experimental models hek293t cell line atcc crl 3216 huh7 cell line jbrc jcrb0403 human primary t cells  (ATCC)

Journal: Journal of Nanobiotechnology

Article Title: Inhibition of METTL14 overcomes CDK4/6 inhibitor resistance driven by METTL14-m6A-E2F1-axis in ERα-positive breast cancer

doi: 10.1186/s12951-024-03021-2

Figure Lengend Snippet: METTL14 was associated with the progression of ERα + breast cancer and reduced the sensitivity of breast cancer cells to CDK4/6i therapy. A Representative image of IHC staining for METTL14 in clinical breast cancer samples. Scale bar = 50 μm. B The present study investigated the potential correlation between the expression of METTL14 in clinical samples of patients with breast cancer and overall survival. C METTL14 expression in tumor tissues (n = 80) and normal tissues (n = 80, t test). D The study explored the potential association between METTL14 expression and breast cancer stage (n = 17, 45 and 18 for histopathological grades I, II and III, respectively) in the aforementioned clinical samples (one-way ANOVA). E Correlation between METTL14 expression in clinical breast cancer samples and lymph node metastasis (n = 48 for nonmetastatic samples and n = 32 for metastatic samples) ( t test). F Representative image of IHC staining for METTL14 in samples from patients with ERα + breast cancer with and without relapse after CDK4/6i therapy. G METTL14 expression in samples from patients with ERα + breast cancer with relapse (n = 4) and without relapse (n = 4) after CDK4/6i therapy ( t test). H T47D-Par cells stably overexpressing METTL14 were generated. Changes in METTL14 expression were confirmed using western blotting. NC: cells transfected with a blank control vector. I The proliferative ability of T47D-Par cells stably overexpressing METTL14 was investigated via assessment of EdU incorporation (green), and representative images are shown. Scale bar = 25 μm. Nuclei were stained with DAPI. Breast cancer cells were treated with a CDK4/6i. J The proliferative ability of T47D-Par cells stably overexpressing METTL14 was investigated via a colony formation assay, and the numbers of colonies were determined (t test). Breast cancer cells were treated with a CDK4/6i. K T47D-Par cells stably overexpressing METTL14 were treated with graded concentrations of a CDK4/6i. The IC50 was measured by a cell counting kit-8 (CCK-8) assay after 48 h. L T47D-Res cells with stable METTL14 knockdown were generated. Changes in METTL14 expression were confirmed using western blotting. M The proliferative ability of T47D-Res cells with stable METTL14 knockdown was investigated via an EdU incorporation assay, and representative images are shown. Scale bar = 25 μm. Breast cancer cells were treated with a CDK4/6i. N The proliferative ability of T47D-Res cells with stable METTL14 knockdown was investigated via a colony formation assay, and the numbers of colonies were determined (one-way ANOVA test). Breast cancer cells were treated with a CDK4/6i. O T47D-Res cells with stable METTL14 knockdown were treated with graded concentrations of a CDK4/6i. The IC50 was measured by a cell counting kit-8 (CCK-8) assay after 48 h. The data are presented as the means ± SDs. ns P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: A human breast cancer cell line (T47D) and human renal TEC line (HEK293T) were purchased from the American Type Culture Collection and cultured following the manufacturer’s instructions.

Techniques: Immunohistochemistry, Expressing, Stable Transfection, Generated, Western Blot, Transfection, Control, Plasmid Preparation, Staining, Colony Assay, Cell Counting, CCK-8 Assay, Knockdown

