Journal: Scientific Reports

Article Title: Synthetic circuits based on split Cas9 to detect cellular events

doi: 10.1038/s41598-023-41367-z

Figure Lengend Snippet: Logic gate to detect cell–cell fusion and schematic diagram of cell–cell electrofusion and hybrid cell formation. Images of admixed transiently transfected cells with complementing Cas9 halves HEK 293 + H1299 and HEK 293 + H2170 after fusion induction by overexpression of Syncytin-1, alone or in pair with electrofusion, or by PEG treatment. Control cells (NC) were transfected with only one half of Cas9 plus the reporter and fusogenic vectors ( A , B ). As we expected admixed cells HEK293 + H1299 expressing either one halve (as a control samples) and electrofused using 300V and 700V do not show any fusion events ( C ), compared to electrofusion of cells expressing both halves where we got results of a few cell fusion ( D ). After enhancement of fusion by electric pulses in pair with Syncytin1 overexpression, we visibly increased occurrence of fusion events ( E ). In control samples of admixed HEK293 + H2170 cells after electrofusion using 300 V and 700 V, also the fusion was undetectable ( F ), in comparison to Syncytin1-induced fusion cells plus electrofusion, where fusion cell were clearly visible ( H ). Images of admixed stable cell lines with complementing Cas9 halves (HEK293, H1299, H2170, HEK293 + H1299, HEK293 + H2170, and H1299 + H2170) after fusion induction by overexpression of Syncytin1, and Syncytin1 plus electrofusion. Control cells (NC) were transfected with only one half of Cas9 plus the reporter and fusogenic vectors. In relation to cells only expressed fusogenic Syncytin1 ( G ), the combination of Syncytin1 and electrical pulses ( H ) greatly enhanced the detection of syncytia. When fusing HEK293 with H1299 or H2170, the syncytia visibly formed contained more nuclei, than lung cancer cells in a mix within the same line. In summary, H1299 cells overexpressing Syncytin1 in pair with electrofusion at 700 V did not show significant fusion events ( H ). Higher efficiency was achieved by expression of Syncytin1 and lower voltage (300 V 500 µs one pulse) in stable expressing complementing Cas9 halves H1299 cells ( I ). Representative large multinucleated syncytia after electrofusion cells overexpressed Syncytin1 H1299 (using 300 V), H2170 cell lines (using 700 V), and mixed HEK 293 with H1299 cell line and HEK293 with H2170 cells (using 700 V) ( J ).

Article Snippet: Cell lines HEK-293, HSF, CCD 841 CoTr, H1299, H2170, SW480, A375, HeLa, U-2 OS, C2C12 and GC-2 were obtained from ATCC; H2170 stable cell line with vimentin reporter (VRCs) was created by us .

Techniques: Electrofusion, Transfection, Over Expression, Expressing, Comparison, Stable Transfection

Journal: bioRxiv

Article Title: Curcumin extends the lifespan of aging postmitotic cells with mitochondrial dysfunction

doi: 10.1101/2023.09.06.556469

Figure Lengend Snippet: The effect of curcumin treatment on the lifespan of human kidney cells (HEK293) postmitotic cells was assessed in D10 medium in a 96-well plate. The survival of aging postmitotic cells was measured using propidium iodide (PI)-fluorescent-based method for the indicated day of analysis. Data are presented as means ± SD (n=2). Statistical significance was determined as follows: *P < 0.05, ****P < 0.0001 and ns (non-significant), based on a two-way ANOVA followed by Šídák’s multiple comparisons test.

Article Snippet: Our study also involved the use of the human embryonic kidney cell line HEK293, obtained from the American Type Culture Collection (ATCC) in Manassas, Virginia, United States.

Techniques:

