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hela human cervical cancer cells (ATCC)

Name: hela human cervical cancer cells
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ATCC hela human cervical cancer cells

(a) Fluorescence confocal images of <t>HeLa,</t> <t>4T1,</t> MCF-7, and NIH 3T3 cells after incubation with Pro-BDP-3 (5.0 μM) for 2 h with or without further incubation with RuL2 or RuL3 (2.5 μM) for a further 4 h (red fluorescence; λ ex = 633 nm, λ em = 650–900 nm). The cells being incubated with BDP-COOH (5.0 μM) for 2 h were used as the positive control. The cell nuclei were stained with Hoechst (1.0 μM) for 15 min (blue fluorescence; λ ex = 405 nm, λ em = 420–500 nm). Scale bar = 20 μm. (b) Corresponding mean red fluorescence intensities quantified by ImageJ. Data are reported as the mean ± standard error of the mean (SEM) for three independent experiments (∗∗∗∗p < 0.0001). (c) Fluorescence confocal images of HeLa, 4T1, MCF-7, and NIH 3T3 cells after the aforementioned treatments and further incubation with H 2 DCFDA (10 μM) for 30 min, followed by light irradiation (λ > 610 nm, 25.8 mW/cm 2 ) for 8 min to give a total fluence of 12 J/cm 2 (green fluorescence; λ ex = 488 nm, λ em = 493–550 nm). Scale bar = 20 μm. (d) Corresponding mean green fluorescence intensities of DCF quantified by ImageJ. Data are reported as the mean ± SEM for three independent experiments (∗∗∗∗p < 0.0001). (e) Dark and photo (λ > 610 nm, 25.8 mW/cm 2 , 12 J/cm 2 ) cytotoxicity of BDP-COOH , Pro-BDP-3 , RuL2 , Pro-BDP-3 + RuL2 , RuL3 , and Pro-BDP-3 + RuL3 against HeLa, 4T1, MCF-7, and NIH 3T3 cells. The cells were incubated with BDP-COOH , Pro-BDP-3 , RuL2 , or RuL3 for 2 h. For Pro-BDP-3 + RuL2 and Pro-BDP-3 + RuL3 , the cells were first incubated with Pro-BDP-3 for 2 h and then with RuL2 or RuL3 (0.5 equiv.) for a further 4 h. Data are expressed as the mean ± SEM of three independent experiments, each performed in quadruplicate. (f) Photocytotoxicity of these agents at 5.0 μM and the combination treatments at 5.0 μM of Pro-BDP-3 against the four cell lines. The rightmost figure compiles the results for Pro-BDP-3 + RuL3 (∗∗∗∗p < 0.0001). Data are expressed as the mean ± SEM of three independent experiments, each performed in quadruplicate. (g) Live/dead cell viability assay using calcein-AM and PI. The cells were treated as described above, followed by incubation with calcein-AM (1 μM) and PI (2 μM) in binding buffer (2 mL) at 37 °C for 30 min. The live cells were indicated by the green fluorescence of calcein-AM (λ ex = 488 nm, λ em = 493–550 nm), while the dead cells were indicated by the red fluorescence of PI (λ ex = 561 nm, λ em = 600–800 nm). Scale bar = 50 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Hela Human Cervical Cancer Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 29054 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Expanding the toolbox of bioorthogonal activation of photosensitizers for precise photodynamic therapy through transition metal-mediated deallylation"

Article Title: Expanding the toolbox of bioorthogonal activation of photosensitizers for precise photodynamic therapy through transition metal-mediated deallylation

