiple algorithms (ΔCt method, BestKeeper, NormFinder, and geNorm). Lower ranking scores indicate higher expression stability, whereas higher values reflect reduced suitability as reference genes. Gene abbreviations are defined in .

Article Snippet: The hepatic parenchymal cell line AML12 (300643, Cytion, Heidelberg, Germany) was maintained under standard culture conditions in a 1:1 mixture of Dulbecco’s modified Eagle’s medium (DMEM) and Ham’s F12 medium (Cytion, Heidelberg, Germany) supplemented with 10% fetal bovine serum (FBS), 1% insulin–transferrin–selenium (ITS, I3146, 100×), 1% antibiotic–antimycotic solution (penicillin, streptomycin, amphotericin B), and dexamethasone (D4902, 40 ng/mL) (all from Sigma-Aldrich Chemie GmbH, Merck KGaA, Darmstadt, Germany) to support hepatocyte-like characteristics.

Techniques: Generated, Derivative Assay, Expressing

Journal: Biomedicines

Article Title: Reference Gene Validation for Quantitative PCR Analysis in 2D and 3D AML12 Hepatocyte Models

doi: 10.3390/biomedicines14010150

Figure Lengend Snippet: Mean Ct values of candidate housekeeping genes in AML12 cells cultured under different 2D and 3D conditions. Panels ( a – i ) correspond to ( a ) Actb, ( b ) Ppia, ( c ) 18S, ( d ) Hprt, ( e ) Hmbs, ( f ) B2M, ( g ) Rplp0, ( h ) Gapdh, and ( i ) Ywhaz. Cells were maintained in four culture setups: 2D standard tissue culture plate (2D-TC); 2D thin-layer gelysate-peptone coating (2D-GelyP); 2D thin-layer collagen type I coating (2D-Coll); 3D collagen type I sandwich culture (3D-Coll). Bars represent mean Ct values. Different letters above the bars indicate statistically significant differences between groups ( p < 0.05).

Article Snippet: The hepatic parenchymal cell line AML12 (300643, Cytion, Heidelberg, Germany) was maintained under standard culture conditions in a 1:1 mixture of Dulbecco’s modified Eagle’s medium (DMEM) and Ham’s F12 medium (Cytion, Heidelberg, Germany) supplemented with 10% fetal bovine serum (FBS), 1% insulin–transferrin–selenium (ITS, I3146, 100×), 1% antibiotic–antimycotic solution (penicillin, streptomycin, amphotericin B), and dexamethasone (D4902, 40 ng/mL) (all from Sigma-Aldrich Chemie GmbH, Merck KGaA, Darmstadt, Germany) to support hepatocyte-like characteristics.

Techniques: Cell Culture

Journal: Biomedicines

Article Title: Reference Gene Validation for Quantitative PCR Analysis in 2D and 3D AML12 Hepatocyte Models

doi: 10.3390/biomedicines14010150

Figure Lengend Snippet: Albumin (Alb) levels in AML12 cells cultured under different 2D and 3D conditions, shown as raw Ct values and after normalization to individual housekeeping genes (HKGs). Panels ( a – j ) represent ( a ) raw Alb Ct mean values and Alb normalized to ( b ) Rplp0, ( c ) Ppia, ( d ) Hprt, ( e ) Actb, ( f ) Ywhaz, ( g ) Gapdh, ( h ) 18S, ( i ) Hmbs, and ( j ) B2M, presented as ΔCt values. Cells were maintained in four culture setups: 2D standard tissue culture plate (2D-TC); 2D thin-layer gelysate-peptone coating (2D-GelyP); 2D thin-layer collagen type I coating (2D-Coll); 3D collagen type I sandwich culture (3D-Coll). Different letters above the data points indicate statistically significant differences between groups ( p < 0.05).

Article Snippet: The hepatic parenchymal cell line AML12 (300643, Cytion, Heidelberg, Germany) was maintained under standard culture conditions in a 1:1 mixture of Dulbecco’s modified Eagle’s medium (DMEM) and Ham’s F12 medium (Cytion, Heidelberg, Germany) supplemented with 10% fetal bovine serum (FBS), 1% insulin–transferrin–selenium (ITS, I3146, 100×), 1% antibiotic–antimycotic solution (penicillin, streptomycin, amphotericin B), and dexamethasone (D4902, 40 ng/mL) (all from Sigma-Aldrich Chemie GmbH, Merck KGaA, Darmstadt, Germany) to support hepatocyte-like characteristics.

