Journal: Journal of Cell Science

Article Title: Phosphorylation of Rab29 at Ser185 regulates its localization and role in the lysosomal stress response in concert with LRRK2

doi: 10.1242/jcs.261003

Figure Lengend Snippet: Lysosomal stress leads to LRRK2-independent phosphorylation of Rab29. (A) Phosphorylation of Rab29. upon CQ treatment in a time-dependent manner in HEK293 cells overexpressing GFP–Rab29. The images are representative of n =4 trials. pRab29 and non-pRab29 indicate phosphorylated and nonphosphorylated Rab29, respectively. Error bars indicate s.e.m.; n =4. ** P <0.01, **** P <0.0001 [one-way ANOVA followed by Dunnett's test against the control (0 h)]. (B) Phosphorylation of endogenous Rab29 in HEK293 and RAW264.7 cell lines. Representative image of n =3 trials. (C) Phosphorylation of a set of Rab proteins with or without CQ in HEK293 cells. A screening of n =1 trial. (D) Comparison of Rab29 phosphorylation upon CQ treatment or by LRRK2 in HEK293 cells. Representative image of n =4 trials. (E) Quantification of the intensity of the lowermost phosphorylated band in D. Error bars indicate s.e.m.; n =4. *** P <0.001 (one-way ANOVA followed by Dunnett's test against the control). (F) Localization of endogenous Rab29 under CQ-treated conditions in RAW264.7 cells. The arrowhead indicates Rab29 colocalization with LAMP1, a lysosomal marker, on enlarged lysosomes. Scale bars: 10 μm. (G) Quantification of lysosomal localization of Rab29 in each field, as shown in F. Error bars indicate s.e.m.; n =3 fields. *** P <0.001 (unpaired, two-tailed Student's t -test against the control). (H) Biochemical detection of endogenous Rab29 in flow through (FT) and lysosome (Lyso) fractions from HEK293 cells treated with or without CQ. LAMP2, α-tubulin and calnexin were also analyzed as markers of lysosome, cytosol and ER membrane, respectively. Representative image of n =3 trials.

Article Snippet: The human embryonic kidney cell line HEK293 (purchased from ATCC, cat. no. CRL-1573), mouse macrophage-like cell line RAW264.7 (purchased from ECACC, cat. no. 91062702), and human tumor-derived cell line HeLaM cells (referred to as HeLa cells throughout this study; RIKEN BRC, cat. no. RCB5388) and human adenocarcinoma derived cell line A549 (purchased from ECACC, cat. no. 86012804) were cultured in Dulbecco's modified Eagle's medium (DMEM; Sigma-Aldrich) with 10% Fetal bovine serum (FBS; Biowest) and 1% penicillin-streptomycin (PS, Gibco) under 5% CO 2 at 37°C.

Techniques: Phospho-proteomics, Control, Comparison, Marker, Two Tailed Test, Membrane

Journal: Journal of Cell Science

Article Title: Phosphorylation of Rab29 at Ser185 regulates its localization and role in the lysosomal stress response in concert with LRRK2

doi: 10.1242/jcs.261003

Figure Lengend Snippet: Phosphorylation of Rab29 could occur on the lysosomal surface. (A) A scheme of the forced localization technique used in this study. Upon treatment with the heterodimerizer AP21967, FKBP-bound Rab29 is anchored away to FRB-positive compartments in the cell. (B) Phosphorylation of FKBP-Rab29 over time upon forced lysosomal localization by AP21967 in HEK293 cells co-expressing LAMP1–FRB. The images are representative of n =4 trials. Error bars indicate s.e.m.; n =4. * P <0.05, ** P <0.01 [one-way ANOVA followed by Dunnett's test against the control (0 h)]. (C,D) Anchoring Rab29 at mitochondria (Fis1) or lysosomes (LAMP1) in HEK293 cells stained with (C) a LAMP1 antibody or (D) MitoTracker Red. Arrowheads indicate Rab29 colocalized with (C) LAMP1 or (D) mitochondria. Scale bars: 10 μm. (E) Phosphorylation of Rab29 upon forced localization to lysosomes (LAMP1) but not to mitochondria (Fis1) in HEK293 cells. Representative image of n =4 trials. (F) Quantification of the phosphorylated bands in E. The percentage of Rab29 phosphorylation (pRab29) was calculated by dividing the intensities of bands indicating pRab29 by the sum of those indicating non-pRab29 and pRab29. n =4. ** P <0.01 (one-way ANOVA followed by Tukey's test).