Journal: Materials Today Bio

doi: 10.1016/j.mtbio.2026.102797

(a) Fluorescence confocal images of HeLa, 4T1, MCF-7, and NIH 3T3 cells after incubation with Pro-BDP-3 (5.0 μM) for 2 h with or without further incubation with RuL2 or RuL3 (2.5 μM) for a further 4 h (red fluorescence; λ ex = 633 nm, λ em = 650–900 nm). The cells being incubated with BDP-COOH (5.0 μM) for 2 h were used as the positive control. The cell nuclei were stained with Hoechst (1.0 μM) for 15 min (blue fluorescence; λ ex = 405 nm, λ em = 420–500 nm). Scale bar = 20 μm. (b) Corresponding mean red fluorescence intensities quantified by ImageJ. Data are reported as the mean ± standard error of the mean (SEM) for three independent experiments (∗∗∗∗p < 0.0001). (c) Fluorescence confocal images of HeLa, 4T1, MCF-7, and NIH 3T3 cells after the aforementioned treatments and further incubation with H 2 DCFDA (10 μM) for 30 min, followed by light irradiation (λ > 610 nm, 25.8 mW/cm 2 ) for 8 min to give a total fluence of 12 J/cm 2 (green fluorescence; λ ex = 488 nm, λ em = 493–550 nm). Scale bar = 20 μm. (d) Corresponding mean green fluorescence intensities of DCF quantified by ImageJ. Data are reported as the mean ± SEM for three independent experiments (∗∗∗∗p < 0.0001). (e) Dark and photo (λ > 610 nm, 25.8 mW/cm 2 , 12 J/cm 2 ) cytotoxicity of BDP-COOH , Pro-BDP-3 , RuL2 , Pro-BDP-3 + RuL2 , RuL3 , and Pro-BDP-3 + RuL3 against HeLa, 4T1, MCF-7, and NIH 3T3 cells. The cells were incubated with BDP-COOH , Pro-BDP-3 , RuL2 , or RuL3 for 2 h. For Pro-BDP-3 + RuL2 and Pro-BDP-3 + RuL3 , the cells were first incubated with Pro-BDP-3 for 2 h and then with RuL2 or RuL3 (0.5 equiv.) for a further 4 h. Data are expressed as the mean ± SEM of three independent experiments, each performed in quadruplicate. (f) Photocytotoxicity of these agents at 5.0 μM and the combination treatments at 5.0 μM of Pro-BDP-3 against the four cell lines. The rightmost figure compiles the results for Pro-BDP-3 + RuL3 (∗∗∗∗p < 0.0001). Data are expressed as the mean ± SEM of three independent experiments, each performed in quadruplicate. (g) Live/dead cell viability assay using calcein-AM and PI. The cells were treated as described above, followed by incubation with calcein-AM (1 μM) and PI (2 μM) in binding buffer (2 mL) at 37 °C for 30 min. The live cells were indicated by the green fluorescence of calcein-AM (λ ex = 488 nm, λ em = 493–550 nm), while the dead cells were indicated by the red fluorescence of PI (λ ex = 561 nm, λ em = 600–800 nm). Scale bar = 50 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Figure Legend Snippet: (a) Fluorescence confocal images of HeLa, 4T1, MCF-7, and NIH 3T3 cells after incubation with Pro-BDP-3 (5.0 μM) for 2 h with or without further incubation with RuL2 or RuL3 (2.5 μM) for a further 4 h (red fluorescence; λ ex = 633 nm, λ em = 650–900 nm). The cells being incubated with BDP-COOH (5.0 μM) for 2 h were used as the positive control. The cell nuclei were stained with Hoechst (1.0 μM) for 15 min (blue fluorescence; λ ex = 405 nm, λ em = 420–500 nm). Scale bar = 20 μm. (b) Corresponding mean red fluorescence intensities quantified by ImageJ. Data are reported as the mean ± standard error of the mean (SEM) for three independent experiments (∗∗∗∗p < 0.0001). (c) Fluorescence confocal images of HeLa, 4T1, MCF-7, and NIH 3T3 cells after the aforementioned treatments and further incubation with H 2 DCFDA (10 μM) for 30 min, followed by light irradiation (λ > 610 nm, 25.8 mW/cm 2 ) for 8 min to give a total fluence of 12 J/cm 2 (green fluorescence; λ ex = 488 nm, λ em = 493–550 nm). Scale bar = 20 μm. (d) Corresponding mean green fluorescence intensities of DCF quantified by ImageJ. Data are reported as the mean ± SEM for three independent experiments (∗∗∗∗p < 0.0001). (e) Dark and photo (λ > 610 nm, 25.8 mW/cm 2 , 12 J/cm 2 ) cytotoxicity of BDP-COOH , Pro-BDP-3 , RuL2 , Pro-BDP-3 + RuL2 , RuL3 , and Pro-BDP-3 + RuL3 against HeLa, 4T1, MCF-7, and NIH 3T3 cells. The cells were incubated with BDP-COOH , Pro-BDP-3 , RuL2 , or RuL3 for 2 h. For Pro-BDP-3 + RuL2 and Pro-BDP-3 + RuL3 , the cells were first incubated with Pro-BDP-3 for 2 h and then with RuL2 or RuL3 (0.5 equiv.) for a further 4 h. Data are expressed as the mean ± SEM of three independent experiments, each performed in quadruplicate. (f) Photocytotoxicity of these agents at 5.0 μM and the combination treatments at 5.0 μM of Pro-BDP-3 against the four cell lines. The rightmost figure compiles the results for Pro-BDP-3 + RuL3 (∗∗∗∗p < 0.0001). Data are expressed as the mean ± SEM of three independent experiments, each performed in quadruplicate. (g) Live/dead cell viability assay using calcein-AM and PI. The cells were treated as described above, followed by incubation with calcein-AM (1 μM) and PI (2 μM) in binding buffer (2 mL) at 37 °C for 30 min. The live cells were indicated by the green fluorescence of calcein-AM (λ ex = 488 nm, λ em = 493–550 nm), while the dead cells were indicated by the red fluorescence of PI (λ ex = 561 nm, λ em = 600–800 nm). Scale bar = 50 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Techniques Used: Fluorescence, Incubation, Positive Control, Staining, Irradiation, Viability Assay, Binding Assay