Techniques: Cell Culture

Journal: Small (Weinheim an Der Bergstrasse, Germany)

Article Title: Reduced Non‐Specific Binding of Super‐Resolution DNA‐PAINT Markers by Shielded DNA‐PAINT Labeling Protocols

doi: 10.1002/smll.202405032

Figure Lengend Snippet: Various labeling approaches applied to COS‐7 cells. a) A primary monoclonal antibody targeting mitochondrial target Tom20 and a commercial Cy3 secondary antibody labels only the target. Note that the nuclear region is clear from fluorescence. b) The commercial secondary antibody is replaced with a Cy3 dye modified DNA‐PAINT oligonucleotide conjugated to a blank secondary antibody. This results in a visibly strong non‐specific nuclear signal. c) Attempting to label the cells with the DNA‐PAINT secondary antibody only shows the same level of non‐specific uptake within the nucleus. d) A fully double‐stranded DNA‐PAINT secondary significantly reduces non‐specific accumulation of markers within the nucleus. Red dashed lines indicate the nuclear perimeter. Scale bar: 5 µm. Images (b–d) taken with antibodies conjugated to docking strand D2 (see Table ).

Article Snippet: COS‐7 cells (Cytion, #605470) were grown at 37 °C in a 5% CO 2 humidified incubator.

Techniques: Labeling, Fluorescence, Modification

Journal: Small (Weinheim an Der Bergstrasse, Germany)

Article Title: Reduced Non‐Specific Binding of Super‐Resolution DNA‐PAINT Markers by Shielded DNA‐PAINT Labeling Protocols

doi: 10.1002/smll.202405032

Figure Lengend Snippet: Pathways leading to non‐specific localization event detection of oligonucleotides. a) A plot of the normalized mean event density (edu) for non‐specific localizations measured across nuclear regions of unlabeled cells for various imagers. Three right‐handed oligonucleotide imager sequences P1 imager, D2 imager, and D3 imager are shown and have decreasing nuclear presence (2.30 ± 0.13, 1.58 ± 0.17, 0.93 ± 0.08 edu respectively). The LP12 imager, a left‐handed non‐natural oligonucleotide, has the least number of detections within the nucleus, 0.27 ± 0.02 edu, (20, 20, 24, and 16 cells, n = 3). b) An example rendered image of a nuclear region in an unlabeled cell exposed to 0.1 n m D3 imager for 5000 frames. c) A COS‐7 cell incubated with a D3 DNA‐PAINT conjugated secondary antibody (2’AB) only, imaged and rendered as in b. d) A plot of normalized mean event localization density for cells labeled non‐specifically in this manner shows an increase in detected events by over an order of magnitude, 15.3 ± 0.6 edu (27 cells, n = 3), when compared to corresponding D3 imager‐only experiments. e) Schematic showing possible routes of single molecule event detections in biological samples. The marker‐based background is caused by the non‐specific attachment of conjugated oligonucleotide ABs during labeling. A much smaller, imager only, background is caused by the non‐specific, stochastic transient trapping of imagers on or inside the cell. All values are given as mean ± standard error of the mean. Boxplot points represent the mean value obtained per measured cell, and the line indicates the median. Independent two‐tailed t‐tests indicate the following levels of significance: ** p ≤ 0.01 and *** p ≤ 0.001. Scale bars: 5 µm.

Article Snippet: COS‐7 cells (Cytion, #605470) were grown at 37 °C in a 5% CO 2 humidified incubator.

Techniques: Incubation, Labeling, Marker, Two Tailed Test

Journal: Small (Weinheim an Der Bergstrasse, Germany)

Article Title: Reduced Non‐Specific Binding of Super‐Resolution DNA‐PAINT Markers by Shielded DNA‐PAINT Labeling Protocols

doi: 10.1002/smll.202405032

Figure Lengend Snippet: Comparison between conventional secondary antibody labeling and Shielded DNA‐PAINT labeling using adapted incubation solutions for reduced non‐specific interactions. a) DNA‐PAINT imagers can freely hybridize to docking sites on single‐stranded (ss) oligonucleotides at target locations. When the strand at the target location is double‐stranded (ds), imagers can no longer bind. In a partially double‐stranded (δds) system DNA‐PAINT super‐resolution measurements are still possible. b) Workflow for labeling biological samples. 1) the sample is fixed, permeabilized, blocked, and labeled with a primary antibody. 2) The sample is then either labeled with a DNA‐PAINT secondary in (a) a normal antibody incubation solution (PBS based) typically containing 137 m m NaCl or incubated in (b) a modified solution containing up to 1M NaCl with complementary “shield” strands that form a partially double‐stranded (δds) system aimed at reducing the non‐specific binding of the secondary antibody during labeling. (3) The sample is washed in imaging buffer, fiducials are added and the sample finally imaged with appropriate imager sequences. c) Widefield images of COS‐7 cells labeled for Tom20 using Cy3 dye modified D3 DNA‐PAINT secondary with the various incubation solutions containing, from left to right: ss and δds at increasing NaCl concentrations (137, 500, 1000 m m ). Normalized mean event densities calculated from localization events detected for the different labeling conditions in d) nuclear regions and e) cytoplasmic regions show decreased levels of non‐specific binding with all δds systems and with 1000 m m NaCl. Target event densities of Tom20, f), showed similar levels in ss and δds configurations. A slight reduction in ss state at 1 m was observed. g) The ratio of nuclear event densities to target showed improved signal to background (S/B) for δds compared to ss, (28, 22, 27, 28, 27, and 24 cells, n = 3). Boxplot points represent the mean value obtained per measured cell, and line indicates the median. Independent two‐tailed t‐tests indicate the following levels of significance: ns p > 0.05, * p ≤ 0.05, ** p ≤ 0.01 and *** p ≤ 0.001. Scale bars: c) 2 µm, inset in d) 5 µm.

