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U2OS cells with inactivation of the WDR75 gene locus and ectopic expression of WDR75 p.T46I present with altered pre-rRNA processing. (A) Sanger sequences of gDNA at the site <t>(guide</t> <t>sequence)</t> of <t>CRISPR/Cas9</t> modification of different U2OS clones ectopically expressing either WDR75 WT or WDR75 p.T46I. (B) Alignments of sequencing profiles from TOPO TA cloned RT-PCR products of U2OS cells modified by CRISPR/Cas9. U2OS cells were treated with CHX 100 µg/ml for 2 h before extracting the RNA. (C) Northern blot analysis of U2OS cells ectopically expressing a pWPI EV, WDR75 WT, or WDR75 p.T46I (M1). The WDR75 gene locus was inactivated (see A and B) in WT- and WDR75 p.T46I-expressing U2OS cells. Indicated radiolabeled probes were used to detect pre-rRNA precursors and mature rRNAs. Statistical analysis presented in C was performed using paired Student’s t test; *P < 0.05; **P < 0.01. Source data are available for this figure: .
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U2OS cells with inactivation of the WDR75 gene locus and ectopic expression of WDR75 p.T46I present with altered pre-rRNA processing. (A) Sanger sequences of gDNA at the site <t>(guide</t> <t>sequence)</t> of <t>CRISPR/Cas9</t> modification of different U2OS clones ectopically expressing either WDR75 WT or WDR75 p.T46I. (B) Alignments of sequencing profiles from TOPO TA cloned RT-PCR products of U2OS cells modified by CRISPR/Cas9. U2OS cells were treated with CHX 100 µg/ml for 2 h before extracting the RNA. (C) Northern blot analysis of U2OS cells ectopically expressing a pWPI EV, WDR75 WT, or WDR75 p.T46I (M1). The WDR75 gene locus was inactivated (see A and B) in WT- and WDR75 p.T46I-expressing U2OS cells. Indicated radiolabeled probes were used to detect pre-rRNA precursors and mature rRNAs. Statistical analysis presented in C was performed using paired Student’s t test; *P < 0.05; **P < 0.01. Source data are available for this figure: .
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U2OS cells with inactivation of the WDR75 gene locus and ectopic expression of WDR75 p.T46I present with altered pre-rRNA processing. (A) Sanger sequences of gDNA at the site <t>(guide</t> <t>sequence)</t> of <t>CRISPR/Cas9</t> modification of different U2OS clones ectopically expressing either WDR75 WT or WDR75 p.T46I. (B) Alignments of sequencing profiles from TOPO TA cloned RT-PCR products of U2OS cells modified by CRISPR/Cas9. U2OS cells were treated with CHX 100 µg/ml for 2 h before extracting the RNA. (C) Northern blot analysis of U2OS cells ectopically expressing a pWPI EV, WDR75 WT, or WDR75 p.T46I (M1). The WDR75 gene locus was inactivated (see A and B) in WT- and WDR75 p.T46I-expressing U2OS cells. Indicated radiolabeled probes were used to detect pre-rRNA precursors and mature rRNAs. Statistical analysis presented in C was performed using paired Student’s t test; *P < 0.05; **P < 0.01. Source data are available for this figure: .