Journal: eLife

Article Title: The ATM-E6AP-MASTL axis mediates DNA damage checkpoint recovery

doi: 10.7554/eLife.86976

Figure Lengend Snippet: ( A ) HEK293 cells were treated with 0.5 µM doxorubicin (DOX) as indicated, cell lysates were collected and analyzed by immunoblotting for MASTL, RPA32, and α-tubulin. ( B ) HEK293 cells were treated with 10 mM hydroxyurea (HU) as indicated, cell lysates were collected and analyzed by immunoblotting for MASTL, RPA32, and α-tubulin. ( C ) HEK293 cells were treated with 10 nM camptothecin (CPT) as indicated, cell lysates were collected and analyzed by immunoblotting for MASTL, RPA32, and α-tubulin. ( D ) HeLa cells were treated with 10 mM HU, and incubated as indicated. Cell lysates were collected and analyzed by immunoblotting for MASTL, phospho-H2AX Ser-139, RPA32, phospho-ATM/ATR substrates, and α-tubulin. ( E ) HeLa cells were treated with or without 0.5 µM DOX, 10 nM CPT, 10 mM HU, and 20 Gy ionizing radiation (IR) for 4 hr. Cell lysates were collected and analyzed by immunoblotting for MASTL, phospho-H2AX Ser-139, phospho-ATM/ATR substrates, and α-tubulin. ( F ) HeLa cells were treated with 0.5 μM DOX, and analyzed by immunoblotting for MASTL, α-tubulin, Aurora A, Aurora B, CDK1, cyclin B1, and phospho-ATM/ATR substrates. ( G ) SCC38 cells were incubated with or without HU and caffeine, as indicated, and analyzed by immunoblotting for MASTL and β-actin.

Article Snippet: Human cervix carcinoma (HeLa) and human embryonic kidney 293 (HEK293) cell lines were obtained and authenticated by ATCC, and maintained in Dulbecco’s modified Eagle medium (DMEM, Hyclone) with 10% fetal bovine serum (FBS, Hyclone).

Techniques: Western Blot, Incubation

Journal: eLife

Article Title: The ATM-E6AP-MASTL axis mediates DNA damage checkpoint recovery

doi: 10.7554/eLife.86976

Figure Lengend Snippet: ( A–C ) HeLa cells expressing CFP-MASTL were treated without or with ionizing radiation (IR). Cells were analyzed by direct fluorescence for CFP-MASTL expression, and by immunofluorescence for the phosphorylation of ATM/ATR substrates. Quantifications are shown in panels B and C (>10 cells/time point). ( D, E ) HEK293 cells were treated without ( D ) or with ( E ) 10 mM hydroxyurea (HU) for 2 hr. These cells were then treated with cycloheximide (CHX, 20 μg/ml) at time 0 to block protein synthesis, and analyzed by immunoblotting for the protein stability of MASTL and α-tubulin.

Article Snippet: Human cervix carcinoma (HeLa) and human embryonic kidney 293 (HEK293) cell lines were obtained and authenticated by ATCC, and maintained in Dulbecco’s modified Eagle medium (DMEM, Hyclone) with 10% fetal bovine serum (FBS, Hyclone).

Techniques: Expressing, Fluorescence, Immunofluorescence, Blocking Assay, Western Blot

Journal: eLife

Article Title: The ATM-E6AP-MASTL axis mediates DNA damage checkpoint recovery

doi: 10.7554/eLife.86976

Figure Lengend Snippet: ( A ) The sequence alignment of the conserved E6AP Ser-218 motif in human, mouse, and Xenopus . ( B ) A phospho-specific antibody recognizing E6AP Ser-218 was generated, as described in Materials and methods. WT or E6AP knockout (KO) HEK293 cells were treated without or with 10 mM hydroxyurea (HU) for 4 hr, and analyzed by immunoblotting for phospho-E6AP Ser-218, E6AP, and α-tubulin. ( C ) HeLa cells were treated without or with 0.5 μM doxorubicin (DOX) and 5 μM KU55933 (ATMi), as indicated, for 1 hr, and analyzed by immunoblotting for phospho-ATM/ATR substrates, phospho-E6AP Ser-218, and α-tubulin. ( D ) HeLa cells were transfected with HA-tagged WT, S218A, or S218D E6AP. Cell lysates were harvested for immunoprecipitation (IP) assays. The input, MASTL IP, and a control IP using empty beads products were analyzed by immunoblotting for MASTL and HA. ( E ) HeLa cells were transfected with HA-tagged WT or S218A E6AP, as in panel D. Cells were treated with or without 0.5 μM DOX for 3 hr, and harvested for IP assays. The input, HA IP, and a control IP using empty beads products were analyzed by immunoblotting for MASTL, phospho-E6AP Ser-218, and HA. ( F ) E6AP KO HeLa cells were transfected with HA-tagged WT or S218A E6AP, as in panel D. Cells were treated with or without 0.5 μM DOX, incubated as indicated, and harvested for immunoblotting for MASTL and α-tubulin.