Journal: Journal of Nanobiotechnology

Article Title: Inhibition of METTL14 overcomes CDK4/6 inhibitor resistance driven by METTL14-m6A-E2F1-axis in ERα-positive breast cancer

doi: 10.1186/s12951-024-03021-2

Figure Lengend Snippet: Studies in ex vivo and in vivo breast cancer models showed that METTL14 promoted CDK4/6i resistance. A – C Biopsy samples from clinical patients who received CDK4/6i therapy were obtained, and breast cancer organoids were generated. Resistant organoids were established by treatment with a CDK4/6i at sequentially increasing concentrations. We subsequently overexpressed METTL14 in parental BC organoids and knocked down METTL14 in CDK4/6i-resistant BC organoids. A Representative images of parental and CDK4/6i-resistant BC organoids. B The percentage growth of parental BC organoids with or without METTL14 overexpression after treatment with a CDK4/6i ( t test). C The percentage growth of CDK4/6i-resistant BC organoids with or without METTL14 knockdown after treatment with a CDK4/6i (one-way ANOVA). D – F T47D-Par cells with or without stable METTL14 overexpression were inoculated into the mammary fat pads of nude mice, and the mice were treated with a CDK4/6i (90 mg/kg, p.o., one injection per day for 2 weeks). Then, the tumors were excised and weighed after the mice were sacrificed. D Representative images of in vivo bioluminescence in each treatment group after the last treatment. E Representative images of IHC staining for METTL14 in tumor sections from the indicated groups. Scale bar = 25 μm. F Data analysis of in vivo bioluminescence in each treatment group was performed at the indicated time points ( t test). G – I T47D-Res cells with or without stable METTL14 knockdown were inoculated into the mammary fat pads of nude mice, which were subsequently treated with a CDK4/6i (90 mg/kg, p.o., one injection per day for 2 weeks). Subsequently, the mice were euthanized, and the tumors were excised and weighed. G Representative images of in vivo bioluminescence in each treatment group after the last treatment. H Representative images of IHC staining for METTL14 in tumor sections from the indicated groups. Scale bar = 25 μm. I Data analysis of in vivo bioluminescence in each treatment group was performed at the indicated time points (one-way ANOVA). J – M MMTV-PyMT-METTL14 ± and MMTV-PyMT-METTL14 +/+ mice were treated with a CDK4/6i (90 mg/kg, p.o., one injection per day for 2 weeks). Then, the tumors were excised and weighed after the mice were sacrificed. J Schematic diagram illustrating the genetic approach used to generate MMTV-PyMT-METTL14 ± mice. K The tumor volume was measured at the indicated time points ( t test). L The appearance of the tumors in the indicated groups. M The weights of the tumors in the indicated groups ( t test). The data are presented as the means ± SDs. ns P > 0.05, * P < 0.05, ** P < 0.01

Article Snippet: A human breast cancer cell line (T47D) and human renal TEC line (HEK293T) were purchased from the American Type Culture Collection and cultured following the manufacturer’s instructions.

Techniques: Ex Vivo, In Vivo, Generated, Over Expression, Knockdown, Injection, Immunohistochemistry

Journal: Journal of Nanobiotechnology

Article Title: Inhibition of METTL14 overcomes CDK4/6 inhibitor resistance driven by METTL14-m6A-E2F1-axis in ERα-positive breast cancer

doi: 10.1186/s12951-024-03021-2

Figure Lengend Snippet: METTL14-mediated m6A modification of E2F1 mRNA maintains its IGF2BP2-dependent stability. A A schematic illustration of the RNA-seq and MeRIP-seq results in T47D-Par cells with or without METTL14 overexpression. B KEGG enrichment analysis of RNA-seq data from T47D-Par cells with or without METTL14 overexpression. The “cell cycle”-related gene signature is labeled in black font. C Density distribution of m6A peaks across mRNA transcripts, as determined by MeRIP-seq. D Venn diagram illustrating the overlapping genes between the differentially expressed genes identified through MeRIP-seq and those identified through RNA-seq. E MeRIP-qPCR analysis was used to investigate changes in m6A modification of the E2F1 gene in T47D-Par cells with or without METTL14 overexpression (IgG was used as a negative control, t test). F Changes in E2F1 protein expression in T47D-Par cells with or without METTL14 overexpression were confirmed by western blotting. G Luciferase reporters containing either the wild-type or mutant E2F1 3′UTR were constructed. H The luciferase reporter assay was used to determine the abundances of transcripts generated from the luciferase vectors containing the wild-type and the mutated E2F1 3′-UTR, and the relative ratio of firefly luciferase activity to Renilla luciferase activity was calculated ( t test). I Decay rate of E2F1 mRNA after treatment with actinomycin D (5 g/ml) in T47D-Par cells with or without METTL14 overexpression ( t test). J Real-time PCR analysis of E2F1 expression in T47D-Par cells after individual knockdown of IGF2BP1, IGF2BP2 or IGF2BP3 (one-way ANOVA). K T47D cells were lysed and incubated with magnetic beads coated with IgG or an antibody specific for IGF2BP2. The RNA‒protein complexes were immunoprecipitated, and the relative interaction between E2F1 and IGF2BP2 was quantified via qPCR (with IgG serving as a negative control; t test). L Correlation between IGF2BP2 and E2F1 expression in ERα + breast cancer tissues from the TCGA database (n = 1226). The data are presented as the means ± SDs. ns P > 0.05, ** P < 0.01