Article Snippet: The human embryonic kidney cell line HEK293 (purchased from ATCC, cat. no. CRL-1573), mouse macrophage-like cell line RAW264.7 (purchased from ECACC, cat. no. 91062702), and human tumor-derived cell line HeLaM cells (referred to as HeLa cells throughout this study; RIKEN BRC, cat. no. RCB5388) and human adenocarcinoma derived cell line A549 (purchased from ECACC, cat. no. 86012804) were cultured in Dulbecco's modified Eagle's medium (DMEM; Sigma-Aldrich) with 10% Fetal bovine serum (FBS; Biowest) and 1% penicillin-streptomycin (PS, Gibco) under 5% CO 2 at 37°C.

Techniques: Phospho-proteomics, Expressing, Control, Staining

Journal: Journal of Cell Science

Article Title: Phosphorylation of Rab29 at Ser185 regulates its localization and role in the lysosomal stress response in concert with LRRK2

doi: 10.1242/jcs.261003

Figure Lengend Snippet: Ser185 residue of Rab29 is phosphorylated under lysosomal stress. (A) A representative spectrum of LC-MS/MS indicating phosphorylation at Ser31 or Ser185. (B) A sequence of Rab29 showing the putative phosphorylation sites Ser31 and Ser185. (C) Reduced phosphorylation of Rab29 upon alanine substitution of Ser185, but not Ser31, under CQ treatment in HEK293 cells. Representative image of n =5 trials. (D) Confirmation of Ser185 phosphorylation by using phospho-specific antibodies against HEK293 cell samples. Representative image of n =3 trials. (E) No changes in CQ-induced phosphorylation of Rab29 by inhibition of LRRK2 in HEK293 cells. Representative image of n =3 trials. (F) An AlphaFold2-generated structural image of Rab29. The pink residue pointed to by the pink arrow is Ser185. The orange and green chains indicate switch 1 and 2, respectively. α-Helices are colored in red and β-sheets in blue.

Article Snippet: The human embryonic kidney cell line HEK293 (purchased from ATCC, cat. no. CRL-1573), mouse macrophage-like cell line RAW264.7 (purchased from ECACC, cat. no. 91062702), and human tumor-derived cell line HeLaM cells (referred to as HeLa cells throughout this study; RIKEN BRC, cat. no. RCB5388) and human adenocarcinoma derived cell line A549 (purchased from ECACC, cat. no. 86012804) were cultured in Dulbecco's modified Eagle's medium (DMEM; Sigma-Aldrich) with 10% Fetal bovine serum (FBS; Biowest) and 1% penicillin-streptomycin (PS, Gibco) under 5% CO 2 at 37°C.

Techniques: Residue, Liquid Chromatography with Mass Spectroscopy, Phospho-proteomics, Sequencing, Inhibition, Generated

Journal: Journal of Cell Science

Article Title: Phosphorylation of Rab29 at Ser185 regulates its localization and role in the lysosomal stress response in concert with LRRK2

doi: 10.1242/jcs.261003

Figure Lengend Snippet: Phosphomimetics of Ser185 alleviate CQ-induced lysosomal enlargement. (A) Lysosome morphology and Rab29 localization in HEK293 cells expressing GFP–Rab29 WT or Ser185 mutants at steady state. (B) Lysosome morphology and Rab29 localization in HEK293 cells expressing GFP–Rab29 WT or mutants upon 8 h of CQ treatment. Arrowheads indicate lysosomes with Rab29 accumulation. Scale bars: 10 μm. (C) Statistical analysis of lysosomal size in A and B. Each shape shows the area of the largest lysosome in each cell, obtained by elliptical approximation of each immunocytochemistry image. Only lysosomes from all of the Rab29-positive cells were included in this analysis (92–172 cells in each condition). The mean is shown by a black horizontal bar in each sample. **** P <0.0001 [one-way ANOVA followed by Dunnett's test against the control (wild type, no CQ) sample].

Article Snippet: The human embryonic kidney cell line HEK293 (purchased from ATCC, cat. no. CRL-1573), mouse macrophage-like cell line RAW264.7 (purchased from ECACC, cat. no. 91062702), and human tumor-derived cell line HeLaM cells (referred to as HeLa cells throughout this study; RIKEN BRC, cat. no. RCB5388) and human adenocarcinoma derived cell line A549 (purchased from ECACC, cat. no. 86012804) were cultured in Dulbecco's modified Eagle's medium (DMEM; Sigma-Aldrich) with 10% Fetal bovine serum (FBS; Biowest) and 1% penicillin-streptomycin (PS, Gibco) under 5% CO 2 at 37°C.