Article Snippet: Human cervix carcinoma (HeLa) and human embryonic kidney 293 (HEK293) cell lines were obtained and authenticated by ATCC, and maintained in Dulbecco’s modified Eagle medium (DMEM, Hyclone) with 10% fetal bovine serum (FBS, Hyclone).

Techniques: Sequencing, Generated, Knock-Out, Western Blot, Transfection, Immunoprecipitation, Incubation

Journal: eLife

Article Title: The ATM-E6AP-MASTL axis mediates DNA damage checkpoint recovery

doi: 10.7554/eLife.86976

Figure Lengend Snippet: ( A ) WT or E6AP knockout HEK293 cells were treated without or with doxorubicin (DOX, 0.5 μM) for 4 hr. Cells were harvested and analyzed by immunoblotting for phospho-E6AP Ser-218 and α-tubulin. ( B ) HEK293 cells were treated without or with DOX (0.5 μM), or ATM inhibitor (KU55933, 10 μM), for 4 hr, and analyzed by immunoblotting. ( C ) HeLa cells were treated with hydroxyurea (HU, 10 mM) combined with ATM/ATR inhibitor (caffeine, 4 mM) or ATM inhibitor (KU55933, 10 μM), for 12 hr, as indicated. Cells were harvested and analyzed by immunoblotting.

Article Snippet: Human cervix carcinoma (HeLa) and human embryonic kidney 293 (HEK293) cell lines were obtained and authenticated by ATCC, and maintained in Dulbecco’s modified Eagle medium (DMEM, Hyclone) with 10% fetal bovine serum (FBS, Hyclone).

Techniques: Knock-Out, Western Blot

Journal: eLife

Article Title: The ATM-E6AP-MASTL axis mediates DNA damage checkpoint recovery

doi: 10.7554/eLife.86976

Figure Lengend Snippet: ( A ) E6AP knockout (KO) HeLa cells were transfected with HA-tagged WT or S218A E6AP, as in . Cells were treated with 0.1 μM etoposide (ETO) for 18 hr, and released in fresh medium for recovery. Cells were then harvested at the indicated time points (after the removal of ETO) for immunofluorescence (IF) using an anti-phospho-Aurora A/B/C antibody. The activation of Aurora phosphorylation (shown in red) and chromosome condensation (in blue) indicated mitosis. The percentages of cells in mitosis were quantified manually and shown. The mean values and standard deviations were calculated from three experiments. An unpaired two-tailed Student’s t test was used to determine the statistical significance (**p<0.01, n>500 cell numbers/measurement). ( B ) E6AP KO HeLa cells expressing HA-tagged WT or S218A E6AP, as in panel A, were treated with 2 mM hydroxyurea (HU) for 18 hr. Cells were then released in fresh medium, and incubated as indicated, for recovery. Cell cycle progression was analyzed by fluorescence-activated cell sorting (FACS). ( C ) WT or S218A E6AP was expressed in E6AP KO HEK293 cells. Cells were treated without or with 0.1 μM ETO for 18 hr, released in fresh medium for recovery, and incubated as indicated. Cells were analyzed by immunoblotting for phospho-cyclin-dependent kinase (CDK) substrates and histone H3. ( D ) WT or S218A E6AP was expressed in E6AP KO HEK293 cells, as in panel C. Cells were treated without or with 1 μM CPT for 90 min, and analyzed by immunoblotting for phospho-ATM/ATR substrates, phospho-SMC1 Ser-957, and α-tubulin.

Article Snippet: Human cervix carcinoma (HeLa) and human embryonic kidney 293 (HEK293) cell lines were obtained and authenticated by ATCC, and maintained in Dulbecco’s modified Eagle medium (DMEM, Hyclone) with 10% fetal bovine serum (FBS, Hyclone).

