Journal: Development (Cambridge, England)

Article Title: Nelfb promotes dermal white adipose tissue formation through RNA polymerase II-mediated adipogenic gene regulation

doi: 10.1242/dev.204976

Figure Lengend Snippet: Nelfb is necessary for adipocyte differentiation by promoting the expression of adipocyte transcription factors. (A) Oil Red O staining of control (CTL: Nelfb fl/wt Pdgfra-Cre + or Nelfb wt/wt Pdgfra-Cre + ) and Nelfb −/− ( Nelfb fl/fl Pdgfra-Cre + ) cells cultured in adipocyte differentiation medium for 14 days ( N =4). (B) Staining of CTL and Nelfb −/− cells with antibodies against perilipin 1 (Plin1; mature adipocyte marker: green) and Hoechst 33342 (nuclei stain: blue) ( N =4). (C) RNA-seq analysis of CTL and Nelfb −/− cells cultured in adipocyte differentiation medium for 14 days. Volcano plot shows 762 genes increased (red) and 358 genes decreased (blue) on a log 2 scale upon Nelfb depletion. N =3. (D) Gene Ontology (GO) terms of the 358 decreased and 762 increased genes using Enrichr. (E) Heat map of the 43 genes (≥2-fold change) found in the top five GO terms of decreased genes in D. (F) CUT&RUN on CTL and Nelfb −/− cells cultured in adipocyte differentiation medium for 14 days using an anti-Nelfb and anti-IgG antibody. qPCR analysis was conducted on transcription start site (TSS) regions of Pparg , Cebpa , Cebpb , Stat3 and Krox20. Signal was calculated as a percent of total input DNA ( N =3). (G,H) CUT&RUN using an anti-RNA Polymerase II (RNA Pol II) antibody and anti-IgG antibody ( N =3). qPCR analysis was performed on the TSS (G) or gene body (H) regions of Pparg , Cebpa , Stat3 and Krox20 . (I,J) CUT&RUN using an anti-RNA Polymerase II Ser2 phosphorylation (Pol II Ser2 Phospho) antibody and anti-IgG antibody ( N =3). qPCR analysis was performed on the TSS (I) or gene body (J) regions of Pparg , Cebpa , Stat3 and Krox20 . (K) CUT&RUN using active chromatin mark anti-H3K4me3 and anti-IgG antibody. qPCR analysis on TSS regions of Pparg , Cebpa , Stat3 and Krox20 . Signal was calculated as a percent of total input DNA ( N =3). Data are mean±s.e.m. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001 (one-way ANOVA followed by Tukey's multiple comparison tests). ns, not significant. Scale bars: 150 μm.

Article Snippet: Quantities used were: Nelfb: 5 μl/reaction (Cell Signaling Technology, #14894S); RNA Pol II: 3 μl/reaction (Active Motif, #39097); RNA Pol II Ser2 Phosphorylation: 2 μl/reaction (Cell Signaling Technology, #13499S); Nelfe: 5 μl/reaction (Proteintech, #10705-1-AP); IgG: 5 μl/reaction (Cell Signaling Technology, #66362); H3K4me3: 5 μl/reaction (Cell Signaling Technology, #9751S); H3K27me3: 2 μl/reaction (Cell Signaling Technology, #9733S); H3K9me3: 2 μl/reaction (Cell Signaling Technology, #13969S); H3K27Ac: 1 μl/reaction (Cell Signaling Technology, #8173S).

Techniques: Expressing, Staining, Control, Cell Culture, Marker, RNA Sequencing, Phospho-proteomics, Comparison

Journal: Molecular cell

Article Title: PAF1C-mediated activation of CDK12/13 kinase activity is critical for CTD phosphorylation and transcript elongation.