Techniques: Expressing, Immunocytochemistry, Control

Journal: Journal of Cell Science

Article Title: Phosphorylation of Rab29 at Ser185 regulates its localization and role in the lysosomal stress response in concert with LRRK2

doi: 10.1242/jcs.261003

Figure Lengend Snippet: PKCs are involved in Ser185 phosphorylation and lysosomal localization of Rab29. (A) In vitro kinase assay using recombinant Rab29 and PKCα or PKCε. (B) Phosphorylation of Rab29 with PMA or Go6983 over time in HEK293 cells. (C) Cells were treated with siRNAs for PKC isozymes that were targeted by both PMA and Go6983, and the phosphorylation of Rab29 upon PMA treatment (24 h) was assessed by Phos-tag or anti-phospho-S185 antibody. Overall PKC activity was additionally monitored by detecting phospho-Ser PKC substrates (pS-PKC substrates). Images in A–C are representative of n =3 trials. (D) Rab29 localization upon PMA treatment in RAW264.7 cells. The arrow indicates colocalization of endogenous Rab29 with LAMP1. Scale bars: 10 μm. (E) Quantification of lysosomal localization of Rab29, as shown in D. Error bars indicate s.e.m.; n =3 fields ** P <0.01 (unpaired two-tailed t -test). (F) Lysosomal localization of endogenous Rab29 upon CQ and Go6983 treatment in RAW264.7 cells. Arrowheads indicate enlarged lysosomes with Rab29 accumulation. Scale bars: 10 μm. (G) Quantification of lysosomal localization of Rab29, as shown in F. Error bars indicate s.e.m.; n =3 fields. **** P <0.0001 (one-way ANOVA followed by Tukey's test). (H) Lysosomal localization of endogenous LRRK2 upon CQ and Go6983 treatment in RAW264.7 cells. Arrowheads indicate enlarged lysosomes with Rab29 accumulation. Images in H are representative of three experiments. Scale bars: 10 μm.

Article Snippet: The human embryonic kidney cell line HEK293 (purchased from ATCC, cat. no. CRL-1573), mouse macrophage-like cell line RAW264.7 (purchased from ECACC, cat. no. 91062702), and human tumor-derived cell line HeLaM cells (referred to as HeLa cells throughout this study; RIKEN BRC, cat. no. RCB5388) and human adenocarcinoma derived cell line A549 (purchased from ECACC, cat. no. 86012804) were cultured in Dulbecco's modified Eagle's medium (DMEM; Sigma-Aldrich) with 10% Fetal bovine serum (FBS; Biowest) and 1% penicillin-streptomycin (PS, Gibco) under 5% CO 2 at 37°C.

Techniques: Phospho-proteomics, In Vitro, Kinase Assay, Recombinant, Activity Assay, Two Tailed Test

Journal: The EMBO Journal

Article Title: YTHDC1 m 6 A-dependent and m 6 A-independent functions converge to preserve the DNA damage response

doi: 10.1038/s44318-024-00153-x

Figure Lengend Snippet: ( A ) Schematic representation of p21 endogenously tagged reporter system in A549 cell line. ( B ) Overview of the CRISPR-screening design. A library containing 1680 guide RNAs was cloned into a lentiviral system and used to infect A549 reporter cells. After 4 days of infection, cells were exposed to Nutlin-3a to allow for p53 protein stabilisation and subsequent reporter gene activation. Cells were sorted by FACS based on the level of reporter gene activation. ( C ) Scatter plot of Log 2 Fold Change for sgRNA enrichment in p53-enhanced-response ( Y axis) and p53-attenuated-response ( X axis) populations. The three top candidates for each population are labelled and highlighted in the plot. ( D ) Functional annotation of the top 50 candidates for p53-enhanced and p53-attenuated populations based on their molecular activity. ( E ) In vitro validation of the top candidate for p53-enhanced and p53-attenuated populations. Each gene was independently silenced by siRNA. After silencing, mVenus signal was taken as readout of p21-reporter gene activation. Statistical analysis was performed by paired two-tailed Student’s t test KD versus scramble (SCR) control. ( F ) Pearson correlation score between p53-downstream-effector misregulation (p53 pathway) compared to the expression of the 407 CRISPR-Screening candidates in Lung carcinoma patients from TCGA (LUAD + LUSC patients) with p53 wild-type genotype versus healthy patients. Data information: All data are shown are representative of at least three independent experiments. Data are presented as mean ± s.d. * P ≤ 0.05, ** P ≤ 0.01, paired two-tailed Student’s t test was performed in ( E ). .