Techniques: Knock-Out, Transfection, Immunofluorescence, Activation Assay, Two Tailed Test, Expressing, Incubation, Fluorescence, FACS, Western Blot

Journal: iScience

Article Title: Cryptotanshinone is a candidate therapeutic agent for interstitial lung disease associated with a BRICHOS-domain mutation of SFTPC

doi: 10.1016/j.isci.2023.107731

Figure Lengend Snippet: High-throughput screening and identification of compounds that reduce ER stress caused by mutant SFTPC (A) Diagram illustrating the construction of XBP1-HiBiT Reporter. It was spliced under ER stress and resulted in the translation of a spliced XBP1(exon 4)-HiBiT fusion protein. The STOP sign indicates the stop codon of the open reading frame of unspliced mRNA. (B) Cellular HiBiT assay using the Nano-Glo HiBiT Lytic Detection System for HEK293 cells transfected with XBP1-HiBiT Reporter and treated with tunicamycin (0.6, 1.25, 2.5 or 5.0 μg/mL) or DMSO (0.05%) for 6 h (n = 6). Data are presented as mean ± SD. ∗∗∗p < 0.001 [One-way analysis of variant (ANOVA) with Tukey’s multiple comparisons test]. (C) Diagram illustrating the screening method. (D) Validation of high-throughput screening using the cellular HiBiT assay. HEK293 cells were co-transfected with XBP1-HiBiT Reporter and empty or L188Q-mutant SFTPC expression vector (L188Q SFTPC). n = 80 (L188Q SFTPC), n = 12 (empty vector). (E) RT-qPCR of the splicing ratio of XBP1-HiBiT Reporter (spliced/total expression) in HEK293 cells subjected to 24 h incubation with co-transfected XBP1-HiBiT Reporter and wild or L188Q SFTPC expression vectors. Data are presented as mean ± SD (n = 3 independent experiments). ∗∗p < 0.01 (Welch’s t test). (F) Scatterplot for relative luminescence units (RLU) (% of DMSO) analyzed by XBP1-HiBiT Reporter co-transfected with L188Q SFTPC expression vector in the screening of 2480 compounds. Data are presented as mean (n = 2). (G) Scatterplot showing pairwise comparisons of the result of WST-8 assay (n = 2) (% of DMSO) versus the result of HiBiT assay (from Figure 1 F) in the screening of the 2480 compounds. Red and blue characters and lines indicate each threshold to identify hit compounds. See also Figure S1 .

Article Snippet: HEK293 human embryonic kidney cell line , ATCC , CRL-1573.

Techniques: High Throughput Screening Assay, Mutagenesis, Transfection, Variant Assay, Expressing, Plasmid Preparation, Quantitative RT-PCR, Incubation

Journal: iScience

Article Title: Cryptotanshinone is a candidate therapeutic agent for interstitial lung disease associated with a BRICHOS-domain mutation of SFTPC

doi: 10.1016/j.isci.2023.107731

Figure Lengend Snippet: Secondary screening for compounds identified in the primary screening (A) RT-qPCR of the splicing ratio of XBP1-HiBiT Reporter (spliced/total expression) in HEK293 cells subjected to 24 h incubation with co-transfected XBP1-HiBiT Reporter and wild or Y104H SFTPC expression vectors. Data are presented as mean ± SD (n = 3 independent experiments). ∗∗∗p < 0.001 (Student’s t test). (B and C) The means of Spot Intensity (B) and Total Spot Area (C) for HEK293 cells transfected with the AcGFP-wild SFTPC or AcGFP-Y104H SFTPC (n = 4, each sample is averaged over 81 field-of-view × 3 well). Data are presented as mean ± SD. ∗∗∗p < 0.001 (Student’s t test). (D) Representative microscopic images of HEK293 cells transfected with the AcGFP-wild SFTPC or AcGFP-Y104H SFTPC. Nuclei were visualized by Hoechst 33342 (blue). Scale bar = 100 μm. (E) Validation of screening using cellular HiBiT assay. HEK293 cells were co-transfected with XBP1-HiBiT Reporter and empty or Y104H-mutated SFTPC expression vector (Y104H SFTPC). n = 80 (Y104H SFTPC), n = 12 (empty vector). (F) Scatterplot showing pairwise comparisons of the result of the means of Spot Intensity (% of DMSO) for HEK293 cells transfected with the AcGFP-Y104H SFTPC (Data are presented as the mean of n = 2, each sample is averaged over 81 field-of-view) versus the means of RLU (% of DMSO) analyzed by XBP1-HiBiT Reporter co-transfected with Y104H SFTPC in the screening of 65 compounds identified in primary screening (n = 2). Red points indicate the compounds which have passed this screening. (G) Analysis of the splicing ratio of XBP1-HiBiT Reporter (spliced/total expression) by RT-qPCR in HEK293 cells subjected to 4 h incubation with co-transfected XBP1-HiBiT Reporter and Y104H SFTPC expression vectors followed by treatment with each of 9 compounds for 24 h at the indicated concentration (n = 2). Data are presented as mean ± SD. Red points indicate the compounds which have passed this screening. The Red line is FC = 0.75. See also Figure S1 .