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ICAM-1- and EGFR-KO iPSCs were established using <t>CRISPR-Cas9</t> system (A) ICAM-1-knockout (KO) and EGFR-KO iPSCs were generated using the CRISPR-Cas9 system. Bar plots represent the indel distribution for ICAM-1 and EGFR. (B) Phase images of wild-type (WT), ICAM-1-KO, and EGFR-KO iPSCs. Scale bars, 100 μm. (C) Immunofluorescence analysis of OCT3/4 (red) expression in WT, ICAM-1-KO, and EGFR-KO iPSCs. Nuclei were counterstained with DAPI (blue). Scale bars, 100 μm. (D) The expression levels of ICAM-1 and EGFR in WT, ICAM-1-KO, and EGFR-KO iPSC-derived respiratory organoids were measured by RT-qPCR. Data are shown as mean ± SD ( n = 3, technical replicates); two-tailed Student’s t test (∗∗ p < 0.01). (E) The expression levels of ICAM-1, EGFR, and β-actin in WT, ICAM-1-KO, and EGFR-KO iPSC-derived respiratory organoids were measured by the capillary-based immunoassay.
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ICAM-1- and EGFR-KO iPSCs were established using <t>CRISPR-Cas9</t> system (A) ICAM-1-knockout (KO) and EGFR-KO iPSCs were generated using the CRISPR-Cas9 system. Bar plots represent the indel distribution for ICAM-1 and EGFR. (B) Phase images of wild-type (WT), ICAM-1-KO, and EGFR-KO iPSCs. Scale bars, 100 μm. (C) Immunofluorescence analysis of OCT3/4 (red) expression in WT, ICAM-1-KO, and EGFR-KO iPSCs. Nuclei were counterstained with DAPI (blue). Scale bars, 100 μm. (D) The expression levels of ICAM-1 and EGFR in WT, ICAM-1-KO, and EGFR-KO iPSC-derived respiratory organoids were measured by RT-qPCR. Data are shown as mean ± SD ( n = 3, technical replicates); two-tailed Student’s t test (∗∗ p < 0.01). (E) The expression levels of ICAM-1, EGFR, and β-actin in WT, ICAM-1-KO, and EGFR-KO iPSC-derived respiratory organoids were measured by the capillary-based immunoassay.
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ICAM-1- and EGFR-KO iPSCs were established using <t>CRISPR-Cas9</t> system (A) ICAM-1-knockout (KO) and EGFR-KO iPSCs were generated using the CRISPR-Cas9 system. Bar plots represent the indel distribution for ICAM-1 and EGFR. (B) Phase images of wild-type (WT), ICAM-1-KO, and EGFR-KO iPSCs. Scale bars, 100 μm. (C) Immunofluorescence analysis of OCT3/4 (red) expression in WT, ICAM-1-KO, and EGFR-KO iPSCs. Nuclei were counterstained with DAPI (blue). Scale bars, 100 μm. (D) The expression levels of ICAM-1 and EGFR in WT, ICAM-1-KO, and EGFR-KO iPSC-derived respiratory organoids were measured by RT-qPCR. Data are shown as mean ± SD ( n = 3, technical replicates); two-tailed Student’s t test (∗∗ p < 0.01). (E) The expression levels of ICAM-1, EGFR, and β-actin in WT, ICAM-1-KO, and EGFR-KO iPSC-derived respiratory organoids were measured by the capillary-based immunoassay.
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ICAM-1- and EGFR-KO iPSCs were established using <t>CRISPR-Cas9</t> system (A) ICAM-1-knockout (KO) and EGFR-KO iPSCs were generated using the CRISPR-Cas9 system. Bar plots represent the indel distribution for ICAM-1 and EGFR. (B) Phase images of wild-type (WT), ICAM-1-KO, and EGFR-KO iPSCs. Scale bars, 100 μm. (C) Immunofluorescence analysis of OCT3/4 (red) expression in WT, ICAM-1-KO, and EGFR-KO iPSCs. Nuclei were counterstained with DAPI (blue). Scale bars, 100 μm. (D) The expression levels of ICAM-1 and EGFR in WT, ICAM-1-KO, and EGFR-KO iPSC-derived respiratory organoids were measured by RT-qPCR. Data are shown as mean ± SD ( n = 3, technical replicates); two-tailed Student’s t test (∗∗ p < 0.01). (E) The expression levels of ICAM-1, EGFR, and β-actin in WT, ICAM-1-KO, and EGFR-KO iPSC-derived respiratory organoids were measured by the capillary-based immunoassay.