Article Snippet: The following human cell lines were employed for this study: A549-p21-Reporter (kindly provided by Dr. Lani F. Wu laboratory), A549 (purchased from ATCC), HeLa (kindly provided by Dr. Tomás Aragon laboratory), HEK293T (purchased from ATCC) and MCF7 (RRID: CVCL_0031) cell lines, which were cultured in DMEM medium (GIBCO), and HCT116 (kindly provided by Dr. Vogelstein’s laboratory) cell line was cultured in RPMI-1640 medium (GIBCO).

Techniques: CRISPR, Clone Assay, Infection, Activation Assay, Functional Assay, Activity Assay, In Vitro, Biomarker Discovery, Two Tailed Test, Control, Expressing

Journal: The EMBO Journal

Article Title: YTHDC1 m 6 A-dependent and m 6 A-independent functions converge to preserve the DNA damage response

doi: 10.1038/s44318-024-00153-x

Figure Lengend Snippet: ( A ) YTHDC1 protein quantification. Cells were transfected with siRNA against TP53 , YTHDC1 or Scramble (SCR) as negative control. After silencing cells were treated to Nutlin-3a, DMSO was used for the untreated condition as negative control. The bar plot shows protein quantification relative to GAPDH level of n = 5 biologically independent experiments. ( B ) c-Myc RNA stability assay. Cells were transfected with siRNA, YTHDC1, or Scramble (SCR) as negative control. After silencing cells were treated with Actinomycin D to stop the transcription. Cells were collected at different time points indicated on the X axis to assess c-Myc RNA level by quantitative RT-qPCR. In vitro transcribed Luciferase RNA was used as spike-in to normalise the signal. ( C ) Comprehensive profile of insertions and deletions (indels) in YTHDC1-KO A549 clone compared to a control A549 cells. ( D ) Representative picture of a Western blot of normal A549 cell line and the YTHDC1-KO A549 clone, together with relative quantification of 3 independent protein extractions from the same clone. ( E ) RNA-level quantification of mature mRNA and pre-mRNA by RT-qPCR for TP53 in A549 cells (grey) and YTHDC1-KO (dark blue). ( F – H ) Correlation plot showing YTHDC1 and TP53 expression (as log2 TPM + 1 pseudocounts) in LUAD samples ( n = 505) ( B ), in LUSC samples ( n = 479) ( C ), and COAD samples ( n = 427) ( D ) from TCGA database (gdc-portal.nci.nih.gov). Correlation P value is calculated using a t-distribution. ( I ) Correlation plot showing YTHDC1 and TP53 expression (as log2 TPM + 1 pseudocounts) in 824 cell lines grouped based on the tissue of origin from DepMap database (Tsherniak et al, , https://depmap.org/portal ). Correlation P value is calculated using a t-distribution. ( J ) RNA-level quantification of mature mRNA by RT-qPCR for TP53 in multiple cell lines. MCF7, HeLa and HCT116 cell lines were transfected with two independent siRNA against YTHDC1 (DC1-1 and -2), or Scramble (SCR) as negative control. ( K ) RNA-level quantification of pre-mRNA by RT-qPCR for TP53 in multiple cell lines. MCF7, HeLa and HCT116 cell lines were transfected with two independent siRNA against YTHDC1 (DC1-1 and -2), or Scramble (SCR) as negative control. Data information: All data are shown are representative of at least three independent experiments. Data are presented as mean ± s.d. ns, not significant P > 0.05, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001, paired two-tailed Student’s t test was performed in ( A , B , D , E , J , K ).

Article Snippet: The following human cell lines were employed for this study: A549-p21-Reporter (kindly provided by Dr. Lani F. Wu laboratory), A549 (purchased from ATCC), HeLa (kindly provided by Dr. Tomás Aragon laboratory), HEK293T (purchased from ATCC) and MCF7 (RRID: CVCL_0031) cell lines, which were cultured in DMEM medium (GIBCO), and HCT116 (kindly provided by Dr. Vogelstein’s laboratory) cell line was cultured in RPMI-1640 medium (GIBCO).