Article Snippet: A human breast cancer cell line (T47D) and human renal TEC line (HEK293T) were purchased from the American Type Culture Collection and cultured following the manufacturer’s instructions.

Techniques: Modification, RNA Sequencing Assay, Over Expression, Labeling, Negative Control, Expressing, Western Blot, Luciferase, Mutagenesis, Construct, Reporter Assay, Generated, Activity Assay, Real-time Polymerase Chain Reaction, Knockdown, Incubation, Magnetic Beads, Immunoprecipitation

Journal: Journal of Nanobiotechnology

Article Title: Inhibition of METTL14 overcomes CDK4/6 inhibitor resistance driven by METTL14-m6A-E2F1-axis in ERα-positive breast cancer

doi: 10.1186/s12951-024-03021-2

Figure Lengend Snippet: CDK4/6i inhibits SPOP-mediated ubiquitination and degradation of METTL14 in CDK4/6i-resistant breast cancer cells. A This figure shows a schematic depiction of the domains of SPOP that interact with METTL14, as well as the mutations frequently associated with breast cancer. B The left panel shows the molecular docking of SPOP and METTL14, with METTL14 represented in yellow and SPOP represented in various colors. The diagram in the right panel highlights the interaction between SPOP and METTL14, which occurs specifically through the MATH domain encompassing amino acids 80–140. The red area indicates the active region of interaction, while the green area indicates the noninteracting regions. C Analysis of whole-cell lysates (WCLs) and SPOP-interacting products derived from T47D-Res cells was conducted using co-IP. Wild-type SPOP and SPOP proteins with mutations frequently associated with breast cancer are shown (IgG served as a negative control). D A cellular ubiquitination assay was performed on WCLs and His precipitates derived from T47D-Res cells transfected with specific constructs. The resulting reaction products were then subjected to immunoblot (IB) analysis. E In vitro ubiquitination assays were carried out using commercial E1, E2, and Ub proteins as well as purified Flag-METTL14 and different HA-SPOP proteins. The reaction products from these assays were also subjected to IB analysis. F T47D-Res cells were subjected to a 24-h treatment with either CDK4/6i or vehicle. Subsequently, cell lysates were collected for immunoblot analysis, which revealed that CDK4/6i treatment resulted in downregulation of SPOP expression and a concurrent increase in the METTL14 protein level

Article Snippet: A human breast cancer cell line (T47D) and human renal TEC line (HEK293T) were purchased from the American Type Culture Collection and cultured following the manufacturer’s instructions.

Techniques: Derivative Assay, Co-Immunoprecipitation Assay, Negative Control, Ubiquitin Assay, Transfection, Construct, Western Blot, In Vitro, Purification, Expressing

Journal: Journal of Nanobiotechnology

Article Title: Inhibition of METTL14 overcomes CDK4/6 inhibitor resistance driven by METTL14-m6A-E2F1-axis in ERα-positive breast cancer