Article Snippet: HEK293 human embryonic kidney cell line , ATCC , CRL-1573.

Techniques: Quantitative RT-PCR, Expressing, Incubation, Transfection, Plasmid Preparation, Concentration Assay

Journal: iScience

Article Title: Cryptotanshinone is a candidate therapeutic agent for interstitial lung disease associated with a BRICHOS-domain mutation of SFTPC

doi: 10.1016/j.isci.2023.107731

Figure Lengend Snippet: CPT reduces aggregates and cell death caused by Y104H SFTPC (A and B) The means of Spot Intensity of AcGFP in each cell (A) and Total Spot Area of AcGFP in each cell (B) were analyzed for A549 cells transfected with the AcGFP-wild SFTPC or AcGFP-Y104H SFTPC for 48 h (n = 3, each sample is averaged over 81 fields of view). Data are presented as the mean ± SD. ∗∗∗p < 0.001 (Student’s t test). (C) Analysis of Spot Intensity and Total Spot Area of A549 cells transfected with the AcGFP-Y104H SFTPC for 4 h and subsequently treated with CPT (1.25, 2.5, or 5 μM) or DMSO (0.1%) for 48 h (n = 3, each sample is averaged over 81 fields of view). Data are presented as the mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 (One-way ANOVA with Tukey’s multiple comparisons test, compared with DMSO control). (D) Representative image of A549 cells transfected with AcGFP-Y104H SFTPC for 4 h and subsequently treated with CPT (2.5 μM) or DMSO (0.1%) for 48 h. Nuclei were visualized by Hoechst 33342 (blue). Scale bar = 50 μm. (E) Representative results of WB using anti-SFTPC antibody on A549 cells transfected with the AcGFP-wild SFTPC or AcGFP-Y104H SFTPC for 4 h and subsequently treated with CPT (1.25, 2.5, or 5 μM) or DMSO (0.1%) for 48 h. α-tubulin was used as a loading control (Left panel). The signal intensities were quantified using densitometry (Right panel). Three independent experiments were conducted, and the data are presented as the mean ± SD. ∗p < 0.05 (Student’s t test, compared with DMSO control). (F) The number of double-positive cells of activated caspase-3 and SFTPC-GFP in A549 cells transfected with the AcGFP-wild SFTPC or AcGFP-Y104H SFTPC for 24 h and calculated by normalizing the number of GFP-positive cells (n = 3, each sample is averaged over 169 fields of view). Data are presented as the mean ± SD. ∗∗p < 0.01 (Student’s t test). (G) The number of double-positive cells of activated caspase-3 and SFTPC-GFP in A549 cells transfected with the AcGFP-Y104H SFTPC (Left graph) or AcGFP-wild SFTPC (Right graph) for 4 h and subsequently treated with 1.25 μM CPT or DMSO (0.1%) for 24 h, and calculated by normalizing the total cell number (n = 3, each sample is averaged over 169 fields of view). Data are presented as the mean ± SD. ∗p < 0.05 (Student’s t test). (H) The number of double-positive cells of activated caspase-3 and SFTPC-GFP in HEK293 cells transfected with the AcGFP-wild SFTPC or AcGFP-Y104H SFTPC for 24 h and calculated by normalizing the number of GFP-positive cells (n = 3, each sample is averaged over 169 fields of view). Data are presented as the mean ± SD. ∗∗∗p < 0.001 (Student’s t test). (I) The number of double-positive cells of activated caspase-3 and SFTPC-GFP in HEK293 cells transfected with the AcGFP-Y104H SFTPC (Left graph) or AcGFP-wild SFTPC (Right graph) for 4 h and subsequently treated with 2.5 μM CPT or DMSO (0.1%) for 24 h, and calculated by normalizing the total cell number (n = 3, each sample is averaged over 169 fields of view). Data are presented as the mean ± SD. ∗∗∗p < 0.001 (Student’s t test). See also Figure S3 .

Article Snippet: HEK293 human embryonic kidney cell line , ATCC , CRL-1573.