Techniques: Transfection, Negative Control, Stability Assay, Quantitative RT-PCR, In Vitro, Luciferase, Control, Western Blot, Quantitative Proteomics, Expressing, Two Tailed Test

Journal: The EMBO Journal

Article Title: YTHDC1 m 6 A-dependent and m 6 A-independent functions converge to preserve the DNA damage response

doi: 10.1038/s44318-024-00153-x

Figure Lengend Snippet: ( A ) Volcano plot showing differentially expressed genes (DEGs) identified in YTHDC1-silenced cells versus scramble (SCR) cells. Genes with log 2 (FC) > 0.5 and Adjusted P value < 0.001, resulting from the DESeq2 DGA, are considered DEGs. Significantly upregulated, downregulated or not changed genes in the YTHDC1 knocked-down cells are labelled in blue, red or grey colour, respectively. ( B ) Schematic representation of truncated-version proteins for ATR, BIRC6 and SETX in negative control condition (grey) and YTHDC1-depleted condition (cyan), due to the emergence of premature stop-codons. ( C ) Schematic representation of primer design for quantitative PCR to detect spliced and unspliced isoforms of ATR, BIRC6 and SETX . An upstream exon-junction region we selected to normalise signal. ( D ) Plot showing the number of differentially retained introns (blue bondi bars) and 1000 random selections of GENCODE v19 introns (grey bars), classified in 5 different groups depending on the proximity to a YTHDC1-CLIP peak (group 1: peak inside the intron; group 2: peak in adjacent exons; group 3: peak in adjacent introns; group 4: peak anywhere inside the gene; group 5: no peak inside the gene). Error bars represent twice the standard deviation. ( E ) Relative distribution of YTHDC1-CLIP (blue bondi) and YTHDC1 ChIP (cyan) peaks over TSS (defined from TSS to 500 bp downstream) and splicing sites (5’ and 3’ splicing sites) along genome. ( F ) RNA-level quantification of intronic retention ratio by RT-qPCR for ATR, BIRC6 and SETX mRNA in A549 cells (grey) and YTHDC1-KO (dark blue). Data information: DESeq2 Wald test P value was calculated in ( A ). Empirical P value for ( D ) was obtained by randomising 100 times the selected introns to compared. Data are presented as mean ± s.d. ns, not significant P > 0.05, **** P ≤ 0.0001, paired two-tailed Student’s t test was performed in ( D , F ).

Article Snippet: The following human cell lines were employed for this study: A549-p21-Reporter (kindly provided by Dr. Lani F. Wu laboratory), A549 (purchased from ATCC), HeLa (kindly provided by Dr. Tomás Aragon laboratory), HEK293T (purchased from ATCC) and MCF7 (RRID: CVCL_0031) cell lines, which were cultured in DMEM medium (GIBCO), and HCT116 (kindly provided by Dr. Vogelstein’s laboratory) cell line was cultured in RPMI-1640 medium (GIBCO).

Techniques: Negative Control, Real-time Polymerase Chain Reaction, Standard Deviation, Quantitative RT-PCR, Two Tailed Test

Journal: The EMBO Journal

Article Title: YTHDC1 m 6 A-dependent and m 6 A-independent functions converge to preserve the DNA damage response