doi: 10.1186/s12951-024-03021-2

Figure Lengend Snippet: Screening of METTL14 inhibitors. A Molecular docking structure of METTL14. B A CCK-8 assay was used to evaluate the therapeutic effects of 70 candidate compounds combined with a CDK4/6i in T47D-Res cells, and 20 candidate compounds (1 μM, 48 h) that could improve the therapeutic effect of CDK4/6i treatment in T47D-Res cells are shown. Compound #43 was WKYMVM-NH2. C Dot blot assay results showing the effect of the 20 candidate compounds in B on the m6A abundance in CDK4/6i-resistant T47D cells. A1-A7, compounds #04, #06, #08, #09, #13, #16, and #43; B1-B7, compounds #25, #28, #32, #33, #40, #48, and #53; C1-C7, compounds #55, #59, #61, #65, #68, and #70 and the blank control (treated with PBS). D Molecular docking analysis results showing the interaction between METTL14 and WKYMVM. E Molecular docking demonstrated that WKYMVM binds to the METTL3 binding domain in METTL14. F For the binding of WKYMVM, significant METTL14-induced fluorescence changes were observed in the MST assay; thus, the resulting dose‒response curves were fitted to a one-site binding model. G A schematic diagram showing the construction of the METTL14 truncation mutants. The wild type contains the full-length amino acid sequence of METTL14. The deletion type 1 and deletion type 2 constructs were designed for the deletion of aa 245–260 and 245–298, respectively, in METTL14. These three sequences were tagged with GST. H WKYMVM tagged with His was incubated with each of the three forms of METTL14 tagged with GST in Glutathione Agarose Resin, and the eluate was analyzed by dot blot, where the protein samples applied onto a nitrocellulose (NC) membrane, followed by incubation with antibodies, and then carrying out a chemiluminescence reaction. I Immunoprecipitation (IP) with an anti-HA antibody (METTL3) followed by immunoprecipitation with an anti-FLAG antibody (METTL14) and subsequent immunoblotting (IB) showed that METTL3 pulled down less METTL14 after WKYMVM treatment (1 μM, 48 h). The data are presented as the means ± SDs. ns P > 0.05, ** P < 0.01

Article Snippet: A human breast cancer cell line (T47D) and human renal TEC line (HEK293T) were purchased from the American Type Culture Collection and cultured following the manufacturer’s instructions.

Techniques: CCK-8 Assay, Dot Blot, Control, Binding Assay, Fluorescence, Sequencing, Construct, Incubation, Membrane, Immunoprecipitation, Western Blot

Journal: Journal of Nanobiotechnology

Article Title: Inhibition of METTL14 overcomes CDK4/6 inhibitor resistance driven by METTL14-m6A-E2F1-axis in ERα-positive breast cancer

doi: 10.1186/s12951-024-03021-2

Figure Lengend Snippet: The effectiveness of CDK4/6i treatment was augmented by the METTL14 inhibitor WKYMVM in vitro and in vivo. A The present study aimed to investigate the proliferative capacity of T47D-Par cells treated with CDK4/6i, as well as T47D-Par cells overexpressing METTL14 treated with CDK4/6i, CDK4/6i + PBS, and the combination of the CDK4/6i (1 μM, 48 h) and WKYMVM (1 μM, 48 h). This was accomplished through colony formation assays, and the resulting colony numbers were subsequently summarized (one-way ANOVA test). B The proliferative ability of T47D-Par cells with and without stable METTL14 overexpression treated with a CDK4/6i, CDK4/6i + PBS, or the combination of the CDK4/6i (1 μM) and WKYMVM (1 μM) was investigated via a CCK-8 assay at 48 h (one-way ANOVA test). C MeRIP-qPCR analysis of alterations in the m6A modification of E2F1 mRNA in T47D-Res cells with or without WKYMVM treatment (1 μM) ( t test). D Changes in E2F1 mRNA expression in T47D-Res cells treated with or without WKYMVM (1 μM, 48 h) were confirmed using qPCR ( t test). E Changes in E2F1 protein expression in T47D-Res cells treated with or without WKYMVM (1 μM, 48 h) were confirmed via western blotting. F Decay rate of E2F1 mRNA after treatment with actinomycin D (5 g/ml) in T47D-Res cells treated with or without WKYMVM (1 μM, 48 h) ( t test). G The proliferation of T47D-Res cells treated with WKYMVM (1 μM, 48 h) alone, a CDK4/6i (1 μM, 48 h) alone, or the combination of WKYMVM and the CDK4/6i was assessed using an EdU incorporation assay. Representative images of the cells are displayed. PBS was utilized as the control treatment. Scale bar = 25 μm. H – K Mice bearing CDK4/6i-resistant PDXs were treated with control, WKYMVM, a CDK4/6i, or the combination of the CDK4/6i and WKYMVM (CDK4/6i, 90 mg/kg, p.o., one injection per day for 2 weeks; WKYMVM, 50 mg/kg, i.p., one injection every two days for 2 weeks). Then, the tumors were excised and weighed after the mice were sacrificed. H Schematic diagram illustrating the workflow for constructing CDK4/6i-resistant PDX models. I The size of the tumors was measured at the indicated time points (one-way ANOVA). J The weights of tumors from the indicated groups were measured (one-way ANOVA). K The appearance of the tumors in the indicated groups. The data are presented as the means ± SDs. ns P > 0.05, * P < 0.05, ** P < 0.01