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ICAM-1- and EGFR-KO iPSCs were established using <t>CRISPR-Cas9</t> system (A) ICAM-1-knockout (KO) and EGFR-KO iPSCs were generated using the CRISPR-Cas9 system. Bar plots represent the indel distribution for ICAM-1 and EGFR. (B) Phase images of wild-type (WT), ICAM-1-KO, and EGFR-KO iPSCs. Scale bars, 100 μm. (C) Immunofluorescence analysis of OCT3/4 (red) expression in WT, ICAM-1-KO, and EGFR-KO iPSCs. Nuclei were counterstained with DAPI (blue). Scale bars, 100 μm. (D) The expression levels of ICAM-1 and EGFR in WT, ICAM-1-KO, and EGFR-KO iPSC-derived respiratory organoids were measured by RT-qPCR. Data are shown as mean ± SD ( n = 3, technical replicates); two-tailed Student’s t test (∗∗ p < 0.01). (E) The expression levels of ICAM-1, EGFR, and β-actin in WT, ICAM-1-KO, and EGFR-KO iPSC-derived respiratory organoids were measured by the capillary-based immunoassay.
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ICAM-1- and EGFR-KO iPSCs were established using <t>CRISPR-Cas9</t> system (A) ICAM-1-knockout (KO) and EGFR-KO iPSCs were generated using the CRISPR-Cas9 system. Bar plots represent the indel distribution for ICAM-1 and EGFR. (B) Phase images of wild-type (WT), ICAM-1-KO, and EGFR-KO iPSCs. Scale bars, 100 μm. (C) Immunofluorescence analysis of OCT3/4 (red) expression in WT, ICAM-1-KO, and EGFR-KO iPSCs. Nuclei were counterstained with DAPI (blue). Scale bars, 100 μm. (D) The expression levels of ICAM-1 and EGFR in WT, ICAM-1-KO, and EGFR-KO iPSC-derived respiratory organoids were measured by RT-qPCR. Data are shown as mean ± SD ( n = 3, technical replicates); two-tailed Student’s t test (∗∗ p < 0.01). (E) The expression levels of ICAM-1, EGFR, and β-actin in WT, ICAM-1-KO, and EGFR-KO iPSC-derived respiratory organoids were measured by the capillary-based immunoassay.
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ICAM-1- and EGFR-KO iPSCs were established using <t>CRISPR-Cas9</t> system (A) ICAM-1-knockout (KO) and EGFR-KO iPSCs were generated using the CRISPR-Cas9 system. Bar plots represent the indel distribution for ICAM-1 and EGFR. (B) Phase images of wild-type (WT), ICAM-1-KO, and EGFR-KO iPSCs. Scale bars, 100 μm. (C) Immunofluorescence analysis of OCT3/4 (red) expression in WT, ICAM-1-KO, and EGFR-KO iPSCs. Nuclei were counterstained with DAPI (blue). Scale bars, 100 μm. (D) The expression levels of ICAM-1 and EGFR in WT, ICAM-1-KO, and EGFR-KO iPSC-derived respiratory organoids were measured by RT-qPCR. Data are shown as mean ± SD ( n = 3, technical replicates); two-tailed Student’s t test (∗∗ p < 0.01). (E) The expression levels of ICAM-1, EGFR, and β-actin in WT, ICAM-1-KO, and EGFR-KO iPSC-derived respiratory organoids were measured by the capillary-based immunoassay.
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Image Search Results


