Fig 2: PTIP inhibits ESCC cell invasion and migration in vivo. (A, B) The effect of PTIP knockdown on tumor growth and metastasis in a mouse model based on tail vein injection. KYSE-150 cells were infected with Scrambled shRNA (shCtrl) or shPTIP#1 lentivirus containing a constitutively expressed luciferase reporter. Then, shScrambled-treated or shPTIP cells were injected into mice via tail veins(n=8), and lung metastases in the two groups were evaluated by an in vivo imaging system(In-Vivo FX PRO). The luciferase signals were compared between the shCtrl group and the shPTIP group. Unpaired, two-tailed Student’s t-test; *P < 0.05. (C) Metastatic lesions in lungs from mice at the 4th week were analyzed by H&E staining. Overall staining patterns were shown at low magnification (original magnification ×5) as a composite figure and zoomed views (original magnification ×20) of the indicated areas (box).

Fig 3: Effect of TENT4A and PAXIP1 on the stability of TLS-related mRNAs and level of TLS-related proteins. (A,B) TENT4A and/or PAXIP1 expression was knocked down in MCF-7 cells for 48 h, after which the cells were treated with 5 µg/mL Actinomycin D for up to 8 h. The expression levels of POLH and RAD18 were each determined by qPCR relative to the expression level at time 0. Half-life was calculated using a non-linear fit one-phase decay curve equation. The significance of the differences was calculated using Student’s t-test (one-tailed); Data are presented as mean ± SEM from three independent experiments. ns, non-significant; p > 0.05. (C) MCF-7 cells were transfected with TENT4A and/or PAXIP1 or non-targeting control siRNA for a final combined concentration of 100 nM. At 65 h post-transfection, the cells were UV-irradiated at 20 J/m2 and harvested 1 or 6 h post-irradiation. Whole-cell extracts were fractionated by SDS-PAGE followed by Western blot with indicated antibodies. Amounts are presented relative to those in extracts of unirradiated cells treated with siControl and the values are shown in the corresponding position of the blots.

Fig 4: MRNIP prevents MRE11 and DNA2-dependent degradation of reversed forks.(A to C) Parental HeLa and HCT116 cells and MRNIP KO CRISPR derivative cell lines were labeled with CldU for 20 min and then IdU for 20 min before the addition of 4 mM HU in the presence or absence of 50 µM mirin for 5 hours (see Fig. 4A). Fork degradation was assessed via the IdU:CldU tract length ratio. A representative set of DNA fibers from each condition is displayed in (A). (D) Parental HeLa and a derivative MRNIP KO cell line were transfected with a nontargeting control siRNA or an siRNA targeting either MRE11 or DNA2. After 48 hours, cells were treated as in (A), and the IdU:CIdU tract length ratio for each condition is shown. (E) MRNIP-A KO or BRCA2 KO HeLa cells were labeled as in (A) and then cotreated with 4 mM HU and either DMSO, PFM39, or DNA2 inhibitor for 5 hours. Fork degradation was assessed via the IdU:CldU tract length ratio. (F and G) MRNIP KO HeLa cells were transfected with a nontargeting control siRNA or siRNAs targeting either PTIP or SMARCAL1. After 48 hours, cells were labeled with CldU for 20 min and then IdU for 20 min before the addition of 4 mM HU for 5 hours. Fork degradation was assessed via the IdU:CldU tract length ratio. (H) Cells were transfected as in (A) and then lysed, and the extracts were resolved by SDS-PAGE followed by blotting with the indicated antibodies. Data represent the mean from three experimental repeats, and the errors displayed represent SD (*P = 0.05 and **P = 0.01 where indicated).

Fig 5: ADINR RNA Regulates Active Chromatin via Binding to PA1(A) RNA pull-down with six different GST-conjugated proteins (WDR5, WDR82, PA1, PTIP, RBBP5, and Menin) from the adipocyte total RNA on day 3. Only GST-PA1 specifically retrieves ADINR RNA.(B) ChIP assay of PA1 in the C/EBPa promoter region. Knockdown of ADINR RNA significantly abrogates the peaks of PA1 occupancy in the region of primers4 and primers5 (see Figure 3A). **p < 0.01, Student’s t test.(C) Affinity of seven truncated ADINR RNA fragments for GST-PA1 (in vitro binding assay). All the panels show means ± SD in three independent experiments.See also Figure S4.