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Journal: Materials Today Bio

Article Title: Expanding the toolbox of bioorthogonal activation of photosensitizers for precise photodynamic therapy through transition metal-mediated deallylation

doi: 10.1016/j.mtbio.2026.102797

Figure Lengend Snippet: (a) Fluorescence confocal images of HeLa, 4T1, MCF-7, and NIH 3T3 cells after incubation with Pro-BDP-3 (5.0 μM) for 2 h with or without further incubation with RuL2 or RuL3 (2.5 μM) for a further 4 h (red fluorescence; λ ex = 633 nm, λ em = 650–900 nm). The cells being incubated with BDP-COOH (5.0 μM) for 2 h were used as the positive control. The cell nuclei were stained with Hoechst (1.0 μM) for 15 min (blue fluorescence; λ ex = 405 nm, λ em = 420–500 nm). Scale bar = 20 μm. (b) Corresponding mean red fluorescence intensities quantified by ImageJ. Data are reported as the mean ± standard error of the mean (SEM) for three independent experiments (∗∗∗∗p < 0.0001). (c) Fluorescence confocal images of HeLa, 4T1, MCF-7, and NIH 3T3 cells after the aforementioned treatments and further incubation with H 2 DCFDA (10 μM) for 30 min, followed by light irradiation (λ > 610 nm, 25.8 mW/cm 2 ) for 8 min to give a total fluence of 12 J/cm 2 (green fluorescence; λ ex = 488 nm, λ em = 493–550 nm). Scale bar = 20 μm. (d) Corresponding mean green fluorescence intensities of DCF quantified by ImageJ. Data are reported as the mean ± SEM for three independent experiments (∗∗∗∗p < 0.0001). (e) Dark and photo (λ > 610 nm, 25.8 mW/cm 2 , 12 J/cm 2 ) cytotoxicity of BDP-COOH , Pro-BDP-3 , RuL2 , Pro-BDP-3 + RuL2 , RuL3 , and Pro-BDP-3 + RuL3 against HeLa, 4T1, MCF-7, and NIH 3T3 cells. The cells were incubated with BDP-COOH , Pro-BDP-3 , RuL2 , or RuL3 for 2 h. For Pro-BDP-3 + RuL2 and Pro-BDP-3 + RuL3 , the cells were first incubated with Pro-BDP-3 for 2 h and then with RuL2 or RuL3 (0.5 equiv.) for a further 4 h. Data are expressed as the mean ± SEM of three independent experiments, each performed in quadruplicate. (f) Photocytotoxicity of these agents at 5.0 μM and the combination treatments at 5.0 μM of Pro-BDP-3 against the four cell lines. The rightmost figure compiles the results for Pro-BDP-3 + RuL3 (∗∗∗∗p < 0.0001). Data are expressed as the mean ± SEM of three independent experiments, each performed in quadruplicate. (g) Live/dead cell viability assay using calcein-AM and PI. The cells were treated as described above, followed by incubation with calcein-AM (1 μM) and PI (2 μM) in binding buffer (2 mL) at 37 °C for 30 min. The live cells were indicated by the green fluorescence of calcein-AM (λ ex = 488 nm, λ em = 493–550 nm), while the dead cells were indicated by the red fluorescence of PI (λ ex = 561 nm, λ em = 600–800 nm). Scale bar = 50 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The HeLa human cervical cancer cells (ATCC, CCL-2), 4T1 murine mammary carcinoma cells (ATCC, CRL-2539), MCF-7 human breast cancer cells (ATCC, HTB-22), and NIH 3T3 murine embryonic fibroblast cells were maintained in Dulbecco's modified Eagle's medium (DMEM, ThermoFisher, cat. no. 11965092) supplemented with fetal calf serum (10 %) and penicillin-streptomycin (100 unit/mL and 100 μg/mL, respectively).