Techniques: Transfection

Journal: iScience

Article Title: Cryptotanshinone is a candidate therapeutic agent for interstitial lung disease associated with a BRICHOS-domain mutation of SFTPC

doi: 10.1016/j.isci.2023.107731

Figure Lengend Snippet:

Article Snippet: HEK293 human embryonic kidney cell line , ATCC , CRL-1573.

Techniques: Recombinant, Plasmid Preparation, Software, Cell Counting, Transfection, Membrane

Journal: Cell Death & Disease

Article Title: LZTR1 deficiency exerts high metastatic potential by enhancing sensitivity to EMT induction and controlling KLHL12-mediated collagen secretion

doi: 10.1038/s41419-023-06072-9

Figure Lengend Snippet: A The results of liquid chromatography-tandem mass spectrometry. B How LZTR1 regulates SEC31A ubiquitination and inhibits collagen secretion through the inhibition of KLHL12 function. C Ascorbate chase analyses were performed by adding ascorbate (0.25 mM ascorbic acid and 1 mM ascorbic acid 2-phosphate) to MEFs from Lztr1 +/+ or Lztr1 −/− mice. Cells were stained with anti-COL1A1 antibody (green) and NucBlue Stain (nuclei, blue). Scale bar, 20 µm. Values are the mean ± SD; ** P ≤ 0.01 (Wilcoxon-Mann-Whitney test, n = 5). D , E HEK293 cells were transfected with the indicated expression plasmids, and the lysates were subjected to co-immunoprecipitation assays using anti-FLAG M2 Magnetic Beads or anti-GFP mAb-Magnetic Beads. The immunoprecipitants were subjected to western blot analyses. F An in vivo ubiquitination assay was performed with anti-HA-tag mAb-Magnetic Agarose. The ubiquitination status of SEC31A was evaluated by western blot.

Article Snippet: The A549 (CRM-CCL-185) and HEK293 (CRL-1573) cell lines were purchased from ATCC (Manassas, VA, USA).

Techniques: Liquid Chromatography, Mass Spectrometry, Inhibition, Staining, MANN-WHITNEY, Transfection, Expressing, Immunoprecipitation, Magnetic Beads, Western Blot, In Vivo, Ubiquitin Assay

Journal: Redox Biology

Article Title: An inducible long noncoding RNA, LncZFHX2, facilitates DNA repair to mediate osteoarthritis pathology

doi: 10.1016/j.redox.2023.102858

Figure Lengend Snippet: mLncZFHX2 regulates osteoarthritis (OA) in vivo and in vitro . ( A ) RT-qPCR of mLncZFHX2 levels in mouse chondrocytes (MCs) under normoxia or hypoxia (n = 6, mean ± SD). ( B ) RT-qPCR of mLncZFHX2 levels in MCs infected with adenovirus (Ad) NC or Ad HIF‐1α (400 MOI) (n = 6, mean ± SD). ( C ) HIF‐1α ChIP assay of mLncZFHX2 promoter region. ( D ) Luciferase activities of HEK-293 cells co-transfected with a luciferase reporter construct containing wild-type or mutant mLncZFHX2 promoter region and Ad NC or Ad HIF‐1α (400 MOI) (n = 6, mean ± SD). ( E ) Representative images of Safranin O/Fast Green and RNA FISH staining of knee joints in CBA/CaCrl and SRT/Ort mice. Degree of OA was determined with OARSI grade (n = 6 per group, mean ± SD). Scale bar, 50 μm and 100 μm. ( F ) RT-qPCR of mLncZFHX2 levels in MCs transfected with ASO mLncZFHX2 or ASO NC (n = 6, mean ± SD). ( G ) Western blotting of MMP13, ADAMTS5, SOX9, ACAN, COL2A1, P16 INK4a and P21 expression in HCs transfected with ASO LncZFHX2 or ASO NC. ( H ) RT-qPCR of mLncZFHX2 (mLnc) levels in MCs transfected with Ad NC (400 MOI) or Ad mLnc (100–400 MOI) (n = 6, mean ± SD). ( I ) Western blotting of MMP13, ADAMTS5, SOX9, ACAN, COL2A1, P16 INK4a and P21 expression in MCs transfected Ad NC (400 MOI) or Ad mLnc (100–400 MOI). ( J ) Schematic of experimental design. Mice (12 and 18 months old) were subjected to intra-articular injections of mLncZFHX2 AAV or control AAV and sacrificed at 24 months. ( K ) Representative images of Safranin O/Fast Green, immunohistochemistry (P21), and RNA FISH (mLncZFHX2) staining of knee joints in aged mouse model. Degree of OA was evaluated with OARSI grade (n = 6 per group, mean ± SD). Scale bar, 50 μm and 100 μm. Graphs show P values from unpaired two-tailed t -test (A, B), one-way ANOVA (D, F, H). OARSI grade was analyzed with Mann-Whitney U tests (E, K), and positive cells were compared with unpaired two-tailed t -test (E), or paired two-tailed t -test (K).