doi: 10.1016/j.molcel.2025.04.012

Figure Lengend Snippet: Figure 5. CDC73-KIM is crucial for CTD phosphorylation, transcript elongation, and cell proliferation (A) Schematic of the switchover system. Following knockdown of endogenous CDC73 (double transfection, 72 and 48 h in total), expression of the recombinant, siRNA resistant, FLAG-tagged CDC73 (FLAG-CDC73*) is induced with doxycycline (Dox; 1 μg/mL) for 24 h. siCDC73, siRNA targeting CDC73; siCon, siRNA targeting control locus. (B) Cell confluence (in %) measured over time using the switchover system. Cell confluence was tracked every 4 h for 5 days. Point indicates mean of technical duplicate and error bars indicate standard deviation. Image is representative of experiment performed in biological triplicate. F, FLAG. (C) Representative images of nascent RNA read counts across SETX and EXT1, with the direction of transcription indicated by arrow. (D) Metagene analysis of non-overlapping coding genes longer than 100 kb (n = 2,217) using library- and spike-in normalized nascent RNA read counts obtained from transient transcriptome chem sequencing (TTchem-seq). Line indicates mean signal of experiment performed in technical triplicate. Shaded area indicates standard error. Si73, siRNA targeting CDC73; CPM, counts per million; TSS, transcription start site; TES, transcription end site; kb, kilobases. (E) Same as (E) except that metagene analysis was performed using non-overlapping coding gene intervals longer than 50 kb (n = 3,860) showing the TSS and first 20 kb. (F) Western blot of chromatin fractions following switchover, measuring total RPB1, Ser2P, Ser5P, and Ser2P-Ser5P signals. Quantification of the normalized ratio of phosphorylated CTD to total RPB1 (NTD) indicated below each blot.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies RPB1 D8L4Y (total, N-terminal) Cell Signaling Technology Cat# 14958S; RRID: AB_2687876 RPB1, serine 2 phosphorylated Kind gift from Dirk Eick 3E10 RPB1, serine 5 phosphorylated Kind gift from Dirk Eick 3E8 RPB1, serine 2 serine5 both phosphorylated Cell Signaling Technology Cat# 13546, D1G3K; RRID:AB_2798253 RPB1, clone 8WG16 The Francis Crick Institute, Cell Services N/A CDK9 (C12F7) Cell Signaling Cat# 2316; RRID:AB_2291505 Cyclin K Bethyl Cat# A301-939A; RRID:AB_1547934 Lamin A Abcam Cat# ab26300; RRID:AB_775965 FLAG (D6W5B) Cell Signaling Cat# 14793; RRID:AB_2572291 GST Abcam Cat# ab19256; RRID: AB_444809 CDK12 Bethyl Cat# A301-679A, RRID:AB_1210919 CTR9 Bethyl Cat# A301-395A, RRID:AB_960973 PAF1 Bethyl Cat# A300-172A, RRID:AB_309394 RTF1 Bethyl Cat# A300-179A, RRID: AB_2185963 Alpha Tubulin Abcam Cat# ab52866, RRID:AB_869989 ATR Santa Cruz Cat# sc-515173, RRID:AB_2893291 FANCD2 (FI17) Santa Cruz Cat# sc-20022, RRID:AB_2278211 CDC73 (Parafibromin) Bethyl Cat# A300-170A, RRID:AB_309449 Anti-Rat IgG (whole molecule)-Peroxidase antibody produced in goat Sigma-Aldrich Cat# A9037, RRID:AB_258429 Peroxidase-AffiniPure Donkey Anti-Mouse IgG Jackson ImmunoResearch Cat# 715-035-150, RRID:AB_2340770 Peroxidase-AffiniPure Donkey Anti-Rabbit IgG Jackson ImmunoResearch Cat# 711-035-152, RRID:AB_10015282 Bacterial and virus strains NEB 5-alpha Competent E. coli One NEB Cat# C2988J DH10Bac Competent Cells Thermo Fisher Scientific Cat# 10361012 Chemicals, peptides, and recombinant proteins Penicillin-Streptomycin (5,000 U/mL) Thermo Fisher Scientific Cat# 15070063 DMEM, GlutaMAX Gibco Cat# 31966-021 Fetal Bovine Serum (FBS), Tetracycline free Biowest Cat# S181T-500 Hygromycin B ENZO life sciences Cat# ALX-380-306-G001 Grace’s Insect Medium Thermo Fisher Scientific Cat# 11595030 Expifectamine Sf transfection reagent Thermo Fisher Scientific Cat# A38915 Sf-900 III insect media Thermo Fisher Scientific Cat# 12658027 InstantBlue® Coomassie Protein Stain (ISB1L) Abcam Cat# ab119211 Doxycycline Clontech Cat# 8634-1 4-thiouridine Glentham Life Sciences Cat# GN6085 MTSEA biotin-XX linker Biotium Cat# BT90066 DynabeadsTM Protein G Invitrogen Cat# 10004D Maxtract high density (200x2ml) Qiagen Cat# 129056 Micro biospin P-30 gel columns Bio-rad Cat# 732-6250 LipofectamineTM 3000 ThermoFisher Scientific Cat# 13000015 Lipofectamine RNAiMAX ThermoFisher Scientific Cat# 13778150 (Continued on next page) e1 Molecular Cell 85, 1952–1967.e1–e8, May 15, 2025

Techniques: Phospho-proteomics, Knockdown, Transfection, Expressing, Recombinant, Control, Standard Deviation, Sequencing, Western Blot

Journal: Molecular cell

Article Title: PAF1C-mediated activation of CDK12/13 kinase activity is critical for CTD phosphorylation and transcript elongation.