Article Snippet: A human breast cancer cell line (T47D) and human renal TEC line (HEK293T) were purchased from the American Type Culture Collection and cultured following the manufacturer’s instructions.

Techniques: In Vitro, In Vivo, Over Expression, CCK-8 Assay, Modification, Expressing, Western Blot, Control, Injection

Journal: Journal of Nanobiotechnology

Article Title: Inhibition of METTL14 overcomes CDK4/6 inhibitor resistance driven by METTL14-m6A-E2F1-axis in ERα-positive breast cancer

doi: 10.1186/s12951-024-03021-2

Figure Lengend Snippet: Targeted delivery of a CDK4/6i combined with WKYMVM sensitized breast cancer cells to CDK4/6i treatment. A A drug delivery system incorporating a CDK4/6i in combination with WKYMVM was developed to target breast cancer cells. Liposomes were designed to contain both a CDK4/6i and WKYMVM, and folate was conjugated to the liposome membrane surface, allowing the liposomes to target breast cancer cells with high folate receptor expression. The CDK4/6i was located between the phospholipid bilayers, while the peptide WKYMVM was located inside the hydrophilic interior of the liposome. B Transmission electron micrograph showing the microstructure of the liposomes in each group. Lipo-V, liposomes containing vehicle. Lipo-I, liposomes containing only the CDK4/6i. Lipo-W, liposomes containing only WKYMVM. Lipo-C, liposomes containing both the CDK4/6i and WKYMVM. C-D The average hydrodynamic size and average zeta potential of the liposomes in each group. E T47D cells were scanned after coculture with Cy3-labeled liposomes (1 µg or 10 µg) for 12 h. F-actin (green) indicates the cytoskeleton, DAPI (blue) indicates the nucleus, and Cy3 (red) indicates the Cy3-labeled liposome. F T47D cells were scanned after coculture with Cy3-labeled liposomes (1 µg) for 1 h with or without a folate receptor blocking antibody (FR Ab). F-actin (green) indicates the cytoskeleton, DAPI (blue) indicates the nucleus, and Cy3 (red) indicates the Cy3-labeled liposome. G The proliferative ability of T47D-Res cells was investigated via colony formation assays, and the numbers of colonies were summarized (one-way ANOVA). H The proliferation of T47D-Res cells treated with different liposomes was tested by a CCK-8 assay at 48 h (one-way ANOVA). I – L Mice bearing CDK4/6i-resistant PDXs were treated with liposomes. Lipo-V, liposomes containing vehicle. Lipo-I, liposomes containing CDK4/6i alone. Lipo-W, liposomes containing WKYMVM alone. Lipo-C-Liposomes containing both CDK4/6i and WKYMVM (all liposomes, 1 g/kg, i.v., one injection every three days for 2 weeks). Then, the tumors were excised and weighed after the mice were sacrificed. I The timeline of treatment with liposomes and tumor measurements in PDX model mice. J The size of the tumors was measured at the indicated time points (one-way ANOVA). K The weights of tumors from the indicated groups were measured (one-way ANOVA). L Representative images of IHC and HE staining of tumor sections from the indicated groups. Scale bar = 25 μm. The data are presented as the means ± SDs. ns P > 0.05, * P < 0.05, ** P < 0.01

Article Snippet: A human breast cancer cell line (T47D) and human renal TEC line (HEK293T) were purchased from the American Type Culture Collection and cultured following the manufacturer’s instructions.