Techniques: Fluorescence, Incubation, Positive Control, Staining, Irradiation, Viability Assay, Binding Assay

Stutter cysteine is sufficient to relocalize YAP1 in transfected HeLa cells. (A and B) Representative images and magnified insets of HeLa cells transfected with mCherry-K5, EGFP-K14, and untagged WT and mutant K15. The mCherry signal is autofluorescence, and YAP1 is visualized through indirect immunostaining. Yellow dashed lines outline nuclei, and green dashed line outline the cell peripheries. Scale bar, 10 µm. (C) Scatter plots representing three pooled independent replicates, displaying mean and standard deviation. Dots represent a sc. Comparisons were made using Mann–Whitney tests. ****P < 0.0001, ***P = 0.0002 and 0.0003, **P = 0.0083.

Journal: The Journal of Cell Biology

Article Title: Keratin 15 promotes a progenitor cell state in basal keratinocytes of skin epidermis

doi: 10.1083/jcb.202503046

Figure Lengend Snippet: Stutter cysteine is sufficient to relocalize YAP1 in transfected HeLa cells. (A and B) Representative images and magnified insets of HeLa cells transfected with mCherry-K5, EGFP-K14, and untagged WT and mutant K15. The mCherry signal is autofluorescence, and YAP1 is visualized through indirect immunostaining. Yellow dashed lines outline nuclei, and green dashed line outline the cell peripheries. Scale bar, 10 µm. (C) Scatter plots representing three pooled independent replicates, displaying mean and standard deviation. Dots represent a sc. Comparisons were made using Mann–Whitney tests. ****P < 0.0001, ***P = 0.0002 and 0.0003, **P = 0.0083.

Article Snippet: HeLa cells were purchased from ATCC and were routinely tested for Mycoplasma using MycoAlert Mycoplasma Detection Kit (LT07-118; Lonza).

Techniques: Transfection, Mutagenesis, Immunostaining, Standard Deviation, MANN-WHITNEY