doi: 10.1016/j.molcel.2025.04.012

Figure Lengend Snippet: Figure 7. Working model for PAF1C-mediated CDK12 activation and generation of CTD marks during transcript elongation Left, RNAPII-associated PAF1C recruits CDK12/13. Middle, recruitment results in increased CTD Ser2 Ser5 di-phosphorylation and a stable, Ser2 Ser5 di-phosphorylated RNAPII elongation complex, capable of efficient transcript elongation across long genes. Right, toward the end of genes, phosphatases such as SSU72 may associate with RNAPII and de-phosphorylate Ser5, leaving Ser2 mono-phosphorylation. Please note that some of the CTD repeats may be pre- phosphorylated at serine 5 (by CDK7 or CDK9), or Ser2 Ser5 di-phosphorylated (by CDK9), already prior to CDK12/13 action. For simplicity, other components of the elongation complex, such as DSIF and SPT6, are not shown.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies RPB1 D8L4Y (total, N-terminal) Cell Signaling Technology Cat# 14958S; RRID: AB_2687876 RPB1, serine 2 phosphorylated Kind gift from Dirk Eick 3E10 RPB1, serine 5 phosphorylated Kind gift from Dirk Eick 3E8 RPB1, serine 2 serine5 both phosphorylated Cell Signaling Technology Cat# 13546, D1G3K; RRID:AB_2798253 RPB1, clone 8WG16 The Francis Crick Institute, Cell Services N/A CDK9 (C12F7) Cell Signaling Cat# 2316; RRID:AB_2291505 Cyclin K Bethyl Cat# A301-939A; RRID:AB_1547934 Lamin A Abcam Cat# ab26300; RRID:AB_775965 FLAG (D6W5B) Cell Signaling Cat# 14793; RRID:AB_2572291 GST Abcam Cat# ab19256; RRID: AB_444809 CDK12 Bethyl Cat# A301-679A, RRID:AB_1210919 CTR9 Bethyl Cat# A301-395A, RRID:AB_960973 PAF1 Bethyl Cat# A300-172A, RRID:AB_309394 RTF1 Bethyl Cat# A300-179A, RRID: AB_2185963 Alpha Tubulin Abcam Cat# ab52866, RRID:AB_869989 ATR Santa Cruz Cat# sc-515173, RRID:AB_2893291 FANCD2 (FI17) Santa Cruz Cat# sc-20022, RRID:AB_2278211 CDC73 (Parafibromin) Bethyl Cat# A300-170A, RRID:AB_309449 Anti-Rat IgG (whole molecule)-Peroxidase antibody produced in goat Sigma-Aldrich Cat# A9037, RRID:AB_258429 Peroxidase-AffiniPure Donkey Anti-Mouse IgG Jackson ImmunoResearch Cat# 715-035-150, RRID:AB_2340770 Peroxidase-AffiniPure Donkey Anti-Rabbit IgG Jackson ImmunoResearch Cat# 711-035-152, RRID:AB_10015282 Bacterial and virus strains NEB 5-alpha Competent E. coli One NEB Cat# C2988J DH10Bac Competent Cells Thermo Fisher Scientific Cat# 10361012 Chemicals, peptides, and recombinant proteins Penicillin-Streptomycin (5,000 U/mL) Thermo Fisher Scientific Cat# 15070063 DMEM, GlutaMAX Gibco Cat# 31966-021 Fetal Bovine Serum (FBS), Tetracycline free Biowest Cat# S181T-500 Hygromycin B ENZO life sciences Cat# ALX-380-306-G001 Grace’s Insect Medium Thermo Fisher Scientific Cat# 11595030 Expifectamine Sf transfection reagent Thermo Fisher Scientific Cat# A38915 Sf-900 III insect media Thermo Fisher Scientific Cat# 12658027 InstantBlue® Coomassie Protein Stain (ISB1L) Abcam Cat# ab119211 Doxycycline Clontech Cat# 8634-1 4-thiouridine Glentham Life Sciences Cat# GN6085 MTSEA biotin-XX linker Biotium Cat# BT90066 DynabeadsTM Protein G Invitrogen Cat# 10004D Maxtract high density (200x2ml) Qiagen Cat# 129056 Micro biospin P-30 gel columns Bio-rad Cat# 732-6250 LipofectamineTM 3000 ThermoFisher Scientific Cat# 13000015 Lipofectamine RNAiMAX ThermoFisher Scientific Cat# 13778150 (Continued on next page) e1 Molecular Cell 85, 1952–1967.e1–e8, May 15, 2025

Techniques: Activation Assay, Phospho-proteomics

Journal: Molecular cell

Article Title: PAF1C-mediated activation of CDK12/13 kinase activity is critical for CTD phosphorylation and transcript elongation.