Article Snippet: COS‐7 cells (Cytion, #605470) were grown at 37 °C in a 5% CO 2 humidified incubator.

Techniques: Comparison, Antibody Labeling, Labeling, Incubation, Modification, Binding Assay, Imaging, Two Tailed Test

Journal: Small (Weinheim an Der Bergstrasse, Germany)

Article Title: Reduced Non‐Specific Binding of Super‐Resolution DNA‐PAINT Markers by Shielded DNA‐PAINT Labeling Protocols

doi: 10.1002/smll.202405032

Figure Lengend Snippet: DNA‐PAINT labeling of nucleolin staining in COS‐7 cells. a) Schematic of ss D3 incubated in 137 m m NaCl. As expected, the DNA‐marker was observed in widefield images, ai, to non‐specifically incorporate itself across the nucleus of the cell and across the cytoplasm. Super‐resolution measurements, aii, of the same region match the non‐specific signal seen in the widefield image. b) Schematic of δds D3 incubated in 1 m NaCl incubation solution where the light‐blue portion of the δds indicates where the imager binds. bi. Widefield images show the nucleoli present within the nucleus of cells with much greater contrast. bii. Super‐resolution detection of single molecule events display well‐defined nucleoli and minimal cytoplasmic localizations. Scale bars: 2 µm.

Article Snippet: COS‐7 cells (Cytion, #605470) were grown at 37 °C in a 5% CO 2 humidified incubator.

Techniques: Labeling, Staining, Incubation, Marker

Journal: Nature Communications

Article Title: Search-and-remove genome editing allows selection of cells by DNA sequence

doi: 10.1038/s41467-025-66896-1

Figure Lengend Snippet: a Strategy to apply SNIPE to kill cancer cells based on cancer specific mutations. Wild type cells are colored in gray, cells with cancer mutations in purple and dying cells in red. b Cell growth curves of human 409B2 iPSCs measured by resazurin assay (upper panel). The lower panel shows NGS read frequency for cancer mutation (purple) and wild type cells (gray) in a mixed population over time. c Killing of TP53 knock-out 409B2 cells by SNIPE. Different amounts of TP53 knock-out cells (purple) were mixed with wild type cells (gray). Indels are colored in blue and wild type with indels in light purple. d Cell survival after selection by SNIPE related to ( c ). e K562 cells carry a Philadelphia chromosome, characterized by a reciprocal translocation of chromosome 9 and 22 resulting in the fusion oncogene BCR-ABL1 . f The BCR-ABL fusion generates a new gRNA recognition site in K562 cells. g Cell survival of THP-1 and K562 cells after targeting the BCR-ABL junction. The label “Cas9 cut” indicates CRISPR targeting without SNIPE. h Agarose gel of PCR products of healthy chromosome 22 (297 bp, gray) and Philadelphia chromosome (267 bp, purple). i Quantification of band intensity for K562 cells of ( h ). j Cell survival of heterozygous cancer cell lines RD-ES, HT-29 and HuCC-T1 after targeting the disease allele with SNIPE. k NGS read frequency of cancer locus alleles in RD-ES, HT-29 and HuCC-T1 for mock and SNIPE edit. The cancer mutation allele is colored in purple, wild type in grey, indels in blue and wild type with indels in light purple. Independent biological replicates were performed ( n = 3 for b , c , d , g , h , i , j , k , n = 2 for RDES in j and k) and are depicted as dots for the cancer mutation allele sequence reads and cell survival. Error bars show the s.e.m.

Article Snippet: Human colorectal adenocarcinoma cells HT-29 (Cytion, catalog no. 300215) were cultured in EMEM (Sigma-Aldrich, catalog no. M4655) supplemented with 10% FBS (Gibco, catalog no. 10270-106) and human CML cells K562 (ECACC, catalog no. 89121407) were cultured in IMDM (Gibco, catalog no. 12440053) with 10% FBS.

Techniques: Resazurin Assay, Mutagenesis, Knock-Out, Selection, Translocation Assay, CRISPR, Agarose Gel Electrophoresis, Sequencing