Analyses of keratin and 14-3-3 protein interactions (complement to ). (A–C′) Lollipop plots displaying the 14-3-3-Pred “consensus score” for all serines and threonines in (A) human KRT14 ; (A′) mouse Krt14 ; (B) human KRT5 ; (B′) mouse Krt5 ; (C) human KRT15 ; (C′) mouse Krt15 . Lollipop height and color both scale to consensus score magnitude. The two residues with the largest positive consensus score are labeled. (D) Lollipop plot illustrates identified phosphorylation sites on K14 protein exhibiting a localization score >0.6 in human N-TERT keratinocytes in culture. The relative occupancy percentage for each phosphorylation site was calculated by dividing the phosphopeptide’s intensity by total intensity of unique K14 peptides. This calculation, shown here as relative phosphorylation site occupancy (%), was performed only for phosphopeptides that are unique to K14 without miscleavages (S44, S281, S435, S437). (D′) Spectra of the phosphopeptides corresponding to the S39 (left) and S44 (right) sites. “Phos” conveys phosphorylation, and “CAM” stands for carbamidomethyl. (E) Representative micrographs of YAP1-14-3-3σ PLA performed on transfected HeLa cells. Cells were transfected with untagged K5 and WT and mutant K14 or K15. Merge panels show PLA punctae (red), EGFP-tagged keratin autofluorescence (green), and DAPI counterstain (blue). Scale bar = 10 µm.

Journal: The Journal of Cell Biology

Article Title: Keratin 15 promotes a progenitor cell state in basal keratinocytes of skin epidermis

doi: 10.1083/jcb.202503046

Figure Lengend Snippet: Analyses of keratin and 14-3-3 protein interactions (complement to ). (A–C′) Lollipop plots displaying the 14-3-3-Pred “consensus score” for all serines and threonines in (A) human KRT14 ; (A′) mouse Krt14 ; (B) human KRT5 ; (B′) mouse Krt5 ; (C) human KRT15 ; (C′) mouse Krt15 . Lollipop height and color both scale to consensus score magnitude. The two residues with the largest positive consensus score are labeled. (D) Lollipop plot illustrates identified phosphorylation sites on K14 protein exhibiting a localization score >0.6 in human N-TERT keratinocytes in culture. The relative occupancy percentage for each phosphorylation site was calculated by dividing the phosphopeptide’s intensity by total intensity of unique K14 peptides. This calculation, shown here as relative phosphorylation site occupancy (%), was performed only for phosphopeptides that are unique to K14 without miscleavages (S44, S281, S435, S437). (D′) Spectra of the phosphopeptides corresponding to the S39 (left) and S44 (right) sites. “Phos” conveys phosphorylation, and “CAM” stands for carbamidomethyl. (E) Representative micrographs of YAP1-14-3-3σ PLA performed on transfected HeLa cells. Cells were transfected with untagged K5 and WT and mutant K14 or K15. Merge panels show PLA punctae (red), EGFP-tagged keratin autofluorescence (green), and DAPI counterstain (blue). Scale bar = 10 µm.

Article Snippet: HeLa cells were purchased from ATCC and were routinely tested for Mycoplasma using MycoAlert Mycoplasma Detection Kit (LT07-118; Lonza).

Techniques: Labeling, Phospho-proteomics, Transfection, Mutagenesis

$SCRAPS motif and the stutter cysteine are required to interconvert YAP1 interaction between K14 and K15. (A) Representative co-immunoprecipitations of HeLa cells transfected with untagged K5 and EGFP-tagged WT K14, K15, and mutant K15. Pull-down of endogenous 14-3-3σ is depicted. (B) Differential levels of WT K15, relative to WT K14 or mutant K15, were observed in the soluble fraction. Transfection of keratin(s) did not affect endogenous 14-3-3σ levels. WB intensity was normalized to a loading control (histone H3). Dots represent three independent replicates, displaying mean and standard error. Comparisons were made using one-way ANOVA, ****P < 0.0001, ns = not significant. (B′) Endogenous 14-3-3σ pulls down more efficiently with WT K14 and mutagenized K15, as compared to WT K15. Equation to calculate 14-3-3σ pull-down efficiency is described in Materials and methods. Dots represent three independent replicates. Comparisons were made using one-way ANOVA, **P = 0.0055, *P = 0.0237, ns = not significant. (C) Representative micrographs of YAP1-14-3-3σ PLA performed on transfected HeLa cells. Cells were transfected with untagged K5 and EGFP-tagged WT and mutant K14 or K15. Merge panels show PLA punctae (red), EGFP-tagged keratin autofluorescence (green), and DAPI counterstain (blue). Scale bar = 10 µm. (D) Scatter plots representing YAP1-14-3-3σ PLA punctae counted per cell in three pooled independent replicates, displaying mean and standard deviation. Dots represent a sc. Comparisons were made using Mann–Whitney tests. ****P < 0.0001, **P = 0.0033, ns = not significant. (E) Transcription of a YAP1 target gene, CYR61 , as measured via RT-qPCR in HeLa cells transfected with untagged K5 and WT and mutant K14 or K15. Dots represent six independent replicates. Comparisons were made using Mann–Whitney tests, **P = 0.0033, ns = not significant. Source data are available for this figure: .$