Techniques: Liposomes, Membrane, Expressing, Transmission Assay, Zeta Potential Analyzer, Labeling, Blocking Assay, CCK-8 Assay, Injection, Staining

Journal: International Journal of Molecular Sciences

Article Title: Zinc Deficiency Exacerbates Lead-Induced Interleukin-2 Suppression by Regulating CREM Expression

doi: 10.3390/ijms26010254

Figure Lengend Snippet: Optimizing zinc conditions and lead exposure. Jurkat T cells were incubated in zinc-adequate (ZA), zinc-deficient (ZD), and zinc-supplemented (ZS) conditions for one week, ( a ) stained with FluoZin-3 AM for intracellular zinc measurements (ZA n = 36, ZD n = 32, ZS n = 6) and ( b ) with propidium iodide (PI) for cell viability (ZA n = 39, ZD n = 27, ZS n = 6). In addition, cells were incubated for 24 h, 48 h, and 72 h without or with 1 µM or 25 µM of lead (Pb) and ( c ) stained with propidium iodide for cell viability to examine the lead-dependent toxicity ( n = 3). All experiments were performed using flow cytometry. Data are presented as mean + SEM. Statistical significance was calculated by ordinary one-way ANOVA with the Holm–Sidak post hoc test ( a , b ) and two-way ANOVA with the Holm–Sidak post hoc test ( c ) with bars not sharing the same letter indicating statistically significant differences ( p < 0.05, ns: not significant).

Article Snippet: The human T lymphocyte cell line Jurkat was cultivated at 37 °C in 5% CO 2 in a cell culture medium containing RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% heat-inactivated FCS, 2 mM L-glutamine, 100 U/mL potassium penicillin,100 µg/mL streptomycin sulfate, 1 mM sodium pyruvate, and 1% 100x nonessential amino acids (all Sigma-Aldrich, St. Louis, MO, USA).

Techniques: Incubation, Staining, Flow Cytometry

Journal: International Journal of Molecular Sciences

Article Title: Zinc Deficiency Exacerbates Lead-Induced Interleukin-2 Suppression by Regulating CREM Expression

doi: 10.3390/ijms26010254

Figure Lengend Snippet: Lead and zinc modulate interleukin-2 (IL-2) production in Jurkat T cells. Jurkat T cells were incubated in ( a ) zinc-adequate (ZA) ( n = 12–14), ( b ) zinc-deficient (ZD) ( n = 6), and ( c ) zinc-supplemented (ZS) (n = 3) conditions for one week and then without or with 1 µM of lead (Pb) for 24 h. Subsequently, the cells were stimulated with ( a – c ) 1 µg/mL phytohaemagglutinin (PHA) or ( d ) left unstimulated ( n = 3), and after 24 h the supernatants were taken. IL-2 production was measured by ELISA assay. The same data as in ( a – c ) are displayed in e with an emphasis on the comparison of the different zinc conditions. Data are presented with mean + SEM. Statistical significance was calculated by a two-tailed paired t -test ( a – d ) and ordinary one-way ANOVA with the Holm–Sidak post hoc test ( e ) (* p < 0.05, ** p < 0.01, *** p < 0.001, ns: not significant).

Article Snippet: The human T lymphocyte cell line Jurkat was cultivated at 37 °C in 5% CO 2 in a cell culture medium containing RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% heat-inactivated FCS, 2 mM L-glutamine, 100 U/mL potassium penicillin,100 µg/mL streptomycin sulfate, 1 mM sodium pyruvate, and 1% 100x nonessential amino acids (all Sigma-Aldrich, St. Louis, MO, USA).