U2OS cells with inactivation of the WDR75 gene locus and ectopic expression of WDR75 p.T46I present with altered pre-rRNA processing. (A) Sanger sequences of gDNA at the site (guide sequence) of CRISPR/Cas9 modification of different U2OS clones ectopically expressing either WDR75 WT or WDR75 p.T46I. (B) Alignments of sequencing profiles from TOPO TA cloned RT-PCR products of U2OS cells modified by CRISPR/Cas9. U2OS cells were treated with CHX 100 µg/ml for 2 h before extracting the RNA. (C) Northern blot analysis of U2OS cells ectopically expressing a pWPI EV, WDR75 WT, or WDR75 p.T46I (M1). The WDR75 gene locus was inactivated (see A and B) in WT- and WDR75 p.T46I-expressing U2OS cells. Indicated radiolabeled probes were used to detect pre-rRNA precursors and mature rRNAs. Statistical analysis presented in C was performed using paired Student’s t test; *P < 0.05; **P < 0.01. Source data are available for this figure: .

Journal: Journal of Human Immunity

Article Title: Ribosomal RNA processing impairments in a B cell immunodeficient patient with WDR75 variants

doi: 10.70962/jhi.20250061

Figure Lengend Snippet: U2OS cells with inactivation of the WDR75 gene locus and ectopic expression of WDR75 p.T46I present with altered pre-rRNA processing. (A) Sanger sequences of gDNA at the site (guide sequence) of CRISPR/Cas9 modification of different U2OS clones ectopically expressing either WDR75 WT or WDR75 p.T46I. (B) Alignments of sequencing profiles from TOPO TA cloned RT-PCR products of U2OS cells modified by CRISPR/Cas9. U2OS cells were treated with CHX 100 µg/ml for 2 h before extracting the RNA. (C) Northern blot analysis of U2OS cells ectopically expressing a pWPI EV, WDR75 WT, or WDR75 p.T46I (M1). The WDR75 gene locus was inactivated (see A and B) in WT- and WDR75 p.T46I-expressing U2OS cells. Indicated radiolabeled probes were used to detect pre-rRNA precursors and mature rRNAs. Statistical analysis presented in C was performed using paired Student’s t test; *P < 0.05; **P < 0.01. Source data are available for this figure: .

Article Snippet: KO score for each sequence was evaluated using Inference of CRISPR Edits (RRID:SCR_024508) software (Performance Analysis, 2019. v3.0; Synthego).

Techniques: Expressing, Sequencing, CRISPR, Modification, Clone Assay, Reverse Transcription Polymerase Chain Reaction, Northern Blot

ICAM-1- and EGFR-KO iPSCs were established using CRISPR-Cas9 system (A) ICAM-1-knockout (KO) and EGFR-KO iPSCs were generated using the CRISPR-Cas9 system. Bar plots represent the indel distribution for ICAM-1 and EGFR. (B) Phase images of wild-type (WT), ICAM-1-KO, and EGFR-KO iPSCs. Scale bars, 100 μm. (C) Immunofluorescence analysis of OCT3/4 (red) expression in WT, ICAM-1-KO, and EGFR-KO iPSCs. Nuclei were counterstained with DAPI (blue). Scale bars, 100 μm. (D) The expression levels of ICAM-1 and EGFR in WT, ICAM-1-KO, and EGFR-KO iPSC-derived respiratory organoids were measured by RT-qPCR. Data are shown as mean ± SD ( n = 3, technical replicates); two-tailed Student’s t test (∗∗ p < 0.01). (E) The expression levels of ICAM-1, EGFR, and β-actin in WT, ICAM-1-KO, and EGFR-KO iPSC-derived respiratory organoids were measured by the capillary-based immunoassay.

Journal: Molecular Therapy. Nucleic Acids

Article Title: Dual inhibition of intercellular adhesion molecule-1 and nucleolin reduces RSV infection efficiency in human respiratory organoids

doi: 10.1016/j.omtn.2026.102932

Figure Lengend Snippet: ICAM-1- and EGFR-KO iPSCs were established using CRISPR-Cas9 system (A) ICAM-1-knockout (KO) and EGFR-KO iPSCs were generated using the CRISPR-Cas9 system. Bar plots represent the indel distribution for ICAM-1 and EGFR. (B) Phase images of wild-type (WT), ICAM-1-KO, and EGFR-KO iPSCs. Scale bars, 100 μm. (C) Immunofluorescence analysis of OCT3/4 (red) expression in WT, ICAM-1-KO, and EGFR-KO iPSCs. Nuclei were counterstained with DAPI (blue). Scale bars, 100 μm. (D) The expression levels of ICAM-1 and EGFR in WT, ICAM-1-KO, and EGFR-KO iPSC-derived respiratory organoids were measured by RT-qPCR. Data are shown as mean ± SD ( n = 3, technical replicates); two-tailed Student’s t test (∗∗ p < 0.01). (E) The expression levels of ICAM-1, EGFR, and β-actin in WT, ICAM-1-KO, and EGFR-KO iPSC-derived respiratory organoids were measured by the capillary-based immunoassay.

Article Snippet: Analysis was done using inference of CRISPR edits (ICE) ( https://www.synthego.com/help/ice ) showed that the KO scores for ICAM-1-KO and EGFR-KO iPSCs were nearly 100 ( A and A).

Techniques: CRISPR, Knock-Out, Generated, Immunofluorescence, Expressing, Derivative Assay, Quantitative RT-PCR, Two Tailed Test