human lung adenocarcinoma cell line nci h1299  (ATCC)

Journal: bioRxiv

Article Title: HIPK2-and IKKβ-dependent phosphorylation stabilizes TAp63α during the oocyte DNA damage response

doi: 10.64898/2026.04.17.719163

Figure Lengend Snippet: (A) Schematic overview of the experimental workflow used to identify TAp63α-interacting proteins from ovarian tissue. (B) Western blot analysis confirming successful immunoprecipitation of endogenous TAp63α from goat ovary lysates. (C) STRING network analysis of proteins identified by mass spectrometry, highlighting predicted interactions between TAp63α and the kinases HIPK2 and IKKβ. (D) Western blot analysis of ovarian lysates from mice of different ages showing that HIPK2 and TAp63α protein levels decrease with age, whereas IKKβ levels remain relatively constant. Two pairs of ovaries were pooled per sample. (E) Co-immunoprecipitation of endogenous TAp63α from mouse ovaries (eight pairs pooled per sample), followed by immunoblot detection of TAp63α and associated IKKβ. (F) Schematic representation of the experimental workflow used for interaction validation in a cell-based system. (G) Co-immunoprecipitation analysis in H1299 cells transiently expressing TAp63α, confirming interaction with HIPK2 and IKKβ. Whole-cell extracts and immunoprecipitates were probed with antibodies against TAp63α, HIPK2, and IKKβ. Due to differences in protein abundance and detection sensitivity, blots were processed and exposed separately to ensure optimal signal detection.

Article Snippet: The human lung adenocarcinoma cell line H1299 (ATCC - CRL-5803) was obtained from ATCC and cultured in RPMI 1640 medium (Cat no. 11875085, Gibco) supplemented with 10% fetal bovine serum (FBS) (Cat no. 10270106, Gibco) and 1× penicillin-streptomycin (Cat no. P4333, Sigma) at 37°C in a humidified incubator with 5% CO2.

Techniques: Western Blot, Immunoprecipitation, Mass Spectrometry, Biomarker Discovery, Expressing, Quantitative Proteomics

Journal: bioRxiv

Article Title: HIPK2-and IKKβ-dependent phosphorylation stabilizes TAp63α during the oocyte DNA damage response

doi: 10.64898/2026.04.17.719163

Figure Lengend Snippet: (A) Schematic overview of the experimental approach used to assess kinase-dependent regulation of TAp63α following DNA damage. (B) Western blot analysis showing a phosphorylation-dependent mobility shift of TAp63α upon doxorubicin treatment. This shift is reduced by CHK2 and CK1 inhibition and is further diminished following calf intestinal phosphatase (CIP) treatment, confirming phosphorylation-dependent modification. (C) Inhibition of HIPK2 or IKKβ, individually or in combination, reduces TAp63α phosphorylation and is associated with decreased protein stability. In contrast, inhibition of CHK2 or CK1 reduces phosphorylation without affecting total TAp63α levels. (D) Schematic representation of the siRNA-mediated knockdown strategy in stable TAp63α-expressing H1299 cells. (E) Knockdown of HIPK2 or IKKβ reduces the phosphorylation-associated mobility shift of TAp63α compared to control and scrambled siRNA conditions. Immunoblotting with phospho-serine/threonine antibodies further confirms a reduction in overall phosphorylation levels upon kinase depletion. (F) Western blot analysis of TAp63α following inhibition of CHK2, HIPK2, and IKKβ under DNA damage conditions. CHK2 inhibition reduces the phosphorylation-associated mobility shift of TAp63α. Inhibition of HIPK2 and IKKβ decreases total TAp63α protein stability. Co-inhibition of CHK2 with HIPK2 and IKKβ restores TAp63α protein levels when applied prior to or concurrently, but not when CHK2 inhibition is applied after HIPK2 and IKKβ inhibition. (G) Schematic representation of the in vivo experimental workflow. (H) Western blot analysis of mouse ovarian lysates showing that cisplatin induces a phosphorylation-associated mobility shift of TAp63α. Co-treatment with HIPK2 or IKKβ inhibitors reduces both phosphorylation and total TAp63α levels. Five pairs of ovaries were pooled per sample. (I) Schematic overview of the experimental approach used for oocyte analysis. (J–K) Representative images showing distinct patterns of TAp63α nuclear distribution in oocytes, along with their quantification. Treatment with HIPK2 or IKKβ inhibitors under DNA damage conditions results in a shift toward reduced nuclear signal intensity. Statistical significance for Class III follicles: doxorubicin + HIPK2 inhibitor (P = 0.0001) and doxorubicin + IKKβ inhibitor (P = 0.0001) compared to doxorubicin alone. Data are presented as mean ± SD; unpaired t-test.

Article Snippet: The human lung adenocarcinoma cell line H1299 (ATCC - CRL-5803) was obtained from ATCC and cultured in RPMI 1640 medium (Cat no. 11875085, Gibco) supplemented with 10% fetal bovine serum (FBS) (Cat no. 10270106, Gibco) and 1× penicillin-streptomycin (Cat no. P4333, Sigma) at 37°C in a humidified incubator with 5% CO2.

