Journal: Genes & Development

Article Title: Plagl1 and Lrrc58 control mammalian body size by triggering target-directed microRNA degradation of miR-322 and miR-503

doi: 10.1101/gad.353138.125

Figure Lengend Snippet: Validation of novel TDMD triggers. ( A ) Schematic of trigger site deletion (Δts) strategy to validate putative TDMD triggers. ( B , D ) Genomic organization of TDMD trigger transcripts with conservation and predicted miRNA base-pairing architecture of the trigger sites. Nucleotides predicted to base-pair with the miRNA are shown in bold. ( C , E ) qRT-PCR analysis of indicated miRNAs, relative to miR-16-5p, in WT and Δts MEFs. Parental MEFs or two independent Δts clones for each trigger site were infected with lentivirus expressing Cas9 and a nontargeting CRISPR guide (sgNT) or Zswim8- targeting guide (sg Zswim8 ). Values were normalized to expression level in sg Zswim8 for each condition. n = 3 technical replicates per clone, with individual data points plotted (mean ± SD shown). P -values were calculated by one-tailed Student's t -test. (*) P < 0.05, (**) P < 0.01, (***) P < 0.001, (****) P < 0.0001. ( F ) qRT-PCR analysis of the indicated miRNAs, relative to U6, in NIH3T3 cells at time points after serum starvation and restimulation. NIH3T3 cells expressing dCas9-KRAB were infected with lentivirus encoding a nontargeting guide (sgNT) or guides targeting Plagl1 or Lrrc58 . Parental NIH3T3 cells were infected with lentivirus expressing Cas9 and a nontargeting guide (sgNT) or Zswim8- targeting guide (sg Zswim8 ). Values were normalized to expression at the 0 h time point for each condition. n = 3 technical replicates per condition, with mean ± SD shown. P -values were calculated by one-tailed Student's t -test comparing sg Plagl1 (dCas9-KRAB) or sg Lrrc58 (dCas9-KRAB) with sgNT (dCas9-KRAB) or comparing sg Zswim8 (Cas9) with sgNT (Cas9). (*) P < 0.05.

Article Snippet: HEK293T and NIH3T3 cells were obtained from ATCC, and immortalized MEFs were generated previously by transfecting primary MEFs with a plasmid expressing SV40 large T antigen ( ).

Techniques: Biomarker Discovery, Quantitative RT-PCR, Clone Assay, Infection, Expressing, CRISPR, One-tailed Test

Journal: Nature Communications

Article Title: Reconstructing epigenomic dynamics through a single-cell multi-epigenome data integration framework

doi: 10.1038/s41467-025-67016-9

Figure Lengend Snippet: a Schematic overview of data integration for multiple sci-mtChIL-seq datasets. RNAPII fragments were counted at the gene level and used as anchors to embed all datasets into a shared low-dimensional space. b Diagram of the skeletal muscle differentiation system using NIH3T3 TetOn3G/MyoD cells. c UMAP visualization of RNAPII counts at the single-cell level. Cells are colored by (left) sampling time point, (middle) paired epigenomic target, and (right) identified cluster. d Cell composition within each cluster. (Top) Distribution by sampling time. (Bottom) Distribution by paired target. e Heatmap of RNAPII accumulation across differentially expressed genes (DEGs). For each cluster, the top 200 cells (ranked by RNAPII count) and the top 50 DEGs (ranked by variance) are shown. f UMAP visualization of RNAPII accumulation levels per cell for representative DEGs. The values indicate niormalized counts of RNAPII for each coding region. g Gene Ontology (GO) enrichment analysis results for each cluster, based on the top 300 DEGs per cluster. h Heatmap showing mean accumulation levels of each epigenomic factor across clusters. i UMAP visualization of the accumulation levels per cell of epigenomic factors for representative DEGs.

Article Snippet: NIH3T3 Tet-On 3G/pCW-MyoD-Blast cells were cultured in DMEM supplemented with 10% Tet-Approved FBS (Takara; 631101).

Techniques: Sampling

Journal: bioRxiv

Article Title: The primary cilium controls programmed cell death via its proteasome-regulating function