doi: 10.1038/s44318-024-00153-x

Figure Lengend Snippet: ( A ) Violin plots of the distribution of expression corrected Pausing Index ratio calculated on the ChIP-seq experiment shown in ( A ). All the actively transcribed genes were divided into four different groups based on the presence or absence of YTHDC1 peaks in the TSS (cyan and blue, respectively) and based on the presence or absence of m 6 A in the mRNA (light and dark red, respectively). Pausing index was calculated as log2 RNAPII promoter density/RNAPII gene body density for both cells transfected with scramble (SCR) siRNA and YTHDC1 siRNA. Data are presented as distribution of Pausing Index ratio for YTHDC1 knockdown versus SCR for each category. ( B ) Plot showing the number of differentially retained introns (dark red bars) and 1000 random selections of GENCODE v19 introns (light bars), classified in five different groups depending on the proximity to a ChrMeRIP peak (group 1: peak inside the intron; group 2: peak in adjacent exons; group 3: peak in adjacent introns; group 4: peak anywhere inside the gene; group 5: no peak inside the gene). Error bars represent twice the standard deviation. ( C , D ) RNA-level quantification of mature mRNA and pre-mRNA of TP53 ( C ) and intron retention of ATR ( D ) by RT-qPCR. Cells were transfected with siRNA against METTL3 (dark red), or Scramble (light grey) as a negative control. Statistical analysis was performed by paired two-tailed Student’s t test METTL3-KD versus scramble (SCR) control. ns, not significant P ≥ 0.05, * P ≤ 0.05. Data are presented as mean ± s.d. ( E ) RNA-level quantification of mature mRNA (orange) and pre-mRNA (light orange) by RT-qPCR for TP53 in A549 cells treated with increasing concentrations of STM2457. ( F ) Intron retention measurement for ATR , BIRC6 and SETX by RT-qPCR, normalising signal from their respective upstream exon junction in A549 cells treated with increasing concentrations of STM2457. ( G ) RNA-level quantification of TP53 mRNA and pre-mRNA levels in A549 cells that has been transfected with siRNA for endogenous YTHDC1 and induced expression of wt and mutant. ( H ) RNA-level quantification of intron retention for ATR , BIRC6 and SETX genes in A549 cells that has been transfected with siRNA for endogenous YTHDC1 and induced expression of wt and mutant. Data information: All data are shown are representative of at least three independent experiments. For ( A ), data are presented as mean ± s.d. ns, not significant P > 0.01, **** P ≤ 0.00001, paired two-tailed Student’s t test was performed. Empirical P value for ( B ) was obtained by randomising 100 times the selected introns to compared. For ( C – H ), data are presented as mean ± s.d. ns, not significant P > 0.05, * P ≤ 0.05, ** P ≤ 0.01, **** P ≤ 0.0001, paired two-tailed Student’s t test was performed. .

Article Snippet: The following human cell lines were employed for this study: A549-p21-Reporter (kindly provided by Dr. Lani F. Wu laboratory), A549 (purchased from ATCC), HeLa (kindly provided by Dr. Tomás Aragon laboratory), HEK293T (purchased from ATCC) and MCF7 (RRID: CVCL_0031) cell lines, which were cultured in DMEM medium (GIBCO), and HCT116 (kindly provided by Dr. Vogelstein’s laboratory) cell line was cultured in RPMI-1640 medium (GIBCO).

Techniques: Expressing, ChIP-sequencing, Transfection, Knockdown, Standard Deviation, Quantitative RT-PCR, Negative Control, Two Tailed Test, Control, Mutagenesis

Journal: The EMBO Journal

Article Title: YTHDC1 m 6 A-dependent and m 6 A-independent functions converge to preserve the DNA damage response

doi: 10.1038/s44318-024-00153-x

Figure Lengend Snippet: ( A ) Genome browser tracks for RNAPII ChIP-seq of cell transfected with scramble siRNA (light grey) or siRNA against YTHDC1 (orange), YTHDC1 ChIP (cyan) YTHDC1-CLIP (blue bondi) and ChrMeRIP input (yellow) and m 6 A specific peaks (dark red), showing reads coverage over TP53 locus. Sequencing data were normalised as Fragments Per Kilobase of transcript per Million mapped reads (FPKM). ( B ) Genome browser tracks for total RNA-seq of cell transfected with scramble siRNA (light grey) or siRNA against YTHDC1 (cyan), YTHDC1-CLIP (blue bondi) and ChrMeRIP input (yellow) and m 6 A specific peaks (dark red), showing reads coverage over ATR , BIRC6 and SETX retained introns. Sequencing data were normalised as Fragments Per Kilobase of transcript per Million mapped reads (FPKM). ( C ) RNA-level quantification of mature mRNA by RT-qPCR for METTL3 . Cells were transfected with two independent siRNA against METTL3 (M3-1 and -2) or Scramble (SCR) as negative control. ( D ) RNA-level quantification of spliced or unspliced for BIRC6 and SETX mRNA by RT-qPCR. Cells were transfected with siRNA against METTL3 (dark red), or Scramble (light grey) as negative control. ( E ) TLC quantification of the ratio between total amount of Adenosine nucleotides ( A ) and N6-methyladenosine nucleotides (m 6 A) of one representative replicate. ( F ) Representative western blot of HA-YTHDC1 wt and mutant versions upon doxycycline treatment with 20 and 200 ng/mL, respectively, from one of the experiments performed in Fig. . ( G ) RNA-level quantification of endogenous YTHDC1 upon 3’-UTR designed siRNA transfection. ( H , I ) Parental A549 cells treated with concentrations of Doxycycline applied for YTHDC1 inducible system. Data information: All data are shown are representative of at least three independent experiments. For ( C , D , G ), data are presented as mean ± s.d. ns, not significant P > 0.05, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, paired two-tailed Student’s t test was performed.