doi: 10.1016/j.molcel.2025.04.012

Figure Lengend Snippet: Figure 5. CDC73-KIM is crucial for CTD phosphorylation, transcript elongation, and cell proliferation (A) Schematic of the switchover system. Following knockdown of endogenous CDC73 (double transfection, 72 and 48 h in total), expression of the recombinant, siRNA resistant, FLAG-tagged CDC73 (FLAG-CDC73*) is induced with doxycycline (Dox; 1 μg/mL) for 24 h. siCDC73, siRNA targeting CDC73; siCon, siRNA targeting control locus. (B) Cell confluence (in %) measured over time using the switchover system. Cell confluence was tracked every 4 h for 5 days. Point indicates mean of technical duplicate and error bars indicate standard deviation. Image is representative of experiment performed in biological triplicate. F, FLAG. (C) Representative images of nascent RNA read counts across SETX and EXT1, with the direction of transcription indicated by arrow. (D) Metagene analysis of non-overlapping coding genes longer than 100 kb (n = 2,217) using library- and spike-in normalized nascent RNA read counts obtained from transient transcriptome chem sequencing (TTchem-seq). Line indicates mean signal of experiment performed in technical triplicate. Shaded area indicates standard error. Si73, siRNA targeting CDC73; CPM, counts per million; TSS, transcription start site; TES, transcription end site; kb, kilobases. (E) Same as (E) except that metagene analysis was performed using non-overlapping coding gene intervals longer than 50 kb (n = 3,860) showing the TSS and first 20 kb. (F) Western blot of chromatin fractions following switchover, measuring total RPB1, Ser2P, Ser5P, and Ser2P-Ser5P signals. Quantification of the normalized ratio of phosphorylated CTD to total RPB1 (NTD) indicated below each blot.

Article Snippet: The membranes were incubated with antibodies against Ser2 (3E8), Ser5 (3E10) (1:1000 dilution, gift from D. Eick), Ser2 + Ser5 di-phosphorylated CTD (D1G3K, Cell Signaling), RPB1 N-terminal domain (D8L4Y, Cell Signaling), CDK9 (C12F7, Cell Signaling), Cyclin K (A301-939A, Bethyl), Lamin A (ab26300, Abcam), FLAG (D6W5B, Cell Signaling), GST (Abcam, ab19256), CDK12 (A301-679A, CrkRS, Bethyl), CTR9 (A301-395A, Bethyl), PAF1 (A300-172A, Bethyl), RTF1 (Bethyl, A300-179A), alpha Tubulin (ab52866, Abcam), ATR (sc-515173, Santa Cruz), FANCD2 (FI17, sc-20022, Santa Cruz) and CDC73 (A300-170A, Parafibromin, Bethyl) (1:1,000) overnight at 4◦C.

Techniques: Phospho-proteomics, Knockdown, Transfection, Expressing, Recombinant, Control, Standard Deviation, Sequencing, Western Blot

Journal: Molecular cell

Article Title: PAF1C-mediated activation of CDK12/13 kinase activity is critical for CTD phosphorylation and transcript elongation.

doi: 10.1016/j.molcel.2025.04.012

Figure Lengend Snippet: Figure 7. Working model for PAF1C-mediated CDK12 activation and generation of CTD marks during transcript elongation Left, RNAPII-associated PAF1C recruits CDK12/13. Middle, recruitment results in increased CTD Ser2 Ser5 di-phosphorylation and a stable, Ser2 Ser5 di-phosphorylated RNAPII elongation complex, capable of efficient transcript elongation across long genes. Right, toward the end of genes, phosphatases such as SSU72 may associate with RNAPII and de-phosphorylate Ser5, leaving Ser2 mono-phosphorylation. Please note that some of the CTD repeats may be pre- phosphorylated at serine 5 (by CDK7 or CDK9), or Ser2 Ser5 di-phosphorylated (by CDK9), already prior to CDK12/13 action. For simplicity, other components of the elongation complex, such as DSIF and SPT6, are not shown.

Article Snippet: The membranes were incubated with antibodies against Ser2 (3E8), Ser5 (3E10) (1:1000 dilution, gift from D. Eick), Ser2 + Ser5 di-phosphorylated CTD (D1G3K, Cell Signaling), RPB1 N-terminal domain (D8L4Y, Cell Signaling), CDK9 (C12F7, Cell Signaling), Cyclin K (A301-939A, Bethyl), Lamin A (ab26300, Abcam), FLAG (D6W5B, Cell Signaling), GST (Abcam, ab19256), CDK12 (A301-679A, CrkRS, Bethyl), CTR9 (A301-395A, Bethyl), PAF1 (A300-172A, Bethyl), RTF1 (Bethyl, A300-179A), alpha Tubulin (ab52866, Abcam), ATR (sc-515173, Santa Cruz), FANCD2 (FI17, sc-20022, Santa Cruz) and CDC73 (A300-170A, Parafibromin, Bethyl) (1:1,000) overnight at 4◦C.

Techniques: Activation Assay, Phospho-proteomics