doi: 10.64898/2026.01.07.698216

Figure Lengend Snippet: ( A-D ) Immunofluorescence on NIH3T3 cells. Cell nuclei are stained in blue by DAPI. ( A ) Cells undergoing PCD are marked in red by CC3. Cells are serum-deprived. The scale bar (in white) represents a length of 20 μm. At least 1600 cells per genotype were used for these measurements, pooled from three replicates. Data shown as mean ± s.e.m. Statistical evaluation was performed by using two-tailed unpaired t test with Welch’s correction. Asterisks denote statistical significance (**** P <0.0001). ( B ) Cells undergoing PCD are marked in red by CC9. Cells are serum-starved. The scale bar (in white) represents a length of 20 μm. At least 450 cells per genotype were used for quantification, pooled from three replicates. Data shown as mean ± s.e.m. Statistical evaluation was performed by using two-tailed unpaired t test with Welch’s correction. Asterisks denote statistical significance (*** P = 0.0001). ( C ) The ciliary axoneme is stained in green by acetylated α-tubulin and the BB in blue by γ-tubulin. Yellow arrows point to cilia. Serum starvation is indicated by -FCS. The scale bar (in white) represents a length of 10 μm. At least 770 cells per genotype and treatment were used for quantification, pooled from three replicates. Data shown as mean ± s.e.m. Statistical evaluation was performed by using Kruskal-Wallis test with Dunn’s multiple comparisons test. Asterisks denote statistical significance ( Rpgrip1l +/+ + FCS vs. Rpgrip1l +/+ - FCS: **** P <0.0001; Rpgrip1l +/+ + FCS vs. Rpgrip1l -/- - FCS: ** P = 0.0014; Rpgrip1l +/+ - FCS vs. Rpgrip1l - /- + FCS: **** P <0.0001; Rpgrip1l +/+ - FCS vs. Rpgrip1l -/- - FCS: ** P = 0.0065; Rpgrip1l - /- + FCS vs. Rpgrip1l -/- - FCS: *** P = 0.0007). ns = not significant. d, Cells undergoing PCD are marked in red by CC3. Serum starvation is indicated by -FCS. The scale bar (in white) represents a length of 30 μm. At least 900 cells per genotype and treatment were used for these measurements, pooled from three replicates. Data shown as mean ± s.e.m. Statistical evaluation was performed by using Kruskal-Wallis test with Dunn’s multiple comparisons test. Asterisks denote statistical significance ( Rpgrip1l +/+ + FCS vs. Rpgrip1l -/- - FCS: **** P <0.0001; Rpgrip1l +/+ - FCS vs. Rpgrip1l -/- - FCS: **** P <0.0001; Rpgrip1l -/- + FCS vs. Rpgrip1l - /- - FCS: ** P = 0.0037). ns = not significant ( Rpgrip1l +/+ + FCS vs. Rpgrip1l +/+ - FCS: P >0.9999; Rpgrip1l +/+ + FCS vs. Rpgrip1l -/- + FCS: P = 0.3974; Rpgrip1l +/+ - FCS vs. Rpgrip1l - /- + FCS: P = 0.1449).

Article Snippet: NIH3T3 cells (#ACC59) and HEK293 cells (#ACC35) were both purchased by the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ).

Techniques: Immunofluorescence, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: The primary cilium controls programmed cell death via its proteasome-regulating function

doi: 10.64898/2026.01.07.698216

Figure Lengend Snippet: ( A-C ) Immunofluorescence on serum-starved NIH3T3 cells. Cell nuclei are stained in blue by DAPI. Cells undergoing PCD are marked in red by CC3. The scale bars (in white) represent a length of 20 μm. ( A ) At least 580 cells per genotype and treatment were used for these measurements, pooled from three replicates. Data shown as mean ± s.e.m. Statistical evaluation was performed by using Brown-Forsythe and Welch ANOVA test with Dunnett’s T3 multiple comparisons test ( Rpgrip1l +/+ vs. Rpgrip1l -/- : **** P <0.0001; Rpgrip1l +/+ vs. Rpgrip1l -/- + DMSO: **** P <0.0001; Rpgrip1l +/+ vs. Rpgrip1l -/- + EUP: ** P = 0.0025; Rpgrip1l +/+ vs. Rpgrip1l -/- + RAP: ** P = 0.0186; Rpgrip1l -/- vs. Rpgrip1l -/- + SFN: **** P <0.0001; Rpgrip1l -/- + DMSO vs. Rpgrip1l -/- + SFN: **** P <0.0001; Rpgrip1l -/- + EUP vs. Rpgrip1l -/- + SFN: ** P = 0.0022; Rpgrip1l -/- + RAP vs. Rpgrip1l -/- + SFN: * P = 0.0174). ns = not significant. ( B ) At least 780 cells per treatment were used for quantification, pooled from three replicates. Data shown as mean ± s.e.m. Statistical evaluation was performed by using ordinary one-way ANOVA with Tukey’s multiple comparison test. Asterisks denote statistical significance ( Rpgrip1l +/+ vs. Rpgrip1l +/+ + MG132: **** P <0.0001; Rpgrip1l +/+ + DMSO vs. Rpgrip1l +/+ + MG132: **** P <0.0001). ns = not significant. ( C ) At least 940 cells per genotype and treatment were used for quantification, pooled from three replicates. Data shown as mean ± s.e.m. Statistical evaluation was performed by using Brown-Forsythe and Welch ANOVA test with Dunnett’s T3 multiple comparisons test. Asterisks denote statistical significance ( Rpgrip1l +/+ vs. Rpgrip1l -/- : **** P <0.0001; Rpgrip1l +/+ vs. Rpgrip1l -/- + DMSO: **** P <0.0001; Rpgrip1l +/+ vs. Rpgrip1l -/- + MG132: **** P <0.0001). ns = not significant.