Techniques: Western Blot, Phospho-proteomics, Mobility Shift, Inhibition, Modification, Knockdown, Expressing, Control, In Vivo

Journal: MedComm

Article Title: Lycorine Derivative Inhibits SARS‐CoV‐2 Replication by Reducing −1 Programmed Ribosomal Frameshifting via Targeting ZAP

doi: 10.1002/mco2.70715

Figure Lengend Snippet: Compound 7 directly binds to ZAP and leads to a decrease in –1PRF. (A) Schematic representation of the dual‐luciferase frameshift reporter construct. The coding sequences for Renilla luciferase and Firefly luciferase were separated by the SARS‐CoV‐2 –1FSE sequence (13460‐13548). (B) Fold change in the Firefly/Renilla (F/R) luciferase ratio after treatment with the indicated drugs (lycorine at 10 µM, compound 7 at 10 µM, merafloxacin at 40 µM). Huh‐7 and H1299 cells transfected with the pHRF‐FSE (–1) luciferase reporter vector were treated with DMSO (Control) or the indicated drugs for 48 h ( n = 6 per group). (C) The frameshift Reporter mRNA containing a 3×FLAG‐tag followed by nucleotides 12686–14190 of the SARS‐CoV‐2 genome was translated in a RRL translation system in the presence of compound 7 or merafloxacin. The 3×FLAG‐tag was introduced at the N‐terminus to facilitate detection. WB analysis of the compound 7 effect on the –1PRF frameshift efficiency (FE) using anti‐flag antibody (Sigma, F3165). BC (blank control), NC (negative control). (D) Relative abundance of Nsp9, Nsp12, and Nsp15 in compound 7 (0, 2.5, and 5 µM) or Mer (merafloxacin, 40 µM)‐treated Vero cells after SARS‐CoV‐2 infection (MOI = 0.05). (E) The cellular target of compound 7 was identified using DARTS technology coupled with LC–MS/MS in H1299 cells. M, marker. (F) Venn diagram between the target proteins of compound 7 and the in vitro RNA antisense purification of the SARS‐CoV‐2 frameshift site from the literature. (G) ZAP protein stability was increased upon compound 7 (10 µM) treatment in H1299 cell lysates. (H) H1299 cells were transfected with the pcDNA3.1‐3×Flag‐Nsp12 plasmid and cultured for 48 h. Cells were then lysed and treated with 10 µM compound 7 (+) or DMSO (–). Recombinant Nsp12 (rNsp12) protein was detected by WB analysis, which was performed using anti‐Flag and anti‐Nsp12 antibodies for detection. (I) CETSA confirmed the binding of compound 7 (50 µM) to ZAP in 293T cells, with GAPDH serving as the internal control. (J) The binding of compound 7 to ZAP was depicted through BLI.

Article Snippet: The human lung adenocarcinoma Calu‐3 cell line (Procell, CL‐0054, Wuhan, China), human hepatoma Huh‐7 cell line (Procell, CL‐0120), human colon Caco‐2 cell line (Procell, CL‐0050), human lung adenocarcinoma H1299 cell line (Procell, CL‐0165), and human embryonic kidney HEK293T cell line (Procell, CL‐0005) were cultured in standard medium at 37°C and 5% CO 2 .

Techniques: Luciferase, Construct, Sequencing, Transfection, Plasmid Preparation, Control, Negative Control, Infection, Liquid Chromatography with Mass Spectroscopy, Marker, In Vitro, Purification, Cell Culture, Recombinant, Binding Assay

Journal: MedComm

Article Title: Lycorine Derivative Inhibits SARS‐CoV‐2 Replication by Reducing −1 Programmed Ribosomal Frameshifting via Targeting ZAP

doi: 10.1002/mco2.70715

Figure Lengend Snippet: Compound 7 exerts antiviral efficacy dependent upon ZAP‐S. (A) Effects of ZAP‐S knockdown on –1PRF followed by DMSO or compound 7 (10 µM) treatment in Huh‐7 and H1299 cells ( n = 3). (B) Compound 7 (10 µM) in combination with ZAP overexpression synergistically decreased –1PRF in H1299 and Huh‐7 cells ( n = 4); (C and D) Antiviral activity of compound 7 (2.5 µM) following ZAP‐S knockdown, or overexpression of ZAP‐S and mutant ZAP‐S. IF visualization of SARS‐CoV‐2 N protein (green) and cell nuclei (blue) in infected Huh‐7 cells at 48 h posttreatment (left). Scale bar: 100 µm. WB of N protein expression in Huh‐7 cells infected with SARS‐CoV‐2 (right).

Article Snippet: The human lung adenocarcinoma Calu‐3 cell line (Procell, CL‐0054, Wuhan, China), human hepatoma Huh‐7 cell line (Procell, CL‐0120), human colon Caco‐2 cell line (Procell, CL‐0050), human lung adenocarcinoma H1299 cell line (Procell, CL‐0165), and human embryonic kidney HEK293T cell line (Procell, CL‐0005) were cultured in standard medium at 37°C and 5% CO 2 .

Techniques: Knockdown, Over Expression, Activity Assay, Mutagenesis, Infection, Expressing