Techniques: Incubation, Enzyme-linked Immunosorbent Assay, Comparison, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: Zinc Deficiency Exacerbates Lead-Induced Interleukin-2 Suppression by Regulating CREM Expression

doi: 10.3390/ijms26010254

Figure Lengend Snippet: Lead does not increase 45/36 kDa cAMP responsive element modulator (CREM). Jurkat T cells were incubated ( a ) long-term in zinc-adequate (ZA) and zinc-deficient (ZD) medium ( n = 5). Cells were lysed for Western blot analysis. ( b ) Jurkat T cells were incubated without or with 1 µM of lead (Pb). After 48 h, lysates were collected ( n = 11) or ( c ) cells were isolated for CREMα RNA analysis by PCR ( n = 3). In experiments a and b, CREM and β-Actin levels were determined by Western blot analysis. CREM was normalized to β-Actin expression, and representative Western blots are shown. CREM and β-Actin were run on different gels. Western blot ( a ) shows one membrane but not adjacent lanes. Data are presented as mean + SEM. Statistical significance was calculated by a two-tailed paired t -test. (* p < 0.05, ns: not significant).

Article Snippet: The human T lymphocyte cell line Jurkat was cultivated at 37 °C in 5% CO 2 in a cell culture medium containing RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% heat-inactivated FCS, 2 mM L-glutamine, 100 U/mL potassium penicillin,100 µg/mL streptomycin sulfate, 1 mM sodium pyruvate, and 1% 100x nonessential amino acids (all Sigma-Aldrich, St. Louis, MO, USA).

Techniques: Incubation, Western Blot, Isolation, Expressing, Membrane, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: Zinc Deficiency Exacerbates Lead-Induced Interleukin-2 Suppression by Regulating CREM Expression

doi: 10.3390/ijms26010254

Figure Lengend Snippet: Zinc supplementation prevents cAMP responsive element modulator (CREM) 100 kDa increase in lead-exposed Jurkat T cells. Jurkat T cells were incubated in ( a ) zinc-adequate (ZA) ( n = 12), ( b ) zinc-deficient (ZD) ( n = 9), and ( c ) zinc-supplemented (ZS) ( n = 6) conditions for one week and then without or with 1 µM of lead (Pb). After 48 h, lysates were collected. CREM and β-Actin levels were determined by Western blot analysis. CREM was normalized to β-Actin expression, and representative Western blots are shown. CREM and β-Actin were run on different gels. The same data as in ( a – c ) are displayed in ( d , e ) to better depict the differences between zinc conditions. Data are presented with mean + SEM. Statistical significance was calculated by a two-tailed paired t -test ( a – d ) and ordinary one-way ANOVA with the Holm–Sidak post hoc test ( e ) (* p < 0.05, ** p < 0.01, *** p < 0.001, ns: not significant).

Article Snippet: The human T lymphocyte cell line Jurkat was cultivated at 37 °C in 5% CO 2 in a cell culture medium containing RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% heat-inactivated FCS, 2 mM L-glutamine, 100 U/mL potassium penicillin,100 µg/mL streptomycin sulfate, 1 mM sodium pyruvate, and 1% 100x nonessential amino acids (all Sigma-Aldrich, St. Louis, MO, USA).

Techniques: Incubation, Western Blot, Expressing, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: Zinc Deficiency Exacerbates Lead-Induced Interleukin-2 Suppression by Regulating CREM Expression

doi: 10.3390/ijms26010254

Figure Lengend Snippet: Overview of lead- and zinc-associated changes in IL-2 production and CREM expression. Lead (Pb) increases CREM expression in zinc-adequate (ZA) Jurkat T cells and concurrently lowers IL-2 production. This effect is enhanced by zinc deficiency (ZD) while zinc supplementation (ZS) can prevent the CREM increase and subsequent IL-2 decrease. This figure was created with BioRender.com.

Article Snippet: The human T lymphocyte cell line Jurkat was cultivated at 37 °C in 5% CO 2 in a cell culture medium containing RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% heat-inactivated FCS, 2 mM L-glutamine, 100 U/mL potassium penicillin,100 µg/mL streptomycin sulfate, 1 mM sodium pyruvate, and 1% 100x nonessential amino acids (all Sigma-Aldrich, St. Louis, MO, USA).

Techniques: Expressing