Journal: The Journal of Cell Biology

Article Title: Keratin 15 promotes a progenitor cell state in basal keratinocytes of skin epidermis

doi: 10.1083/jcb.202503046

Figure Lengend Snippet: SCRAPS motif and the stutter cysteine are required to interconvert YAP1 interaction between K14 and K15. (A) Representative co-immunoprecipitations of HeLa cells transfected with untagged K5 and EGFP-tagged WT K14, K15, and mutant K15. Pull-down of endogenous 14-3-3σ is depicted. (B) Differential levels of WT K15, relative to WT K14 or mutant K15, were observed in the soluble fraction. Transfection of keratin(s) did not affect endogenous 14-3-3σ levels. WB intensity was normalized to a loading control (histone H3). Dots represent three independent replicates, displaying mean and standard error. Comparisons were made using one-way ANOVA, ****P < 0.0001, ns = not significant. (B′) Endogenous 14-3-3σ pulls down more efficiently with WT K14 and mutagenized K15, as compared to WT K15. Equation to calculate 14-3-3σ pull-down efficiency is described in Materials and methods. Dots represent three independent replicates. Comparisons were made using one-way ANOVA, **P = 0.0055, *P = 0.0237, ns = not significant. (C) Representative micrographs of YAP1-14-3-3σ PLA performed on transfected HeLa cells. Cells were transfected with untagged K5 and EGFP-tagged WT and mutant K14 or K15. Merge panels show PLA punctae (red), EGFP-tagged keratin autofluorescence (green), and DAPI counterstain (blue). Scale bar = 10 µm. (D) Scatter plots representing YAP1-14-3-3σ PLA punctae counted per cell in three pooled independent replicates, displaying mean and standard deviation. Dots represent a sc. Comparisons were made using Mann–Whitney tests. ****P < 0.0001, **P = 0.0033, ns = not significant. (E) Transcription of a YAP1 target gene, CYR61 , as measured via RT-qPCR in HeLa cells transfected with untagged K5 and WT and mutant K14 or K15. Dots represent six independent replicates. Comparisons were made using Mann–Whitney tests, **P = 0.0033, ns = not significant. Source data are available for this figure: .

Article Snippet: HeLa cells were purchased from ATCC and were routinely tested for Mycoplasma using MycoAlert Mycoplasma Detection Kit (LT07-118; Lonza).

Techniques: Transfection, Mutagenesis, Control, Standard Deviation, MANN-WHITNEY, Quantitative RT-PCR