Article Snippet: NIH3T3 cells (#ACC59) and HEK293 cells (#ACC35) were both purchased by the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ).

Techniques: Immunofluorescence, Staining, Comparison

Journal: bioRxiv

Article Title: The primary cilium controls programmed cell death via its proteasome-regulating function

doi: 10.64898/2026.01.07.698216

Figure Lengend Snippet: ( A ) Coimmunoprecipitation in NIH3T3 cells. MYC-tagged MOAP1 full-length protein and FLAG-tagged RID (domain of RPGRIP1L) were transiently overexpressed and tested for interaction by coimmunoprecipitation from total cell lysates. Immunoprecipitation assays were performed by using an anti-FLAG antibody. IP, immunoprecipitation; WB, western blot. ( B ) In situ proximity ligation assay ( in situ PLA) on serum-starved NIH3T3 cells. Cell nuclei are stained in blue by DAPI and cilia are marked by the product of a transiently transfected IFT88-EYFP construct. The PLA+ signal is shown in red. The scale bar (in white) represents a length of 2 μm. ( C ) Immunofluorescence on serum-starved NIH3T3 cells. Super-resolution image was acquired by using Airyscan microscopy. The ciliary axoneme is labelled in green by detyrosinated α-tubulin, the basal body is marked in blue by γ-tubulin. MOAP1 is shown in red. The scale bar (in white) represents a length of 5 μm. ( D ) Immunofluorescence on serum-starved NIH3T3 cells. Super-resolution image was acquired by using dSTORM. The ciliary axoneme is labelled in green by detyrosinated α-tubulin, the basal body is marked in turquoise by γ-tubulin. MOAP1 is shown in magenta. The scale bar (in white) represents a length of 1 μm. The depicted cilium is shown in three different modes of representation – the point cloud mode (on the left), the cluster mode (in the middle) and point splatting mode (on the right). In the point splatting mode, the yellow arrow points to the portion of MOAP1 that protrudes into the basal body up to the transition zone (shown in white). ( E ) Immunofluorescence on serum-starved NIH3T3 cells. The ciliary axoneme is stained in green by detyrosinated α-tubulin and the BB in blue by γ-tubulin. The scale bar (in white) represents a length of 1 μm. 20 cilia per genotype were used for quantification, pooled from three replicates. Data shown as mean ± s.e.m. Statistical evaluation was performed by using Kruskal-Wallis test with Dunn’s multiple comparisons test. Asterisks denote statistical significance ( Rpgrip1l +/+ vs. Rpgrip1l -/- : **** P <0.0001; Rpgrip1l +/+ vs. Rpgrip1l -/- + DMSO: **** P <0.0001; Rpgrip1l -/- vs. Rpgrip1l -/- + SFN: **** P <0.0001; Rpgrip1l -/- + DMSO vs. Rpgrip1l -/- + SFN: **** P <0.0001). ns = not significant. ( F ) Immunofluorescence on serum-starved NIH3T3 cells. The ciliary axoneme is stained in green by detyrosinated α-tubulin and the BB in blue by γ-tubulin. The scale bar (in white) represents a length of 1 μm. 20 cilia per genotype were used for quantification, pooled from three replicates. Data shown as mean ± s.e.m. Statistical evaluation was performed by using Kruskal-Wallis test with Dunn’s multiple comparisons test. Asterisks denote statistical significance ( Rpgrip1l +/+ vs. Rpgrip1l -/- : **** P <0.0001; Rpgrip1l +/+ vs. Rpgrip1l -/- + siRNA Ctrl.: **** P = 0.0009; Rpgrip1l -/- vs. Rpgrip1l -/- + siRNA Moap1 : **** P <0.0001; Rpgrip1l - /- siRNA Ctrl. vs. Rpgrip1l -/- + siRNA Moap1 : **** P <0.0001). ns = not significant; Ctrl., control. ( G ) Immunofluorescence on serum-starved NIH3T3 cells. Cell nuclei are stained in blue by DAPI. Cells undergoing PCD are marked in red by CC3. The scale bar (in white) represents a length of 10 μm. At least 800 cells per genotype and treatment were used for quantification, pooled from three replicates. Data shown as mean ± s.e.m. Statistical evaluation was performed by using Brown-Forsythe and Welch ANOVA test with Dunnett’s T3 multiple comparisons test. Asterisks denote statistical significance ( Rpgrip1l +/+ vs. Rpgrip1l -/- : **** P <0.0001; Rpgrip1l +/+ vs. Rpgrip1l -/- + siRNA Ctrl.: *** P = 0.0009; Rpgrip1l -/- vs. Rpgrip1l - /- + siRNA Moap1 : **** P <0.0001; Rpgrip1l -/- + siRNA Ctrl. vs. Rpgrip1l -/- + siRNA Moap1 : ** P = 0.0013). ns = not significant; Ctrl., control.