Article Snippet: The following human cell lines were employed for this study: A549-p21-Reporter (kindly provided by Dr. Lani F. Wu laboratory), A549 (purchased from ATCC), HeLa (kindly provided by Dr. Tomás Aragon laboratory), HEK293T (purchased from ATCC) and MCF7 (RRID: CVCL_0031) cell lines, which were cultured in DMEM medium (GIBCO), and HCT116 (kindly provided by Dr. Vogelstein’s laboratory) cell line was cultured in RPMI-1640 medium (GIBCO).

Techniques: ChIP-sequencing, Transfection, Sequencing, RNA Sequencing, Quantitative RT-PCR, Negative Control, Western Blot, Mutagenesis, Two Tailed Test

Journal: The EMBO Journal

Article Title: YTHDC1 m 6 A-dependent and m 6 A-independent functions converge to preserve the DNA damage response

doi: 10.1038/s44318-024-00153-x

Figure Lengend Snippet: ( A ) Representative images of COMET assay. Cells were transfected with two independent siRNA against YTHDC1 (DC1-1 and -2), siRNA against TP53 , siRNA against ATR or Scramble (SCR) as negative control. After silencing cells were treated with cisplatin to induce DNA damage. One cell per condition was selected as representative of the total cells analysed. 100 µm are represented in scale bars. ( B ) Quantification of COMET assay experiments. Boxplot showing the percentage of DNA in the tail for the conditions described in ( A ). ( C ) Immunofluorescence quantification of γ-H2AX. Cells were transfected with two independent siRNA against YTHDC1 (DC1-1 and -2), siRNA against TP53 , or Scramble (SCR) as negative control. After silencing cells were treated with cisplatin to induce DNA damage. After γ-H2AX immunostaining the cells were imaged. The boxplots represent the number of foci per area obtained analysing 100 images per replicate. ( D ) Flow cytometry quantification of γ-H2AX. Cells were transfected with siRNA against YTHDC1 , siRNA against ATR , or Scramble (SCR) as negative control. After silencing cells were transfected with a plasmid expressing ATR to rescue the phenotype, or an empty plasmid as a control, then treated with cisplatin to induce DNA damage. After γ-H2AX immunostaining the cells were analysed by flow cytometry. The bar plot represents the average delta signal of γ-H2AX of treated versus cisplatin-treated cells. ( E ) Cell proliferation determined by MTS assay for cells transfected with two independent siRNA against YTHDC1 (DC1-1 and -2), or Scramble (SCR) as negative control. After silencing, viability was measured by absorbance at days indicated on the X axis. ( F ) Colony formation assay in cells transfected with siRNA against YTHDC1 , or Scramble (SCR) as negative control. The bar plot represents the average number of colonies obtained. ( G ) Volume of tumours obtained after subcutaneous injection of A549 transfected with siRNA against YTHDC1 , or Scramble (SCR) as negative control in immune-compromised mice. Twenty-seven days post injection the mice were sacrificed and the relative volume was quantified by imaging, image of tumours above of graph. The bar plot represents the average relative size of n = 6 samples for each condition. Data information: All data are shown are representative of at least three independent experiments, except for ( C ). Data are presented as mean ± s.d. ns, not significant P > 0.05, * P ≤ 0.05, ** P ≤ 0.01, **** P ≤ 0.0001, paired two-tailed Student’s t test was performed in ( B – G ). Boxplots represent the 25th and 75th percentiles, with mean as wider line, and outer line encompass values not considered as outlier, in ( B , C ). .

Article Snippet: The following human cell lines were employed for this study: A549-p21-Reporter (kindly provided by Dr. Lani F. Wu laboratory), A549 (purchased from ATCC), HeLa (kindly provided by Dr. Tomás Aragon laboratory), HEK293T (purchased from ATCC) and MCF7 (RRID: CVCL_0031) cell lines, which were cultured in DMEM medium (GIBCO), and HCT116 (kindly provided by Dr. Vogelstein’s laboratory) cell line was cultured in RPMI-1640 medium (GIBCO).