Functional investigation of the new MCTS1 variants. (A) Predicted structure of the MCTS1 protein with the domain of unknown function 1947 (DUF1947) and the pseudouridine synthase and archaeosine transglycosylase (PUA) domains for WT protein, the synthetic LOF variant p. A109D, and the patient variants p.L170* (P1), p.W175* (P2), and predicted p.E60Kfs5* (P4). The numbers annotated for the WT MCTS1 protein correspond to the first and last amino acids of the MCTS1 protein, the DUF1947, and PUA domains. The variants of MCTS1 are shown in red. (B) Western blot of WT and MCTS1-KO HeLa cells after transfection with EV, WT MCTS1 (pWT), or the MCTS1 variants p.E60Kfs5* (pE60Kfs5*), p.L170* (pL170*), p.W175* (pW175*), and p.A109D (pA109D). Details are provided in . Non-transfected HeLa cells (“−”) were used as a negative control. (C) Schematic diagram of the Fluc and lamin B + stuORF reporters used to assess MCTS1 activity. stuORF, synthetic strong Kozak uORF. Modified from . (D) Activity of the MCTS1 variants in the luciferase translation reinitiation assay after the transfection of WT and MCTS1-KO cells with EV, WT MCTS1 or the various MCTS1 variants (p.E60Kfs5* p.L170*, W175*, and p.A109D; see details in ). Bars: Mean and standard deviation of three technical replicates, represented as turquoise dots. This experiment is representative of the three biological replicates. The asterisks indicate the level of significance, as assessed in a one-way ANOVA with Tukey’s test for multiple comparisons and adjustment for multiple testing (*P < 0.05; **P < 0.005). (E) Sanger sequencing confirmation of KI of the MCTS1 W175* variant and silent L174L variants in HEK293T cells. (F) Western blot of HEK293T cells subjected to genome editing to introduce MCTS1 KO in a pooled manner or MCTS1 W175* KI with the indicated single-cell derived clones shown. (G) Quantitation of three independent western blots of the indicated cell lines. Statistical significance was assessed using two-sided Mann–Whitney tests. Source data are available for this figure: .

Journal: Journal of Human Immunity

Article Title: Complete and partial forms of X-linked MCTS1 deficiency in patients with mycobacterial disease

doi: 10.70962/jhi.20250073

Figure Lengend Snippet: Functional investigation of the new MCTS1 variants. (A) Predicted structure of the MCTS1 protein with the domain of unknown function 1947 (DUF1947) and the pseudouridine synthase and archaeosine transglycosylase (PUA) domains for WT protein, the synthetic LOF variant p. A109D, and the patient variants p.L170* (P1), p.W175* (P2), and predicted p.E60Kfs5* (P4). The numbers annotated for the WT MCTS1 protein correspond to the first and last amino acids of the MCTS1 protein, the DUF1947, and PUA domains. The variants of MCTS1 are shown in red. (B) Western blot of WT and MCTS1-KO HeLa cells after transfection with EV, WT MCTS1 (pWT), or the MCTS1 variants p.E60Kfs5* (pE60Kfs5*), p.L170* (pL170*), p.W175* (pW175*), and p.A109D (pA109D). Details are provided in . Non-transfected HeLa cells (“−”) were used as a negative control. (C) Schematic diagram of the Fluc and lamin B + stuORF reporters used to assess MCTS1 activity. stuORF, synthetic strong Kozak uORF. Modified from . (D) Activity of the MCTS1 variants in the luciferase translation reinitiation assay after the transfection of WT and MCTS1-KO cells with EV, WT MCTS1 or the various MCTS1 variants (p.E60Kfs5* p.L170*, W175*, and p.A109D; see details in ). Bars: Mean and standard deviation of three technical replicates, represented as turquoise dots. This experiment is representative of the three biological replicates. The asterisks indicate the level of significance, as assessed in a one-way ANOVA with Tukey’s test for multiple comparisons and adjustment for multiple testing (*P < 0.05; **P < 0.005). (E) Sanger sequencing confirmation of KI of the MCTS1 W175* variant and silent L174L variants in HEK293T cells. (F) Western blot of HEK293T cells subjected to genome editing to introduce MCTS1 KO in a pooled manner or MCTS1 W175* KI with the indicated single-cell derived clones shown. (G) Quantitation of three independent western blots of the indicated cell lines. Statistical significance was assessed using two-sided Mann–Whitney tests. Source data are available for this figure: .

Article Snippet: WT HeLa cells (Cat# CRM-CCL-2; ATCC, RRID: CVCL_0030) and MCTS1 KO HeLa cells ( ) were cultured in Dulbecco/Vogt modified Eagle’s minimal essential medium (DMEM, Gibco) with 10% fetal-calf serum (FCS) and 100 IU/ml penicillin/streptomycin (Cat# 15140122; Gibco).

Techniques: Functional Assay, Variant Assay, Western Blot, Transfection, Negative Control, Activity Assay, Modification, Luciferase, Standard Deviation, Sequencing, Introduce, Derivative Assay, Clone Assay, Quantitation Assay, MANN-WHITNEY

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