Article Snippet: NIH3T3 cells (#ACC59) and HEK293 cells (#ACC35) were both purchased by the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ).

Techniques: Immunoprecipitation, Western Blot, In Situ, Proximity Ligation Assay, Staining, Transfection, Construct, Immunofluorescence, Microscopy, Control

Journal: bioRxiv

Article Title: The primary cilium controls programmed cell death via its proteasome-regulating function

doi: 10.64898/2026.01.07.698216

Figure Lengend Snippet: ( A, B ) Proteasomal activity was quantified in total cell lysates of serum-starved NIH3T3 cells. The activity of the 20S proteasomal subunit was measured without ATP. The activity of the 20S proteasomal subunit and the 26S proteasome together was quantified after ATP addition. The activity of the 26S proteasome was determined by subtracting the value measured for the activity of the 20S proteasomal subunit from the value measured for the activity of the 20S proteasomal subunit and the 26S proteasome together. ns = not significant. ( C ) Fluorescence-based proteasome activity assay on serum-starved NIH3T3 cells. The ciliary axoneme is stained in green by acetylated α-tubulin and the BB in blue by γ-tubulin. The ZsProSensor-1 protein signal shines green. The scale bar (in white) represents a length of 0.5 μm.

Article Snippet: NIH3T3 cells (#ACC59) and HEK293 cells (#ACC35) were both purchased by the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ).

Techniques: Activity Assay, Fluorescence, Staining

Journal: bioRxiv

Article Title: The primary cilium controls programmed cell death via its proteasome-regulating function

doi: 10.64898/2026.01.07.698216

Figure Lengend Snippet: ( A, B ) Immunofluorescence on serum-starved NIH3T3 cells. Cell nuclei are stained in blue by DAPI. Cells undergoing PCD are marked in red by CC3. The scale bars (in white) represent a length of 20 μm. ( A ) At least 650 cells per genotype and treatment were used for these measurements, pooled from three replicates. Data shown as mean ± s.e.m. Statistical evaluation was performed by using Kruskal-Wallis test with Dunn’s multiple comparisons test. Asterisks denote statistical significance ( Tctn +/+ vs. Tctn1 -/- : **** P = 0.0001; Tctn1 +/+ vs. Tctn1 -/- + DMSO: **** P <0.0001; Tctn1 -/- vs. Tctn1 -/- + SFN: **** P <0.0001; Tctn1 -/- + DMSO vs. Tctn1 -/- + SFN: **** P <0.0001). ns = not significant. ( B ) At least 790 cells per genotype and treatment were used for these measurements, pooled from three replicates. Data shown as mean ± s.e.m. Statistical evaluation was performed by using Kruskal-Wallis test with Dunn’s multiple comparisons test. Asterisks denote statistical significance ( Cep290 +/+ vs. Cep290 -/- : **** P <0.0001; Cep290 +/+ vs. Cep290 -/- + DMSO: **** P <0.0001; Cep290 -/- vs. Cep290 -/- + SFN: **** P <0.0001; Cep290 -/- + DMSO vs. Cep290 -/- + SFN: **** P <0.0001). ns = not significant. ( C ) TUNEL assay on control + DMSO ( n = 35), control + SFN ( n = 39), cep290 MO + DMSO ( n = 25) and cep290 MO + SFN ( n = 31) Xenopus embryos at stage 34, pooled from three replicates. Data shown as mean ± s.e.m. Statistical evaluation was performed by using two-tailed Mann-Whitney U test. Asterisks denote statistical significance (control + DMSO vs. cep290 MO + DMSO: **** P <0.0001; cep290 MO + DMSO vs. cep290 MO + SFN: **** P <0.0001). ns = not significant.

Article Snippet: NIH3T3 cells (#ACC59) and HEK293 cells (#ACC35) were both purchased by the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ).

Techniques: Immunofluorescence, Staining, TUNEL Assay, Control, Two Tailed Test, MANN-WHITNEY