Techniques: Single Cell Gel Electrophoresis, Transfection, Negative Control, Immunofluorescence, Immunostaining, Flow Cytometry, Plasmid Preparation, Expressing, Control, MTS Assay, Colony Assay, Injection, Imaging, Two Tailed Test

Journal:

Article Title: Comparison of Multiplex PCR Assay with Culture for Detection of Genital Mycoplasmas

doi: 10.1128/JCM.42.4.1528-1533.2004

Figure Lengend Snippet: Nucleotide sequences of primers and probes used

Article Snippet: The following organisms were purchased from the American Type Culture Collection (ATCC): Ureaplasma urealyticum (ATCC 27618), M. genitalium (ATCC 33530), M. hominis (ATCC 23114), M. arthritidis (ATCC 14152), M. salivarium (ATCC 14277), M. fermentans (ATCC 15474), M. ovale (ATCC 23714D), M. penetrans (ATCC 55252), Acholeplasma oculi (ATCC 27350) Chlamydia trachomatis (ATCC VR-902B), Chlamydia pneumoniae (ATCC VR-1310), Candida albicans (ATCC 14000), Escherichia coli (ATCC 25922), Gardnerella vaginalis (ATCC A2508), Neisseria gonorrhoeae (ATCC 49981), Staphylococcus aureus (ATCC 25923), Staphylococcus epidermidis (ATCC 27336), Streptococcus pneumoniae (ATCC 27336), Streptococcus pyogenes (ATCC 19615), and Haemophilus influenzae (ATCC 9006).

Techniques: Sequencing, Multiplex Assay

Journal:

Article Title: Comparison of Multiplex PCR Assay with Culture for Detection of Genital Mycoplasmas

doi: 10.1128/JCM.42.4.1528-1533.2004

Figure Lengend Snippet: Performance of multiplex PCR in mixed specimens. Amplification of DNA from 100 CFU of U. urealyticum, M. hominis, and M. genitalium, either individually or in combination, was carried out. The results are shown on a ethidium bromide-stained agarose gel, and the OD490 was determined by ELOSA.

Article Snippet: The following organisms were purchased from the American Type Culture Collection (ATCC): Ureaplasma urealyticum (ATCC 27618), M. genitalium (ATCC 33530), M. hominis (ATCC 23114), M. arthritidis (ATCC 14152), M. salivarium (ATCC 14277), M. fermentans (ATCC 15474), M. ovale (ATCC 23714D), M. penetrans (ATCC 55252), Acholeplasma oculi (ATCC 27350) Chlamydia trachomatis (ATCC VR-902B), Chlamydia pneumoniae (ATCC VR-1310), Candida albicans (ATCC 14000), Escherichia coli (ATCC 25922), Gardnerella vaginalis (ATCC A2508), Neisseria gonorrhoeae (ATCC 49981), Staphylococcus aureus (ATCC 25923), Staphylococcus epidermidis (ATCC 27336), Streptococcus pneumoniae (ATCC 27336), Streptococcus pyogenes (ATCC 19615), and Haemophilus influenzae (ATCC 9006).

Techniques: Multiplex Assay, Amplification, Staining, Agarose Gel Electrophoresis

Journal:

Article Title: Comparison of Multiplex PCR Assay with Culture for Detection of Genital Mycoplasmas

doi: 10.1128/JCM.42.4.1528-1533.2004

Figure Lengend Snippet: Confirmatory PCR discordant result analysis

Article Snippet: The following organisms were purchased from the American Type Culture Collection (ATCC): Ureaplasma urealyticum (ATCC 27618), M. genitalium (ATCC 33530), M. hominis (ATCC 23114), M. arthritidis (ATCC 14152), M. salivarium (ATCC 14277), M. fermentans (ATCC 15474), M. ovale (ATCC 23714D), M. penetrans (ATCC 55252), Acholeplasma oculi (ATCC 27350) Chlamydia trachomatis (ATCC VR-902B), Chlamydia pneumoniae (ATCC VR-1310), Candida albicans (ATCC 14000), Escherichia coli (ATCC 25922), Gardnerella vaginalis (ATCC A2508), Neisseria gonorrhoeae (ATCC 49981), Staphylococcus aureus (ATCC 25923), Staphylococcus epidermidis (ATCC 27336), Streptococcus pneumoniae (ATCC 27336), Streptococcus pyogenes (ATCC 19615), and Haemophilus influenzae (ATCC 9006).

Techniques: Multiplex Assay