Article Snippet: The human HEK-293 cell line was obtained from the American Type Culture Collection (ATCC, VA, USA), and the mice ATDC5 cell line was obtained from the Cell Engineering Division (Tsukuba, Japan) and maintained in DMEM containing 10% FBS (Gibco, Grand Island, NY, USA) at 37 °C in a 5% CO 2 atmosphere.

Techniques: In Vivo, In Vitro, Quantitative RT-PCR, Infection, Luciferase, Transfection, Construct, Mutagenesis, Staining, Western Blot, Expressing, Immunohistochemistry, Two Tailed Test, MANN-WHITNEY

Journal: Redox Biology

Article Title: An inducible long noncoding RNA, LncZFHX2, facilitates DNA repair to mediate osteoarthritis pathology

doi: 10.1016/j.redox.2023.102858

Figure Lengend Snippet: mLncZFHX2 regulates RIF1 expression via recruiting KLF4 onto RIF1 promoter. ( A ) Venn diagram of overlaps between ChIRP-seq (probe mLnc/input) and RNA-seq (Ad mLnc/Ad NC). ( B ) Heatmap of 10 genes in MCs treated with Ad mLnc or Ad NC. ( C ) RT-qPCR of mRNA levels of 10 genes in MCs treated with Ad mLnc or Ad NC (n = 6, mean ± SD). ( D ) MCs cultured in hypoxia for 48 h and subjected to ChIRP assay using biotin-labeled probes against mLncZFHX2. Precipitated DNA fragments were extracted for RT-qPCR to determine mLncZFHX2 enrichment on the RIF1 promoter (n = 6, mean ± SD). ( E ) Complementarity between the RIF1 promoter (−1323 to −1313) with the mLncZFHX2 sequence (82 to −92). ( F ) Venn diagram of overlap between probe mLnc specifically conjugated proteins and mouse transcription factors from CIS-BP. ( G ) RNA pulldown assay on biotin-labeled fragments of mLncZFHX2 from MC lysates. ( H ) Computed electrostatic potentials of mLncZFHX2 and KLF4 from molecular docking experiments. ( I ) A RIP assay of KLF4 and its mutants interacting with mLncZFHX2 was conducted using anti-Flag (n = 6, mean ± SD). ( J ) EMSA using biotin-labeled mLncZFHX2 and recombinant KLF4 with or without anti-KLF4. ( K ) Schematic of putative KLF4-binding sites within the RIF1 promoter region (2000 bp). KLF4 ChIP assay of the RIF1 promoter. ( L ) Luciferase reporter assay using RIF1 promoters with mLncZFHX2 or KLF4 overexpression in HEK-293 cells (n = 6, mean ± SD). ( M ) Luciferase assay of RIF1 wild-type and mutant promoter activity under mLncZFHX2 and KLF4 co-overexpression in HEK293 cells. ( N ) MCs were transfected with Ad NC or Ad mLnc and then subjected to KLF4 ChIP assay targeting the RIF1 promoter. ( O ) Schematic of mLncZFHX2 functional mechanisms. Graphs show P values from unpaired two-tailed t -test (C, D), one-way ANOVA (I, L, M) and two-way ANOVA (N).

Article Snippet: The human HEK-293 cell line was obtained from the American Type Culture Collection (ATCC, VA, USA), and the mice ATDC5 cell line was obtained from the Cell Engineering Division (Tsukuba, Japan) and maintained in DMEM containing 10% FBS (Gibco, Grand Island, NY, USA) at 37 °C in a 5% CO 2 atmosphere.

Techniques: Expressing, RNA Sequencing Assay, Quantitative RT-PCR, Cell Culture, Labeling, Sequencing, Recombinant, Binding Assay, Luciferase, Reporter Assay, Over Expression, Mutagenesis, Activity Assay, Transfection, Functional Assay, Two Tailed Test